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Scientific Working Group

Report on African trypanosomiasis (sleeping sickness)

4-8 June 2001 Geneva, Switzerland

www.who.int/tdr

TDR/SWG/01 Original: English Report of the Scientific Working Group meeting on African trypanosomiasis Geneva, 4-8 June, 2001 TDR/SWG/01

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Message from the Executive Director, CDS ...... vi

Message from the Director, TDR ...... vii

Preface ...... viii

1 Executive summary ...... 1

2 Overview and objectives ...... 3

3 Epidemiology, disease surveillance and control EMERGENCE AND RE-EMERGENCE ...... 7 EPIDEMIOLOGY ...... 8 Reservoir Studies ...... 8 Incidence and Prevalence ...... 9 SURVEILLANCE AND INTERVENTION FOR CONTROL ...... 9 Diagnostics ...... 9 Vectors ...... 10 Changing Institutional Environment ...... 10 SOCIOECONOMIC AND BEHAVIOURAL ASPECTS ...... 11 RECOMMENDATIONS ...... 12

4 Drug development, preclinical and clinical studies, and drug resistance PRECLINICAL STUDIES Resistance to Arsenicals and Diamidine ...... 13 Blood–Brain Barrier ...... 14 Role of CNS Trypanosomes ...... 15 Drug Discovery and Drug Targets ...... 15 RECOMMENDATIONS ...... 16

CLINICAL STUDIES ...... 17 African trypanosomiasis Clinical Aspects of Treatment Failure and Monitoring ...... 17 Application of Existing Drugs ...... 17 Early-stage drugs ...... 17 Late-stage drugs ...... 17 Other potential drugs ...... 17 New Compounds ...... 17

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 iii Combination Therapy ...... 18 Follow-up of Treatment ...... 18 Prevention and Management of Encephalopathy Syndromes ...... 19 Coordination of Clinical Trials ...... 19 Recommendations ...... 20

5 Pathogenesis, genomics and applied genomics PATHOGENESIS Background ...... 21 Trypanosome Biological Phenotype ...... 22 RECOMMENDATIONS ...... 22 APPLIED GENOMICS ...... 23 Trypanosoma brucei Genomics ...... 23 RECOMMENDATIONS ...... 23 TSETSE GENETICS ...... 24 RECOMMENDATIONS ...... 25

6 Cross-cutting issues RESOURCE FLOW ...... 27 ADVOCACY AND MARKETING FOR SLEEPING SICKNESS ...... 28 INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING ...... 28 SOUTH-SOUTH COLLABORATION ...... 29 RECOMMENDATIONS ...... 30

Annex 1 ...... 31 RESOURCE FLOW FOR AFRICAN TRYPANOSOMIASIS ...... 32

Annex 2 ...... 33 A POSITION PAPER ON AFRICAN TRYPANOSOMIASIS ...... 34

African trypanosomiasis Annex 3 EMERGENCE AND RE-EMERGENCE OF HUMAN AFRICAN TRYPANOSOMIASIS ...... 42 I The situation in Angola ...... 43 II The situation in Tanzania ...... 45 III The situation in Uganda and Sudan ...... 47

iv Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Annex 4 EPIDEMIOLOGY, DISEASE SURVEILLANCE AND CONTROL, AND VECTOR CONTROL ...... 54 I Epidemiology and control of human African trypanosomiasis ...... 55 II Vector control in relation to human African trypanosomiasis ...... 73 III A basis for financial decision-making on strategies for the control of human African trypanosomiasis ...... 77

Annex 5 DRUG DEVELOPMENT, PRECLINICAL AND CLINICAL STUDIES, AND DRUG RESISTANCE . . . . . 90 I Drug development for African trypanosomiasis ...... 91 II Drug resistance in sleeping sickness ...... 96 III Human African trypanosomiasis ...... 112 IV Treatment and clinical studies: Working paper for the Scientific Working Group on Human African Trypanosomiasis ...... 114

Annex 6 PATHOGENESIS, GENOMICS AND APPLIED GENOMICS ...... 120 I Discussion document on pathogenesis/applied genomics ...... 121 II Applied genomics: Prospects for control of African trypanosomiasis via the tsetse vector ...... 140 III Applied genomics and bioinformatics ...... 150

Annex 7 INSTITUTIONAL CAPABILITY STRENGTHENING ...... 163 Institutional development and capacity building in countries endemic for sleeping sickness ...... 164

Annex 8 LIST OF PARTICIPANTS ...... 167 African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 v Message from the Executive Director, Communicable Diseases

Following the appointment of Dr Brundtland as Director-General of the World Health Organization (WHO) in 1998, functional restructuring placed both the control of infectious diseases and tropical diseases research under one cluster. This has permitted better identification of priorities and the linking of research with prevention and control activities, resulting in joint fundraising. The recent agreement with the drug industry on chemotherapy of African trypanosomiasis is a product of such joint activities. It is hoped that other agreements emphasizing sleeping sickness will also be signed.

In addition, there has been a concerted effort to move infectious diseases higher in the economic development agenda in various world economic fora, resulting in political commitment by governments of both developed and disease endemic countries, and in financial commitment by members of the G8, in particular France and the United States of America, to the Global Fund for AIDS and Health. Currently, the Global Fund is directed at the three major (based on morbidity and mortality data) infectious diseases – namely malaria, tuberculosis and AIDS. This will lead to improved health delivery systems that will be better placed to deal with other diseases such as sleeping sickness. It is envisaged that the health delivery systems will undergo diversification, allowing governments, NGOs and the private sector to work together to deliver health services more effectively. This will be of particular importance for diseases such as sleeping sickness, where NGOs have had to be depended on for continued support during periods of decreased resources.

This meeting aims to draw up a well thought out research agenda, provide data that can be used to convince policy-makers to place infectious diseases at the forefront of development activities, and show the world that the job on infectious diseases is not yet done.

David L Heymann

African trypanosomiasis World Health Organization Geneva, June 2001

vi Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Message from the Director, TDR

In 1998, the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) underwent an external review. It was noted that, with the reorganization of the Programme into functional units, certain aspects of disease focus had been neglected. It was decided, therefore, that TDR should hold scientific working group (SWG) meetings to address each of the ten diseases handled by the Programme. Two of these SWG meetings, this SWG on African trypanosomiasis being the first, will be held each year in order that all ten diseases are covered in five years. It is expected that this first meeting will set a research agenda for African trypanosomiasis, closely linked to control needs and open to the opportunities that science and technology can provide, which will act as a guide not only to TDR but also to other parties interested in research on African trypanosomiasis.

Funding through TDR is on the increase, and a full-time disease research coordinator for African trypanosomiasis is to be recruited. This is a reflection of growing donor interest in the disease, for which a significant amount of funding has already been assured. However, the funding available is largely restricted to particular projects and limited in geographical coverage to a small proportion of the 250 known foci of sleeping sickness in Africa. Furthermore, the number and mix of donors is limited, as is the period covered by the current funding agreement, which is only for five years. Securing resources for the period following these five years is therefore a priority. Strategies to attract more funding will include creating a supportive environment, joining forces with others of like mind, and using the voices of affected countries. The current matrix approach to programme management in TDR lays great emphasis on accountability and gives a certain amount of funding security to diseases that are unlikely to receive additional funding from alternative sources.

Carlos M Morel Director, TDR Geneva, June 2001 African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 vii Preface

During the last ten years, sleeping sickness has only been marginally recognized as a health problem and a research priority, but the recent re-emerging outbreaks and increasing drug resistance have painfully demonstrated the potential danger in any endemic area. Although sleeping sickness was brought to low endemic levels practically everywhere in Africa during the sixties, since then, most national sleeping sickness control programmes have gradually been sacrificed in favour of more prominent health needs. As a result, the core of national know-how simply disappeared while dependency on external support was rendered even greater. Recent outbreaks have opened eyes worldwide as to how serious the situation is, and increasing awareness is manifest in scientific publications, the layman’s press, and television programmes. The donor community, as well as industry, has been alerted, and is now providing unprecedented levels of support to both control and research. As this report states, it was an opportune time to convene a Scientific Working Group.

This TDR Scientific Working Group was asked to provide technical guidance to donors, responsible officers at national level, and research institutions as to the concrete directions research should take, in the Group’s opinion, and where resources should be made available. The relatively large group of approximately 30 participants permitted wide geographical representation and ample coverage in terms of scientific discipline. In order to guarantee that the recommendations issuing from the meeting were critical, the SWG decided to restrict these to the two of highest priority for each subsection of the report.

This report briefly reviews the recent emergence and re-emergence of trypanosomiasis, and elaborates on as yet unanswered questions and issues such as the role of animal reservoirs in T. b. gambiense transmission, the need for epidemiological indicators for monitoring control interventions, and the lack of a “gold standard” for new and existing diagnostic tests. One research area pertinent to the identification of the reservoir of infection and transmission patterns would be the improvement and validation of the current bloodmeal analysis technology. New tools for vector control per se are not considered a priority at this moment – “the tool box is full” – but research into the question of why relatively little use is being made of the currently available tools is indicated. African trypanosomiasis In the past, practically everywhere, trypanosomiasis control was in the hands of vertical programmes, which meant a great deal of adaptation was needed, both mentally and logistically, to integrate personnel and equipment into the general health services. Although in a few countries this took place gradually and more or less satisfactorily, in countries with a large number of endemic foci and where large numbers of personnel and facilities were involved, integration was, and remains, a complicated process. In such cases, local studies on new delivery pathways are urgently needed.

viii Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 The disability adjusted life year (DALY) has finally found grace in socioeconomic reasoning and is now being applied in several economic analyses for trypanosomiasis control. However, the data collected so far indicate that further refinement is needed to be able to compare the cost-effectiveness of the different control approaches. Also, little is yet known of the personal costs a patient spends on seeking care, his/her participation in cost sharing, or the costs of death and incapacity for the family.

The arsenal of trypanocidal drugs, being scarce already, is becoming more and more limited due to increasing drug resistance. As an immediate relief measure, applied research to help develop standard protocols for optimal use of combinations of currently available compounds is needed. As a long-range objective, the highest priority should be given to laboratory research to develop new molecules.

When Mott published, in 1899, his classic report demonstrating perivascular infiltration to be the main histopathological characteristic of sleeping sickness, he probably would not have imagined how, a good hundred years later, specialists would still be busy following up the pathogenesis of these brain lesions. Although only a few research groups have been involved up till now, this field of interesting research is relevant to the possible development of non- invasive tests for determining the stage of disease. Pathogenesis research could also lead to prevention and treatment of the fatal reactive encephalopathies which occur during treatment.

Much attention has been paid in this report to genomics as a means of refining parasite identification, and to genetics for studies on the vectorial capacity of tsetse flies. The T. brucei genome network of collaborating centres, initiated by TDR jointly with other institutions, is expected to strengthen functional links at an international level. Emphasis is placed on strengthening laboratories in Africa for participation in the work on bioinformatics and genomics.

As a result of the lack of career openings, a high proportion of ex-trainees has left the trypanosomiasis field. One of the recommendations of the Group is to improve the position of

local scientists in African research institutes by allowing for salary supplements. Making African trypanosomiasis research careers more attractive and stable should eventually result in establishing an accept- able core of scientists in the endemic countries. Although the drain of specific knowledge and experience to elsewhere has been disappointing, the Group has no doubt that continued training is the only solution to filling the gap in availability of scientists and technicians in endemic countries.

Considering the improved funding perspectives for both control and research, it is now opportune to make efforts to enhance collaboration between the two. In order to be able to generate

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 ix valid disease data from each endemic country, it is recommended that a research nucleus group be identified and that inter-country collaboration and comparison of results between endemic countries be strongly advocated.

With this up-to-date review of the present epidemiological situation and the actual control needs, the Scientific Working Group calls for a response from the scientific community all over the world. The improved funding possibilities that exist at the present time provide the necessary momentum for introducing new research and control projects and for strengthening relevant ongoing programmes.

Peter de Raadt, SWG Chairperson African trypanosomiasis

x Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 1Executive summary

The Scientific Working Group (SWG) meeting It was noted that recent scientific advances in on African trypanosomiasis brought together a applied genomics and bioinformatics provide multidisciplinary group of scientists, partners opportunities that can be exploited to provide and collaborators from academia, public and new tools for control of disease, and recent sup- private sectors, sleeping sickness control pro- port from the pharmaceutical industry and a pri- grammes, and both disease endemic and disease vate foundation has given impetus to tackling the non-endemic countries. The objectives of the problem of African trypanosomiasis. However, meeting were to chart out a global research the challenges of obtaining adequate donor sup- agenda on African trypanosomiasis, closely port and commitment of governments of endemic linked to control needs and open to opportunities countries, and of personnel recruitment and arising from basic science, to guide TDR and retention, are daunting. Research on African try- other parties interested in research on African panosomiasis is an international effort and the trypanosomiasis and provide data that can be need for partnerships cannot be overemphasized. used in advocacy to convince policy-makers and donor agencies to place control of this disease The meeting provided an opportunity to identify higher on their agenda. knowledge that could be exploited for developing new, and improving existing, tools for manage- The Group reviewed the current status of know- ment of disease and vectors, and to determine the ledge and made recommendations on what tools needs for research capability strengthening in are needed for appropriate and effective manage- basic sciences in disease endemic countries. ment and control of sleeping sickness. Three TDR’s comparative advantage in enhancing exist- broad areas were considered: ing, and developing new partnerships for maxi- • Epidemiology, disease surveillance and mal application of knowledge was highlighted. control. The following are the highest priority recommen- • Drug development, preclinical and clinical dations made by the SWG. The SWG: studies, drug resistance. • noted with concern that sleeping sickness is a • Pathogenesis and applied genomics. re-emerging disease which is not given due attention by governments of endemic countries Considerable progress has been achieved in or the international donor community until it research on African trypanosomiasis in the fol- attains epidemic proportions, and recommend- lowing areas: diagnosis and development of ed that the disease burden and cost effective- diagnostic tests; epidemiology, host-parasite-

ness of control strategies be calculated to show African trypanosomiasis vector relationships, animal reservoirs; develop- that the social and economic consequences of ment of tsetse traps and screens; understanding epidemics outweigh the cost of maintaining of the pathology of the disease; and, drug target- surveillance; ed biochemistry of trypanosomes. However, this progress has not been matched in control of the • noted that lack of appropriate field applicable disease, due to lack of capacity to sustain diagnostic tools for disease detection, and improved interventions and to civil disorder in stage of disease, critically affect the control of some endemic countries. sleeping sickness, and recommended that simple non-invasive, single-format, field-applicable tests for diagnosis and determination of stage of disease be developed and validated;

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 1 • considered the small number of anti-try- • noted the possible co-existence of T. b. rhode- panosomal drugs available, and recommended siense and T .b. gambiense sleeping sickness that synthetic and natural product libraries be patients in foci where refugees are settled, integrated for use in drug development; the difficulties in differentiating the two • acknowledged that the development of drugs trypanosome species, and the different treat- for late-stage disease is hindered by the blood- ment schedules for both forms of the disease, brain barrier, which prevents the delivery of and recommended that genomics be applied drugs to the central nervous system (CNS), to comparing T. brucei sub species, strains and and recommended that CNS-penetration mod- life cycle stages for their differentiation and els be used in drug development strategies for disease management; human African trypanosomiasis, and that • appreciated the important role that vector strategies which facilitate the delivery of drugs control plays in reducing the transmission of across the blood brain barrier be developed; vector borne diseases, and recommended that • took into account the limited evidence suggest- tsetse-trypanosome interactions be investigated ing that combination therapy with late-stage to determine the molecular basis of refractori- drugs has an additive effect, and, in view of ness for trypanosome transmission and mecha- the urgent need to have alternative treatments nisms for driving desirable genes into vector for melarsoprol refractory patients, recom- populations; mended that combination chemotherapy using • noted with concern the absence, within TDR, late-stage drugs be optimized; of a full-time staff member responsible for • acknowledged the difficulties associated with research activities on African trypanosomiasis, the treatment of sleeping sickness patients, and and recommended recruitment of such a the lengthy post-treatment follow-up, and rec- person; ommended investigating and applying new • recognized the inadequacy of infrastructure for information on immune parameters to (i) the research in different endemic countries, and determination of stage of disease, (ii) the pre- recommended networking and cross country vention and/or amelioration of the encephalitis comparison of research progress to assist in and encephalopathy associated with the dis- capacity building and stimulate cross border ease and its treatment respectively, and (iii) the interest and advocacy; development and validation of a non-invasive • noted with concern the low priority given to protocol for determining cure and shortening African trypanosomiasis by governments of the duration of after-treatment follow-up; endemic countries, and recommended strong African trypanosomiasis • expressed concern that the limited number of advocacy to persuade disease endemic country suitable centres in Africa created a situation governments to accord priority attention to where research was increasingly compartmen- research and control of African trypanosomia- talized, and recommended that the capacity of sis amidst their other health priorities. laboratories/centres within Africa be strengthened in the basic sciences, including in bioin- Other high priority recommendations, with sug- formatics, genomics and applied genomics, gestions for studies and/or actions that should be drug discovery and development; undertaken to meet the objectives of the meeting, are listed in the text.

2 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 2 Overview and objectives

African trypanosomiasis is caused by protozoan trypanosomes, and significantly, in T. b. parasites, trypanosomes, which are transmitted rhodesiense sleeping sickness, domestic and by tsetse flies (of the genus Glossina). The dis- wild animals. In T. b. gambiense disease, the ease occurs in two forms: a chronic form caused classical human-fly-human transmission cycle by Trypanosoma brucei gambiense, which occurs occurs in both endemic and epidemic situations. in West and Central Africa; and an acute form, caused by T. b. rhodesiense, which occurs in Sleeping sickness is a re-emergent disease, Eastern and Southern Africa. The chronic infec- but does not get due attention, probably because tion lasts for years, whilst the acute disease may its impact is regional. The disease occurs in last for only weeks before death occurs, if treat- 36 sub-Saharan countries, within the area of ment is not administered. distribution of the tsetse fly. Over 60 million people living in some 250 foci within this region The epidemiology of sleeping sickness is com- are at risk of contracting the disease (see figure 1). plex and transmission cycles are subject to interactions between humans, tsetse flies and

Figure 1 - The focal distribution of human African trypanosomiasis African trypanosomiasis

Left of dotted line: Trypanosoma brucei gambiense foci in West and Central Africa

Right of dotted line: T. b. rhodesiense foci in Eastern and South Africa Source: WHO picture library

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 3 The number of cases reported annually is over The social and economic impact of sleeping 40 000, but this is highly underestimated, due sickness is often underestimated. During epi- to difficulties in diagnosis and remoteness of demics, large proportions of communities are affected areas. It has been estimated that the affected, with loss of life and untold suffering. actual number of cases is at least 300 000, the These have serious social and economic conse- vast majority of whom are not diagnosed or quences, which far outweigh the cost of main- treated (WHO 1998). These figures are relatively taining surveillance. The disease has been a small compared to other tropical diseases, but major cause of depopulation of large tracts of African trypanosomiasis, without intervention, Africa. The fear it causes has led to abandon- has the propensity to develop into epidemics, ment of fertile lands, and is an impediment to making it a major public health problem. development. Some affected countries have agri- Furthermore, the case fatality rate in untreated culture-based economies, and workers in cocoa patients is 100%. This fact, combined with the and coffee plantations are always at risk of con- focal nature of the disease, means that the dis- tracting the disease, consequently decreasing the ability adjusted life years (DALYs) averted per labour force. This is reinforced by the fact that infection cured or prevented are very high. As a the disease mainly strikes the active adult popu- result, control of this disease in areas at risk is lation. highly cost-effective, falling well below the accepted threshold value for money of US$25 Regular medical surveillance, involving accurate per DALY averted (Dr A. Shaw, personal case detection and appropriate treatment, and communication, see attached working document, tsetse control where applicable, is the backbone Shaw and Cattand, Annex 4 section III). of the strategy for the control of sleeping sickness (WHO, 1998). With the available tools, con- At the beginning of the last century, huge sleep- trol is a continuing effort rather than eradication. ing sickness epidemics devastated large areas of Experience has shown that where control is inter- the continent. In the 1960s, the prevalence of the rupted, for a variety of reasons, resurgence of the disease had been successfully reduced to less disease occurs sooner or later. than 0.1% in all endemic countries, through historic campaigns by the former colonial powers. Over recent years, human trypanosomiasis has Soon after independence, however, national gov- been the subject of renewed interest among the ernments failed to sustain such programmes due donor community and scientists. Substantial vol- to lack or diversion of resources to other more untary contributions have been made by Belgium pressing health problems. Breakdown of special- and France for research and control in the African trypanosomiasis ized mobile teams and health facilities in several endemic countries, as well as by the pharmaceu- countries, as a consequence of war and civil tical industry, but these contributions only partly strife or change in health policy, resulted in dra- cover the current needs, and the number of matic resurgence of the disease, the distribution donors is still very limited. Nongovernmental of which corresponds closely with that of major organizations (NGOs) have clearly committed conflicts in sub-Saharan Africa. efforts to participate in control. Special articles on the trypanosomiasis problem have appeared

4 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 in both the scientific press and public media (Figaro, June 2001; New York Times, 9 February 2001).

The objectives of the scientific working group (SWG) were to: • Identify areas where there are gaps in knowledge, and studies that are necessary to fill these gaps. • Identify research that is directly relevant to control programmes and treatment centres as a priority. • Promote development of new tools for control and new methods for use of old tools, and effective approaches to disease control. • Set objectives for research capability strengthening for basic science, genomics and applied genomics, drug discovery and development in disease endemic countries. African trypanosomiasis

References WHO Expert Committee on Control and Surveillance of African Trypanosomiasis. Geneva, World Health Organization, 1998 (WHO Technical Report Series, No. 881).

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 5

3 Epidemiology, disease surveillance and control

EMERGENCE AND RE-EMERGENCE

The persistence and re-emergence of sleeping may increase human-fly contact and hinder sickness in Africa is attributable to various regular medical surveillance of the population factors including lack of surveillance, shortage at risk. In rhodesiense sleeping sickness, cattle of drugs, and several other determinants which movements also increase the risk of infection. operate at different levels. Means for regular Agro-ecological changes may alter tsetse habitat surveillance are often inadequate. At the individ- and increase human-fly contact. A significant ual and family levels, there may be inadequate resurgence of the disease has occurred, notably knowledge about disease symptoms, transmis- in Angola, the Democratic Republic of the sion dynamics and treatment. Population move- Congo, Sudan and Uganda. New foci have also ments, such as seasonal migration and refugees, emerged in recent years.

Table 1 - Number of sleeping sickness patients treated by year per country

Sleeping sickness Country 1995 1996 1997 1998 1999 2000

Angola 6 786 8 275 7 373 5 351 4 546 D.R. Congo 18 158 19 342 25 200 26 318 T. b. gambiense Sudan 157 737 1 800 18 684 16 975 N.W. Uganda 1 062 980 1 069 967 1 020 S.E. Uganda 497 271 287 299 T. b. rhodesiense Tanzania 400 531 421 588 627

Source: Compiled from the working documents of the various countries.

Ministries of health, research organizations Central governments often accord sleeping and services often lack, or do not have, adequate sickness a low priority, until it assumes epidemic economic resources for sleeping sickness proportions. In addition, political upheavals, civil control programmes due to competing health pri- strife and wars lead to the breakdown of health orities. Recruitment of medium-level personnel services and of control programmes. African trypanosomiasis is inhibited by lack of incentives and career prospects. Ministries may lack funds for the The international community is prepared to purchase of diagnostic tests and drugs, except mobilize resources when epidemics occur but as part of externally-funded programmes. is often unable to provide long-term support for surveillance and preventive measures in Other factors that increase the risk of infection endemic situations. Until recently, the continued and human-fly contact include agricultural devel- production of anti-trypanosomal drugs was in opment such as coffee and cocoa plantations, and question. This problem has been resolved for the tourism industry. the next five years through generous donations

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 7 by pharmaceutical companies. However, arrange- networks, such as the Programme Against ments need to be made to ensure a sustained African Trypanosomiasis (PAAT) managed by supply beyond this period. the Food and Agriculture Organization (FAO), Organization of African Unity (OAU) and In order to ensure that research results are com- WHO, and new networks, such as the WHO parable over a wide area, advantage should be surveillance network, should be supported. taken of the potential of networking (for more general data collection) and using consortia of investigators, as appropriate, including public/ private partnerships. Avenues should be explored as to how to make better use of existing

EPIDEMIOLOGY

Reservoir Studies

Reservoir hosts play an important part in sustaining endemicity and in the re-emergence of • Assessment of the epidemiological sig- epidemics. In the case of T. b. rhodesiense nificance of an animal reservoir for sleeping sickness, several wild and domestic gambiense sleeping sickness using new animal reservoir hosts have been identified and approaches such as the molecular certain outbreaks brought under control by treat- approach. ing nearby cattle. Although natural infections • Assessment of the epidemiological and with T. b. gambiense have been reported in clinical significance of “unconfirmed domestic animals such as pigs, dogs, sheep and cases”, defined as individuals with clin- cattle, and may occur in wild animals as well, ical signs, or as cases where indirect the role of an animal reservoir in the epidemiol- evidence of infection such as sero-posi- ogy of T. b. gambiense remains as yet undeter- tivity or polymerase chain reaction mined. Another important group of reservoir (PCR)-positivity exists but where the hosts, especially in T. b. gambiense epidemiolo- African trypanosomiasis parasite cannot be demonstrated by gy, are the human carriers: infected individuals microscopy. who remain undiagnosed due to inadequate surveillance and/or the limitations of current diagnostic tools in demonstrating low levels of trypanosome infections. The following areas for research were identified:

8 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Incidence and Prevalence

To design optimal control strategies and The validation of diagnostic tests is needed estimate the burden of disease, knowledge of to obtain more accurate estimates of the real current prevalence and the impact of control disease prevalence. measures is required. This could be obtained by:

• Standardizing and extending reporting systems using geographic information systems (GIS). • Collating and analysing existing epidemiological data on the impact of past control schemes on prevalence. • Introducing a standardized protocol for monitoring the impact of control measures on epidemiological indicators. • Epidemiological modelling to better understand and predict the disease’s behaviour, e.g. to obtain better estimates of the ratio of unreported to reported cases, or of the basic reproductive number, Ro.

SURVEILLANCE AND INTERVENTION FOR CONTROL

Diagnostics African trypanosomiasis Currently, the screening tests used are only avail- tics based on molecular techniques (e.g. PCR) able in bulk presentation, which limits their use for epidemiological and clinical studies is strong- where small numbers of people or individuals ly recommended. The need for field applicable, need to be tested, e.g. in rural health centres, for non-invasive diagnostic methods is recognized, surveillance on a small scale. There is a need for especially to avoid lumbar punctures. further development of simple, field applicable, single test formats (such as dipsticks), with high The main constraint to the development and vali- specificity. A multicentre validation of diagnos- dation of diagnostic tests is the lack of a gold

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 9 standard. Statistical methods exist for the evalua- Changing Institutional Environment tion of diagnostic tests in the absence of a gold standard, but so far these have not been applied Far-reaching institutional changes have occurred to the diagnosis of human African trypanosomia- during the last decade which have had a major sis. It is recommended that the use of these impact on the organization and implementation methods should be explored. of sleeping sickness control. These include decentralization, integration of human African For stage determination and follow-up, the trypanosomiasis (HAT) control into the primary following investigations should be undertaken: health care (PHC) system, cost recovery/sharing, and varying degrees of community involvement in surveillance and control. Capacity building in • Multicentre validation of latex/IgM policy and health systems, to help identify and and PCR tests on cerebrospinal fluid. remedy the changes that have adversely affected • Identification of new markers of disease control, is recommended. The following neuropathogenesis, e.g. cytokines. areas of research were identified: • Evaluation of the use of antigen detection tests for follow-up. • Evaluation of the impact of changes in delivery pathways for sleeping sickness control on the effectiveness of control Vectors measures. • Investigation of the effects of changes in cost-sharing arrangements on disease A range of tools for the control of tsetse flies detection rates, compliance, and effec- have been developed over the last twenty years, tiveness of control. some of which can be applied by communities. However, their adoption and application by communities in endemic areas has not been sustainable. The exact role of the vector in relation to The monitoring protocols recommended above transmission of the disease from the various ani- should be applied here. mal reservoir hosts is unclear. The following research areas were identified: African trypanosomiasis

• The constraints affecting sustained use of vector control tools by affected communities. • Comparison, validation and improvement of available tools for blood-meal analysis.

10 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 SOCIOECONOMIC AND BEHAVIOURAL ASPECTS

Controlling sleeping sickness is a highly cost- It is recommended that social science research effective intervention, with the cost per DALY address the following issues: comparing very favourably with other health interventions, and falling well below the accepted value of US$25 per DALY averted • Costing of the different control strategies (Dr A. Shaw, personal communication). This to cover both endemic and epidemic sit- reflects the focal nature of the disease, and the uations, NGO and national programmes, fact that the case fatality rate in untreated and a range of countries. patients is 100%. Although the funding situation • The direct and indirect costs to patients has improved somewhat, and greater awareness and their families of obtaining diagnosis, of sleeping sickness as a public health priority treatment, hospitalization, and follow-up exists, it is vital to reinforce and extend this by examinations, as well as the costs of generating appropriate socioeconomic informa- permanent disability. tion in order to: • Refinement of the work done so far on Determine the financial resources that are • calculation of DALYs, and its extension required for control. to other settings. • Choose the most appropriate and cost-effec- • Clarification of issues influencing tive control strategies. community and individual support of, • Promote advocacy through better understand- and involvement in, control measures ing of the economic burden of the disease. including: • Provide guidelines for allocating resources - The development of approaches for amongst competing health needs. enhancing and sustaining community participation in the control and surveillance of sleeping sickness in endemic areas. - The possible existence of gender issues in the diagnosis and treatment of sleeping sickness, focusing on knowledge, practice and health care seeking patterns. African trypanosomiasis

When combined with the epidemiological information outlined in the section above (on epidemiology), such studies would enable the burden of disease to be estimated and the cost- effectiveness of different interventions to be calculated and compared.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 11 RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority • Development and validation of non-invasive, field-applicable, single-test format diagnostics tests, including tests for disease-stage determination. • Calculation of burden of disease and cost-effectiveness of control strategies.

High priority • Assessment of the epidemiological and clinical significance of ‘unconfirmed suspects’. • Systematic monitoring of disease incidence and prevalence, especially in relation to control measures. • Identification of issues influencing individual and community participation in control measures. African trypanosomiasis

12 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 4 Drug development, preclinical and clinical studies and drug resistance

PRECLINICAL STUDIES

The frequency and extent of use of the standard with little success. But with the advent of com- drugs against African trypanosomiasis, melarso- binatorial chemistry and the sequencing of the prol and pentamidine, is likely to lead to the trypanosome genome, new techniques can now development of resistance. Indeed, there has be applied to the development of novel, specific been an increase of late-stage cases refractory and non-toxic agents for HAT. In addition, to to melarsoprol treatment in the past decade foster continuation of research and development (Legros et al, 1999). The availability of these of new drugs after the end of the currently agents, and of eflornithine (DFMO), was not assured five-year period, capacity strengthening assured until recently. Even now, it is only of laboratories and/or centres within Africa for assured for the next five years. With the excep- drug discovery and development is recommend- tion of a new pentamidine-related drug (DB ed. 289), which is due to enter phase II clinical trials in 2001, no new candidate agents are currently in the advanced stage of development. Resistance to Arsenicals All the drugs are expensive and require hospital- and Diamidines ization for administration by the parenteral route. In addition, melarsoprol is associated with a 10% incidence of reactive encephalopa- Laboratory studies have identified the P2 thy that is fatal in up to 5% of the victims. amino-purine transporter as one route of entry Therefore, there is an urgent need for novel, into trypanosomes for both melarsoprol and safe, rapidly-acting and inexpensive agents for pentamidine. Loss or alterations to this trans- the treatment of human African trypanosomiasis porter, caused by genetic modifications to the (HAT) in the 21st Century. TbAT1 gene, can contribute to the development of resistance. However, other transporters have During the late stages of African trypanososmia- been implicated in the uptake of pentamidine, sis, parasites lodge in privileged sites within the and possibly other analogues, since parasites central nervous system, causing encephalitis. lacking the P2 transporter have been shown to The biological nature of such parasites, the be sensitive to pentamidine. Moreover, geneti- mechanisms by which they, and the drugs that cally modified parasites, lacking the TbAT1 cure late-stage infections, cross the blood-brain gene, are only marginally less sensitive to barrier, are unknown. There is evidence that melarsoprol than wild type trypanosomes. African trypanosomiasis eflornithine acts additively with non-permeating drugs in late-stage infections and suppresses the Treatment failure with melarsoprol may also be encephalitis. The nature of these drug interac- attributed to the level of melarsoprol permeating tions is not known but understanding them is into the cerebrospinal fluid (CSF), which varies critical to the development of new approaches to considerably between individuals. Concentra- chemotherapy. tions of this drug within the central nervous system (CNS) are close to the minimum In the past decade, drug discovery has proceed- inhibitory concentration (MIC) of the drug ed along biochemical target-based approaches against T. b. gambiense. Hence, modest increas-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 13 es in MIC values in parasites can increase the of drugs for the treatment of Alzheimer`s dis- proportion of late-stage patients showing refrac- ease, brain tumours and neuro-psychiatric con- toriness to treatment with melarsoprol. ditions, where novel in vitro methods to screen Therefore, the tripartite relationship between for compounds that cross the BBB or deliver drug, host and parasite, and cross-resistance drugs across the BBB have been utilized. It is between veterinary trypanocides, e.g. dimi- recommended that the relevance of these nazene aceturate, and HAT drugs, needs consid- approaches in the treatment of HAT be investi- eration in determining treatment failure in the gated and applied as the first approach in the field. drug screening pathway, followed by more focused animal model studies. Three areas of research are recommended: The following investigations are recommended:

• Identification of modes of uptake, and of action and mechanisms of resistance • The use of models of CNS penetration to arsenicals and diamidines in veteri- (BBB models) for inclusion in the HAT nary and human use. The influence of drug development pathway, in particu- both parasite and host factors on treat- lar: ment failure requires consideration. - In silico models that use the physico- • Comparison of the mechanisms under- chemical properties of compounds to lying treatment failure in field isolates determine which are most likely to of the parasite with the mechanisms distribute from the blood to the brain underlying resistance in laboratory (primary screen). strains of the parasite in which resist- - In vitro cell culture models (e.g. ance has been induced. MDCK and endothelial cells) that • Reliable diagnosis of drug resistance in enable the study of permeation of the field. drugs through a cell layer that has "tight" junctions (secondary screen). • Strategies that facilitate the delivery of drugs across the blood-brain barrier in animal models using known trypanocides, including:

African trypanosomiasis Blood-Brain Barrier - A bradykinin agonist (RMP-7, Cereport) that is on clinical trial for The development of therapeutic drugs for the delivery of anticancer and antiviral treatment of late stage HAT is limited by the drugs into the brain. blood-brain barrier (BBB) that prevents the free distribution of drugs into the CNS. An essential - The role of drugs that modulate the component in the development of drugs for the CNS inflammatory response (for exam- treatment of late stage HAT is to design or ple, DFMO and azathioprine) on drug select compounds that can cross the BBB. This access and activity in animal models. approach has been a priority in the development

14 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Role of CNS Trypanosomes drugs without support for the synthesis and testing of new molecules. The use of contemporary Drug action and efficacy depend on the physio- drug discovery methods should be encouraged to logical/metabolic state of the trypanosome, which find new molecules for the treatment of HAT. may be different for stages in the CNS and CSF These methods should follow current drug dis- from those in the bloodstream. Metabolically covery practices utilized by the pharmaceutical inactive and non-dividing forms tend to be less industry. Building facilities and training scientists sensitive to drugs, and, depending on the mode of to carry out drug discovery and development action of the drug, can even be completely insen- research in endemic countries should be strongly sitive. Apart from parasites in the brain and CSF, encouraged. trypanosomes in other tissue niches that are less accessible to the drugs, must be taken into The following areas of research are recommended: account. Research therefore should be directed at: • Drug discovery efforts which integrate synthetic and natural product libraries • Development of suitable models for stud- with structure-based modelling, computa- ies on CNS and CSF trypanosomes, tional chemistry and high-throughput including in vitro models and animal screening (HTS) both for efficacy and models. toxicity. Target based libraries will initial- • Studies on the biology of CNS and CSF ly require specific biochemical assays parasites, their localization and means of while diverse synthetic and natural prod- entry from the vascular sites, and their uct libraries will require whole cell inter-exchange. assays. • Studies on the metabolic state, and • High-throughput screening of synthetic susceptibility to drugs, of CSF and CNS combinatorial libraries designed based on trypanosomes. existing leads or obtained from existing combinatorial libraries in pharmaceutical companies. Drug Discovery and Drug Targets • Investigation of target enzymes or pathways which, when inhibited, render parasites non-viable. Examples of such tar- While agencies funding scientific research devote gets are S-adenosylmethionine decar- considerable resources to carrying out research African trypanosomiasis boxylase, myristate metabolism and vari- on the basic biology of trypanosomes, limited able surface glycoprotein (VSG) synthe- funding goes to drug discovery and development sis, and farnesyltransferase. projects. Likewise, drug discovery efforts by the pharmaceutical industry, directed specifically • Identification, validation by genetic towards new agents for the treatment of HAT, are manipulation, and production of new tar- nearly non-existent. A wealth of knowledge about gets based on exploitation of information the biology of the organism, and the promise of from the genome project. Validated tar- new drug targets resulting from genome research, gets should enter HTS screens. will have little impact on the discovery of new

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 15 RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority • Drug discovery efforts which integrate synthetic and natural product libraries with structure- based modelling, computational chemistry and high-throughput screening, and whole cell assays, both for efficacy and toxicity. • Investigation of the use of models of CNS penetration for inclusion in the HAT drug development pathway, and development of strategies that facilitate the delivery of drugs across the blood-brain barrier.

High priority • Characterization of modes of uptake and action of melarsoprol and diamidines, and identification of mechanisms of treatment failure with these drugs in the field. • Studies on the biology of CNS and CSF parasites, their localization and means of entry from the vascular site, and their inter-exchange. African trypanosomiasis

16 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 CLINICAL STUDIES

Clinical Aspects of Treatment Failure 2. Eflornithine and Monitoring The ongoing pharmacokinetic study of oral eflornithine monotherapy should be completed, and the results used in planning further develop- Treatment failures with melarsoprol have been ment. In addition, development of a new route observed in up to 30% of patients in some foci of synthesis should be considered in view of the in north-western Uganda, southern Sudan and technical difficulties with the current route. northern Angola (Legros, 1999). The WHO coordinated Sleeping Sickness Treatment and 3. Nifurtimox Drug Resistance Network is conducting sentinel Nifurtimox is being used on a compassionate surveillance for treatment failures. Reasons basis for the treatment of melarsoprol refractory underlying treatment failures should be investi- cases. However, it is not registered for treatment gated. of sleeping sickness. Complete development of the drug up to registration is recommended. Application of Existing Drugs Currently available data suggest that nifurtimox may be more suitable for use in combination Early-stage drugs than as monotherapy. 1. Pentamidine Recent data from pharmacokinetic studies Other potential drugs suggest that the half-life of pentamidine is suff- 1. Diminazene aceturate iciently long to allow shorter treatment regimens. Diminazene aceturate has been used by sleeping 2. Suramin sickness control programmes in several coun- The efficacy of a shorter course for the treatment tries, but has not been registered for human use. of early stage T. b. rhodesiense should be Development for human use as well as an oral explored. preparation should be considered. 2. Benznidazole Late-stage drugs Limited experimental data suggest that 1. Melarsoprol benznidazole has some activity against strains Currently, the 10-day (concise) melarsoprol of T. brucei. It has an established safety record regimen is being reviewed in 17 centres in in humans in the treatment of American 7 countries. A full report is expected at the end trypanosomiasis (Chagas disease). It should be of 2002. considered as a possible alternative compound for use in combination therapy of sleeping Preclinical and clinical evaluation of the concise sickness. melarsoprol regimen for treatment of T. b. African trypanosomiasis rhodesiense infections is recommended. New Compounds

The possibilities of a new formulation for melar- 1. DB 289 soprol should be explored in order to avoid the An international consortium is conducting clini- adverse extravascular effects at the site of admin- cal research to develop DB 289 for oral use istration caused by the currently used solvent against first-stage sleeping sickness. Phase I tri- propylene glycol. CNS penetration of new for- als have been concluded and a Phase IIa (proof- mulations should be equivalent or superior to the of-principle) trial is planned. Results of the current formulation. investigations will be made available to TDR.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 17 2. Megazol • Combination of melarsoprol and nifur- Limited studies in animal models suggest that timox vs. monotherapy with each of the megazol is effective as monotherapy in first- drugs. Preliminary results show that the stage sleeping sickness and in combination with combination therapy with low-dose con- suramin in second-stage T. gambiense infections. secutive melarsoprol combined with Toxicity studies should be completed. Should the short-duration nifurtimox was superior to data from these studies be favourable, further monotherapy with either melarsoprol or investigations on the efficacy of monotherapy in nifurtimox. first- and second-stage disease, using appropriate • Trials comparing melarsoprol and nifur- animal models, should be carried out. timox, melarsoprol and intravenous (iv) eflornithine, iv eflornithine and nifurtimox, have recently started.

Combination Therapy

In the longer term, combination treatment regi- There is experimental and limited clinical evi- mens should be optimized (i.e. maximum effica- dence suggesting that combinations of presently cy, minimum toxicity, shortest possible duration, available late-stage drugs, melarsoprol, eflor- simplicity, with preferably oral administration, nithine and/or nifurtimox, act additively and minimal cost). Further pharmacological and (Jennings, 1988, 1993). The current simultaneous preclinical investigations are necessary, including availability of these drugs gives an unprecedent- experimental studies on the optimal proportions ed opportunity to establish optimal combination of drugs in combination treatment regimens. treatment regimens. Although there are indica- Those investigations should be followed by tions that the early-stage drug suramin, in combi- appropriate clinical trials, preferably including nation with several other compounds, can cure pharmacokinetic data collection. Oral eflor- sleeping sickness, priority should be given to nithine would be preferred for combination ther- studies of combinations of late-stage drugs. apy as the current complicated iv regimen of eflornithine is not suitable for large-scale treat- In view of the urgent need to have available alter- ment in rural areas. Drug combinations with native treatments for melarsoprol refractory melarsoprol should be based on the concise patients, short-term as well as longer-term solu- melarsoprol treatment regimen. tions are needed. To identify alternative treatment African trypanosomiasis for melarsoprol-refractory patients, the following clinical trials are currently being conducted:

18 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Follow-up of Treatment

One of the major problems of clinical trials and case management is the long two-year follow-up period, requiring multiple lumbar punctures. Appropriately planned studies to determine the interval between treatment and relapses, which may allow a reduction of the follow-up period, are recommended. Research on less invasive markers for stage determination and cure should be given high priority, with emphasis on their applicability in the field.

Prevention and Management of Encephalopathy Syndromes

Encephalopathy syndromes, which occur during administration of late-stage drugs, are a major problem in the treatment of sleeping sickness. Research into the underlying mechanisms is critical for the development of improved strategies for prevention and management of the complications of treatment.

Coordination of Clinical Trials

The current number of suitable centres (with adequate equipment, trained staff, accessibility and security) to conduct clinical trials in the field of sleeping sickness is very limited. In view of the anticipated number of clinical trials, coordination African trypanosomiasis will be necessary. It is recommended that a clinical trial group be created within the WHO sleeping sickness treatment and drug resistance network. This group should coordinate clinical trials and provide appropriate training (good clinical practice and ethics).

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 19 RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority • Development and optimization of a protocol for combination therapy using late-stage drugs. • Development of tools for shortening the duration of after-treatment follow-up and for disease-stage determination.

High priority • Development of nifurtimox up to registration for use against African trypanosomiasis. • Preclinical evaluation of the 10-day concise melarsoprol regimen for treatment of T. b. rhodesiense (evaluation in an appropriate monkey model). • If the toxicological data are favourable, appropriate preclinical studies on the efficacy of megazol in first- and second-stage infections. • Elucidation of the underlying mechanisms of encephalopathy syndromes in view of their prevention and management. • Exploration of the possibility of better formulations of melarsoprol.

References Legros D et al. [Therapeutic failure of melarsoprol among patients treated for late stage T. b. gambiense human African trypanosomiasis in Uganda]. Bulletin de la Société de Pathologie Exotique, 1999, 92(3):171-2 [in French].

Jennings FW. Chemotherapy of trypanosomiasis: the potentiation of melarsoprol by concurrent difluoromethylornithine (DFMO) treatment. Transactions of the Royal Society of Tropical Medicine and Hygiene,

African trypanosomiasis 1988, 82(4):572-3.

Jennings FW. The potentiation of arsenicals with difluoromethylornithine (DFMO): experimental studies in murine trypanosomiasis. Bulletin de la Société de Pathologie Exotique et de ses Filiales, 1988, 81(3 Pt 2):595-607.

Jennings FW. Combination chemotherapy of CNS trypanosomiasis. Acta Tropica, 1993, 54(3-4):205-13.

20 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 5 Pathogenesis and applied genomics

PATHOGENESIS

Background is typified by a loss of Th1 cells secreting IFNg and the production of very high levels of the Proper and safe chemotherapy of trypanosomia- anti-inflammatory cytokine IL-10. The high IL- sis is acutely dependent upon accurately identify- 10 levels are associated with loss of tissue resist- ing the clinical stage of infection (staging). ance and spread of trypanosomes to the CNS. Staging has been difficult in the past because of a lack of accurate, sensitive and relatively non- Early- and late-stage trypanosomiasis can there- invasive clinical or parasitological tools for diag- fore be characterized by the levels of specific nosis. However, recent advances in the immunol- cytokines in patients. High IFNg but low IL-10 ogy of trypanosomiasis and in parasite cell biolo- levels are associated with the early non-invasive gy suggest new avenues for accurate staging. disease, and high IL-10 but low IFNg levels are Immunological results from both experimental associated with late-stage invasive disease. Since and clinical trypanosomiasis studies have shown preliminary clinical measurements show that that infection may be broken down into three rel- there is concordance with respect to serum and atively well-defined stages: early innate CSF levels of these cytokines, the prospects for response; early acquired immune response; late determining early- or late-stage disease by test- acquired immune response. Each of these ing serum for IFNg or IL-10 levels, respectively, responses exhibits distinguishing prognostic is clear. Furthermore, examination for cytokine characteristics. levels, which are very short-lived in serum and CSF, may help clarify whether a seropositive The early innate response is triggered by the individual has had a recurrence of disease. presence of trypanosomes within tissues, in suf- Moreover, information concerning immune status ficiently high numbers, leading to the release of and trypanosome-derived inflammatory factors pro-inflammatory mediators including the (e.g. glycosyl phosphatidyl inositol) may impact cytokines interleukin-1 (IL-1), IL-6, IL-12 and on how patients are treated for late-stage disease. tumour necrosis factor alpha (TNFa) as well as However, the picture is clouded by new informa- nitric oxide (NO). The early release of these pro- tion about the trypanosome itself. The following inflammatory factors helps set the stage for early areas of study were identified: acquired immune responses to the parasite variant surface glycoprotein (VSG). The early • Validation of experimental and prelimi- release of IL-12 promotes T helper (Th) to African trypanosomiasis release cytokines, particularly interferon-gamma nary clinical results suggesting that stage (IFNg), which is responsible for host resistance of disease can be determined by the against parasites spreading throughout host tis- presence of specific cytokines including sues. This may be more important than anti-VSG IFNg, IL-10 and TNF. antibodies which are only found within blood • Definition of the pathogenesis of the vessels. This early resistance is, however, super- encephalitis associated with late-stage seded by later immune responses that are associ- sleeping sickness and the encephalopa- ated with the spread of trypanosomes to the thy associated with treatment, and CNS. The late-stage acquired immune response

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 21 Trypanosome Biological Phenotype investigation of ways to prevent or ameliorate these phenomena using in vitro Trypanosomes have evolved mechanisms to reg- and in vivo disease models. ulate their biological virulence phenotype at the • Utilization of the data and technical clonal level. There is evidence that clonally approaches of the human genome project derived “virulent variants” are capable of enter- to unravel the composite immune ing the CNS at an accelerated rate in non-human response to trypanosomes at each stage primates, suggesting the possibility for develop- of the disease process. ment of discrete potential for CNS invasion within the host. The molecular mechanisms behind such clonal changes are unknown. This information might be useful in choosing the appropriate clinical treatment at any stage of disease. For example, the presence of trypanosomes expressing “CNS invasion markers” at any stage of disease might be used to initiate late-stage chemotherapy. In addition, such information has the potential to detect drug resistant variants in patients and aid the choice of drugs used to treat such patients.

RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

African trypanosomiasis Highest priority • Identification of parameters within the host immune system for disease-stage determination and improved management of post treatment encephalopathy.

High priority • Determination of the biological phenotypes of trypanosomes lodging in different tissue compartments to identify tissue tropism and drug-resistance characteristics.

22 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 APPLIED GENOMICS

Trypanosoma brucei Genomics and functional analysis of genes. Two resource centres have been established: DNA-based The T. brucei genome network was formed by resources in Cambridge, and derivative and TDR with a brief to coordinate the analysis and mutant lines of trypanosomes in Glasgow. These sequencing of the nuclear genome. This huge centres are funded to the end of the sequencing task can progress most efficiently if the commu- phase, at which time a re-appraisal of resource nity works together with open sharing of data and provision will be required. resources. The network has sufficient funds (from the Wellcome Trust UK and the US The TDR planning meeting for the Parasite National Institute of Allergy and Infectious Genome Initiative assigned some activities to Diseases/National Institutes of Health be undertaken in Africa, but these activities were [NIAID/NIH] USA) to complete the sequencing not sustained for various reasons which included of the megabase chromosomes, where the majori- reduced funding for African trypanosomiasis ty of genes are located, which will take until by TDR and changes in available technologies 2004. Approximately 85-90% of the genome is and priorities within the genome networks. The currently available as fragmentary sequences in involvement of African scientists and TDR is databases in the sequencing centre websites, and crucial in ensuring the application of information will ultimately be available in public web-based from genomics into disease control. African insti- databases. tutions must develop the capacity to fully exploit the resources from the genome projects. It is The Wellcome Trust has provided funds to estab- anticipated that capacity strengthening in African lish a genome database, at the Sanger Centre, institutions will impact on the applications from that will contain all data related to sequencing both the parasite and the vector genome projects.

RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority • Strengthening of capacity of research laboratories/centres in Africa in bioinformatics, genomics and applied genomics. • Application of genomics to comparison of Trypanosoma brucei subspecies, strains, and life cycle stages. African trypanosomiasis

High priority • Identification, application and database collation of DNA-based polymorphic markers for species differentiation, reservoir identification and determination of drug resistance. • Application of bioinformatics and experimental methods of determination of gene function to identify novel drug targets. • Use of resources available from the human genome project to investigate host response to infection.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 23 TSETSE GENETICS

Recent advances in molecular technologies, and their application to insects, are being widely Tsetse-trypanosome interactions explored because they have the potential to • Determination of the molecular result in the development of novel strategies for basis of refractoriness for try- control of vector borne diseases. The technolo- panosome transmission. gies needed to undertake such studies have been • Engineering of trypanosome refrac- developed for other insect vectors and thus can tory traits into strains of tsetse fly. facilitate rapid application to tsetse biology. • Investigation of gene spreading mechanisms that can be used for Limited field data indicate extensive genetic population replacement studies, e.g. sub-structuring in populations, which display cytoplasm incompatibility mediated differences in vectoral capacity. Information on by Wolbachia symbionts of tsetse. population genetics would facilitate more effective and targeted disease management strategies by identifying sub-populations of flies responsi- Population genetics of tsetse ble for disease transmission. There exists a mid- • Development of molecular polymor- gut symbiont-based genetic transformation sys- phic DNA markers and their appli- tem which allows expression of gene products cation to field flies to understand that confer trypanosome refractory phenotypes. the extent of genetic sub-structuring Candidate genes with either anti-trypanosomal existing in tsetse populations. traits or which, when expressed, can block para- • Investigation of mating incompati- site transformation and /or differentiation, bilities existing among field popula- should be identified for expression in the tsetse tions. mid-gut. The eventual genetic engineering of anti-trypanosomal traits into tsetse populations • Investigation of differences in vector would result in new vector control strategies. competence abilities of genetically The laboratory and field-based research recom- isolated sub-populations of flies. mended below is necessary to develop the applied approaches that can lead to novel vector- based disease-control strategies. African trypanosomiasis

24 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

Highest priority • Determination of the molecular basis of refractoriness for trypanosome transmission and development of mechanisms for driving the desired genes/traits into tsetse populations.

High priority • Development and application of molecular markers to determine genetic sub-structuring and mating incompatibilities in tsetse populations, and vector competence of genetically isolated sub-populations. • Development of a tsetse-parasite genome network to obtain and coordinate information on expressed sequence tags (EST), genomic sequences, physical map locations of selected genes, and eventual proteomics approaches. • Coordination of the network by TDR, whose comparative advantage is evidenced by the successful management of the mosquito-parasite genome networks. African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 25

6 Cross-cutting issues

RESOURCE FLOW

Funding support for control, and for research The SWG was held at an opportune time when and development, requires long-term commit- the funding level through TDR is increasing and ment. Over the past 20 years, many countries when African trypanosomiasis is re-surfacing in have provided support for control of, and the global health agenda. The funding and assur- research on, African trypanosomiasis; in particu- ance of a five-year drug supply by the private lar the governments of Belgium, France and UK sector provides a unique opportunity to deal with are major supporters. The European Union, the current epidemic in Africa as well as to USA, Canada, the Organization of Petroleum develop protocols for combination chemotherapy Exporting Countries (OPEC) Fund and China are in the face of increased melarsoprol refractori- also partners. A list of countries and institutions ness. In addition, the five years provides a period (by no means complete) that provide financial of grace during which TDR and the scientific support is attached (Annex 1). community can lay the foundations for ensuring continued availability of medicaments beyond Considerable sums of money are invested in the five years by investing in research and capa- basic research on trypanosomes by institutions in bility strengthening within Africa for drug dis- USA, and EU countries, because the try- covery and development as well as for genomics, panosome is a good model for basic research on applied genomics and proteomics. The SWG cell biology. Consequently, there is probably acknowledged the comparative advantage that more information on the cellular structure, bio- TDR has in coordinating and networking the chemistry and molecular biology of try- activities of various scientific groups, laborato- panosomes than any other non-mammalian cell ries and centres in different parts of the world. type, and a great deal is known about the differ- Consequently, the group recommended recruit- ences between trypanosomes and mammalian ment of a full-time professional staff member cells. However, only a small amount of this within TDR to coordinate the various activities knowledge is being applied directly in the man- on African trypanosomiasis. agement and control of the disease. Some of the knowledge is exploitable for development of new tools for disease and vector control as well as for improved patient management. The SWG meeting provided an opportunity to identify the knowledge that could be exploited for develop- African trypanosomiasis ment of new tools and improvement of existing ones for disease and vector management, as well as to determine the needs for research capability strengthening in disease endemic countries for basic sciences. TDR’s comparative advantage in enhancing existing, and developing new, partnerships for maximal application of the available knowledge was highlighted.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 27 ADVOCACY AND MARKETING FOR SLEEPING SICKNESS

The SWG noted with appreciation the increase in NGOs and national systems). A need to enhance funding coming to TDR for African trypanosomi- collaboration between the groups working in asis. However, there was concern that, in the pre- research and those working in control, and also vious six years or so, there had been little interest between groups working in disease and non-dis- from the donor community to fund activities on ease endemic countries, was identified. This African trypanosomiasis. A need to present the would increase the much needed credibility when disease in a way that changes donor perception of requesting donor support. The SWG noted that the problem was identified. The Group was scientists working in disease endemic countries informed that one way of changing is to present often work in isolation and have difficulties projects that are convincing, focused and concise, developing the required credibility for donor sup- emphasizing donor/researcher partnerships in port, and identified a need to support good ideas project management and follow-up and the bene- from young scientists, enabling them to seek fits to the target communities. Networking independent funding, i.e. to support them to a appears to be more attractive to donors than sin- point where donors have confidence in them. In gle, isolated projects. addition, a mechanism to assist young scientists to develop their ideas into fundable proposals, The Group indicated a number of networks where their ideas are likely to provide vital infor- already existed, both formal and informal (with mation, should be put in place.

INSTITUTIONAL DEVELOPMENT AND CAPACITY BUILDING

Given the re-emergent nature of African try- try, was recommended. A need to train technical panosomiasis, it is necessary to identify and staff for specific needs in research, disease sur- strengthen a nucleus of research groups and veillance and management was also identified. institutions in different endemic countries to The Group further noted the difficulties of sus- generate data, on disease burden and surveil- taining trained staff within the field of African lance, for advocacy. Where whole institutions trypanosomiasis in disease endemic countries, are difficult to maintain as disease specific and suggested lack of career development research institutes, their disease mandates could opportunities as one of the factors contributing

African trypanosomiasis be expanded to enable them to add on other dis- to this shortage. ease research activities while retaining core activities in the area of sleeping sickness. Staff retention is a global problem, not peculiar to the field of African trypanosomiasis. Those The SWG commended TDR for the great num- working in this field are in the public sector, ber of scientists it has supported for post-gradu- where salaries are not attractive and career ate training. However, continued training of advancement is slow. Lack of resources to carry research personnel in African trypanosomiasis, out research, and to attend short-term training and linking of this training to specific techno- courses, is a disincentive to remain in the sector. logical needs, which vary from country to coun- Funding for research, on a more sustainable

28 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 basis and for longer periods, is a necessity; while disease endemic areas have identified scientists linking of national institutions with overseas or technicians in scientific institutions as a ones, with donor support, is an area for possible national resource with unique operational needs. expansion. It was agreed that advocacy would be crucial, and international forums, such as the The SWG welcomed the recent TDR initiative of Organization of African Unity (OAU) institutional strengthening grants; a call for let- International Scientific Council for ters of intent was already issued. TDR is also Trypanosomiasis Research and Control (ISC- working on strategies for strengthening whole TRC), would be used to send the message. The national health research systems in countries that presence of TDR at such forums, and in other do not yet have a strong research culture, and a African national systems, would also be useful. mechanism is being worked out to give priority At the same time, there is a need to ensure to funding of research-strengthening proposals. appropriate networking in order to avail the necessary assistance in writing good proposals. The The commitment of African governments to sup- possibility of providing salary support with port trypanosomiasis research and control, with research grants should also be given considera- improved terms of service for those working in tion by TDR. the sector, is important. Very few countries in the

SOUTH-SOUTH COLLABORATION

In March 2001, WHO held a meeting in Harare, America, was emphasized. This networking will Zimbabwe, to discuss an initiative aimed at enhance the application of molecular biology increasing discussion and scientific interaction techniques and genomics (functional and between investigators in disease endemic coun- applied) to developing solutions to public health tries. The meeting was attended by scientists problems. Initiatives for multicentre projects, from several countries in Africa, Asia and Latin which incorporate capacity building and training, America, who discussed ways of increasing the will be developed. Additionally, a number of collaboration that already exists between investi- short-term training courses in basic and applied

gators in the South and collaborators in more biology, that incorporate the application of the African trypanosomiasis advanced laboratories of the North. The need to latest technologies, are planned. It is anticipated develop more sustainable pathways for training, that the results of this network will include that will make more efficient use of available process indicators (academic qualifications, resources by exploiting opportunities for the highest quality publications), products (e.g. exchange of complementary expertise present tools, chemotherapeutic agents), and an impact within laboratories in Africa, Asia and Latin on alleviating disease.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 29 RECOMMENDATIONS

The following are the highest and high priority recommendations from this section:

• Recruitment by TDR of a full-time professional staff member to coordinate African trypanosomiasis activities. • Identification and strengthening of a nucleus of research groups and institutions in different endemic countries to generate data on disease surveillance and advocacy. • Networking and cross-country comparison of research progress to assist in capacity building and stimulate cross border interest and advocacy. • Strong advocacy to persuade DEC governments to give priority to research and control of African trypanosomiasis amidst other health priorities. • Payment of a salary component within TDR funded grants to enhance retention of disease endemic country scientists in the field. African trypanosomiasis

30 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Annex 1 RESOURCE FLOW FOR AFRICAN TRYPANOSOMIASIS African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 31 BCT Bureau Central de la LIRI Livestock Research Institute Trypanosomiose MEMISA Medische Missie Samenwerking BMS Bristol-Myers Squibb MSF Médecins sans Frontières CAR Central African Republic NIAID National Institute of Allergy and DFID Department for International Infectious Diseases Development OAU Organization of African Unity DRC Democratic Republic of Congo OCCGE Organisation de Coordination EU European Union et de Coopération pour la lutte contre les Grandes Endemies FAO Food and Agriculture Organization OCEAC Organisation de Coordination pour la lutte contre les FITCA Farming in Tsetse Controlled Endemies en Afrique centrale Areas PATT Programme against African FOMETRO Fonds Médicale Tropicale Trypanosomiasis IBAR InterAfrican Bureau for Animal STI Swiss Tropical Institute Resources UN United Nations ICCT Instituto de Combate e Controlo das Trypanosomiases UNC University of North Carolina IPMP Instituto Português de Medecina UNDP United Nations Development Preventiva Programme IRD Institut de Recherche et WHO World Health Organization Développement ITM Institut Tropical de Médecin KETRI Kenya Trypanosomiasis Research Institute

32 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Annex 2 POSITION PAPER African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 33 A POSITION PAPER ON AFRICAN should be explored for drug discovery and develop- TRYPANOSOMIASIS ment of tools for trypanosomiasis control and patient management. Felix A.S. Kuzoe 5. Institutions in the endemic countries should be WHO/TDR, Geneva strengthened to enable implementation research. Clinical studies should be conducted according to the concept of good clinical practice (GCP). A few clinical MEETING THE CHALLENGE research centres or treatment centre networks should 1. New tools are needed for the control of African try- be identified and strengthened and clinical investiga- panosomiasis (sleeping sickness); however, these on tors given appropriate training. their own will not improve the trypanosomiasis situation. Factors that militate against the effective use of existing tools, such as continued worsening economies and structural adjustment programmes in the affected SUMMARY countries, will remain. This position paper provides an overview of the • Operational research should be conducted on the main features of African trypanosomiasis and the effective use of available tools with existing capaci- problems relating to its control. It gives a historical ties, such as the health services, strong control pro- account of the disease, its epidemiology and socioe- grammes in other diseases, NGOs, etc. • Development of simple surveillance systems with conomic impact. It describes the strategy for control, available tools that can be integrated into available namely case detection, chemotherapy and vector capacities to improve case detection and active control, and the limitations. It discusses global fund- screening of populations at risk will permit early ing of basic research on African trypanosomes, and diagnosis of cases and increase the number of peo- the pivotal role of WHO/TDR in facilitating the ple under medical surveillance. • Long-term commitment by the international com- evaluation of any spin-offs from the research munity to national sleeping sickness control pro- towards development of public health tools. grammes, rather than support for crisis manage- Considerable progress has been achieved in ment, will ensure sustainability. research on African trypanosomiasis, but this 2. Social, economic and cultural factors enhance or progress has not been reflected in the field due to hinder efforts to control sleeping sickness. There is lack of interest in development of new tools and need for studies on social, socio-cultural and anthro- pological aspects of endemicity of sleeping sickness. lack of sustainability of control methods, resulting The effects of decentralization of health services on largely from inadequate resources for public health African trypanosomiasis need to be studied for and wars and civil strife in some endemic countries. improved management of control programmes. With the decrease in resources for African try- 3. The private sector has the expertise for drug devel- panosomiasis in TDR, which started in 1994, there opment. However, for diseases such as African trypanosomiasis that have no market for drugs, develop- have been grave consequences in endemic coun- ment must be based on public sector financing. tries, both in terms of reduction in human resources • The recent award of US$ 15 million by the Bill & and degrading of facilities for research and evalua- Melinda Gates Foundation to a consortium of scien- tion of new tools for African trypanosomiasis. To tists towards the development of drugs for African meet the challenge of African trypanosomiasis in the trypanosomiasis and Leishmaniasis is a welcome 21st century, this position paper makes a number of development that has no precedence in the history of African trypanosomiasis. Furthermore, the World proposals. Health Organization (WHO) and Aventis Pharma have announced a major initiative to control African AFRICAN TRYPANOSOMIASIS AS trypanosomiasis, whereby Aventis Pharma has com- A PUBLIC HEALTH PROBLEM mitted US$25 million to support WHO’s activities in At the beginning of the last century, sleeping sick- this field for a period of five years. • TDR has links with the outside world with special ness was perceived by the colonial powers as by far reference to product development. Over its lifetime, the most important public health problem in Africa. it has generated multiple partnerships for product Huge epidemics devastated large areas of the conti- development and has gained considerable experi- nent. In the 1960s, the prevalence of sleeping sick- ence. Thus, it has been successful and taken some ness was successfully reduced in all endemic coun- products up to registration in collaboration with the private sector, for example, mefloquine, eflornithine, tries to less than 0.1%, through historic campaigns AmBisome and artemether. More partners are need- by the former colonial powers. Soon after indeed in drug discovery research and drug develop- pendence, however, national governments were ment for African trypanosomiasis. either lacking in resources or had diverted 4. Basic research on trypanosomes continues to be resources to other pressing health problems. funded with considerable sums of money in the North. Breakdown of specialized mobile teams and health No drugs have yet come out of basic research by rational drug design. Functional genomics, bioinfor- facilities in several countries, as a consequence of matics and proteomics provide new opportunities that war and civil strife or change in health policy, resulted in dramatic resurgence of African try-

34 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 panosomiasis, the distribution of which corre- T. b. rhodesiense, which occurs in Central and sponds closely with that of major conflicts in sub- Southern Africa. The chronic infection lasts for Saharan Africa. In the Democratic Republic of the years, whilst the acute infection may last only for Congo (DRC), the number of cases being reported weeks. yearly has now reached levels comparable to the 1930s, and may result in as many deaths in adults Other forms of trypanosomiasis, called nagana, as HIV/AIDS (Ekwanzala et al 1996). And yet affect livestock, and are considered the most impor- African trypanosomiasis is curable. Other countries tant infectious disease holding back the develop- affected by the resurgence/epidemics are Angola, ment of livestock production in much of Africa. The the DRC, Central African Republic, Sudan and annual losses in meat production attributed to try- Uganda. Sleeping sickness due to T. b. rhodesiense panosomiasis are estimated to be US$5 billion seems to be quiescent at present and less wide- (ILRAD 1993). No new drug has entered the market spread, with active foci occurring in Tanzania and for over 30 years because the disease does not affect Uganda. African trypanosomiasis is a re-emergent livestock in Western developed countries. disease, but does not get due attention, probably because its impact is regional. Epidemiology The epidemiology of sleeping sickness is complex The disease occurs in some 36 sub-Saharan coun- and the transmission cycles are subject to interac- tries, within the area of distribution of the tsetse fly. tions between humans, tsetse flies, trypanosomes Over 60 million people living in some 250 foci with- and, significantly in T. b. rhodesiense sleeping sick- in this region are at risk of contracting the disease. ness, domestic and wild animals. In T. b. gambiense The number of cases reported annually is over 40 disease, the classical human-fly-human transmis- 000, but this is highly underestimated due to the dif- sion cycle occurs in both endemic and epidemic sit- ficulty of diagnosis and remoteness of affected uations. Humans are the important reservoir and areas. It has been estimated that the actual number hence it is possible to reduce transmission through of cases is about 300 000 (WHO 1998). Though these diagnosis and treatment of the infected population. figures are relatively small compared to other tropi- Although it has been demonstrated by biochemical cal diseases, African trypanosomiasis, without inter- and DNA techniques that trypanosomes identical to vention, has the propensity to develop into epi- those which cause T. b. gambiense disease in humans demics. This characteristic makes it a major public occur in domestic animals and some game, the sig- health problem. During epidemics, large propor- nificance of these potential reservoir hosts on trans- tions of communities are affected with great loss of mission is not clear. In T. b. rhodesiense disease, on life and untold human suffering. Epidemics have the other hand, it has been recognized that infec- serious social and economic consequences, which tions in humans in endemic situations are acquired far outweigh the cost of maintaining surveillance. from savannah species of tsetse flies, all of which Sleeping sickness has perhaps been a major cause of feed preferentially on a wide variety of game. The depopulation of large tracts of Africa, and fear of the game-fly-human cycle is typical. Endemic T. b. disease has led to abandonment of fertile lands and rhodesiense situations are sporadic in nature and is an impediment to development. The World Bank patchy in distribution, and adult men tend to be Report (1993) estimated that, in 1990, there were 55 predominantly infected. In epidemic T. b. rhode- 000 deaths due to African trypanosomiasis and 1.8 siense, however, human-fly-human or domestic ani- million disability adjusted life years (DALYs) lost mal-fly-human cycles predominate. There is equal due to the disease. More recent estimates are rather distribution of infection among men, women, and similar, with 2.1 million DALYs lost and 66 000 children. For T. b. rhodesiense disease, chemoprophy- deaths in 1999. As a comparison, the number of mil- laxis of the domestic animal reservoir has been rec- lions of DALYs lost is estimated at 45.0 for malaria, ommended as a new approach to control. Available 4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9 evidence suggests that HIV infection has had little for schistosomiasis, 1.1 for onchocerciasis and 0.7 for impact on the epidemiology of T. b. gambiense Chagas disease (WHO 2000). African trypanosomiasis (Louis et al 1991; Pepin et al 1992; Meda et al 1995). THE DISEASE African trypanosomiasis is caused by protozoan Medical Surveillance haemoflagellates, trypanosomes that are transmit- Regular medical surveillance, involving case detected by tsetse flies (Glossina spp). The disease occurs tion and treatment, and tsetse fly control, where in two forms: the chronic form caused by applicable, is the backbone of the strategy for con- Trpanosoma brucei gambiense, which occurs in West trol of sleeping sickness (WHO, 1998). With avail- and Central Africa; and the acute form, caused by able tools, control is a continuing effort rather than

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 35 eradication. Experience over the last 50 years has T. b. rhodesiense sleeping sickness. All these drugs shown that, where control efforts are interrupted, have adverse effects, melarsoprol causing reactive e.g. due to civil strife, political upheavals, economic encephalopathy in 5-10% of patients with a fatal constraints, or out of complacency, sooner or later, outcome in 1-5%. Increasing numbers of patients - there will be resurgence of the disease. between 20-25% in certain foci - do not respond to melarsoprol treatment, probably due to resistance of Diagnosis the parasite to the drug (Legros et al 1999). The term For unequivocal diagnosis in humans, it is essential ‘drug resistance’ covers host and parasite-related to demonstrate trypanosomes in lymph node aspi- factors. Host related factors include poor distribu- rate, blood or cerebrospinal fluid (CSF). A number tion of drug to infected tissues and intracellular of tools exist for the diagnosis of patients. In T. b. sites, variation in drug metabolism between indi- gambiense areas, the Card Agglutination Test for viduals, and diminished activity of drug. Parasite Trypanosomiasis (CATT), a serological test detect- related factors that contribute to resistance include ing antibodies, is used for mass screening. reduced drug accumulation in the parasite, a change Serologically positive cases are then confirmed in enzyme target through increase in enzyme levels, using parasitological tests. Since parasitemia varies increase in metabolite production or retention, or between foci and disease stage, it is necessary to use of alternative pathways to bypass the site of adopt blood concentration techniques, such as the inhibition. A number of these factors could be Haematocrit Centrifugation technique (HCT), the involved and therefore the mechanism of resistance Miniature Anion Centrifugation Technique needs to be elucidated and strategies to combat it, (MAECT), or the Quantitative Buffy Coat (QBC) such as the use of drug combinations, developed. technique. A study on the treatment of serologically The development of a simple test to quickly diag- positive patients who are parasitologically negative nose resistance in parasite isolates is also needed for with one injection of pentamidine is in progress in successful chemotherapy. Clinical trials with a com- DRC. The use of the CATT for screening popula- bination of melarsoprol and eflornithine, whose tions in T. b. gambiense areas has greatly improved synergism has been demonstrated in the mouse the potential for community diagnosis. Despite the model, should be given priority (Jennings 1988). advances, techniques, especially the CATT, have Combinations of other drugs are also envisaged. rarely been put into practice in endemic areas except The varying treatment schedules of available drugs as part of externally funded programmes. This were developed empirically and, therefore, opti- relates in part to the costs (Smith et al 1998). The mization of current treatment regimens is needed. Card Indirect Agglutination Test for Burri et al (2000) have shown that, with a new regi- Trypanosomiasis (CIATT) is another serological test men of melarsoprol, it is possible to reduce the dura- which detects antigens and therefore active infection of treatment from 40 days to 10 days. A study tion. However, the very high frequency of positive sponsored by TDR on treatment with pentamidine, results in low endemic areas, indicates that it may 7 days versus 3 days, in DRC has been interrupted not be suitable for screening in control programmes. due to rebel forces taking over that part of the coun- The role of the polymerase chain reaction (PCR) in try. This occurrence underscores the difficulties of diagnosis remains to be determined. A test is need- carrying out field research on African trypanosomi- ed to diagnose late-stage disease, which presently asis. relies on lumber puncture that is painful and not well accepted by people. A test is also needed to Eflornithine, developed in 1990, is the only available determine cure after chemotherapy. The current alternative drug to treat T. b. gambiense patients who requirement for a 2-year period of follow-up of do not respond to melarsoprol. It is not effective in treated patients is cumbersome, costly, and leads to T. b. rhodesiense sleeping sickness. The drug has loss of many patients. many drawbacks: a complicated mode of administration (intravenously every 6 hours at a dose of Chemotherapy 100mg/kg/day for 14 days), which limits its use to The chemotherapy of African trypanosomiasis is a hospital setting; it cannot be used for mass treat- unsatisfactory, relying on a few drugs which have ment; and the cost of treatment is US$300-500 per adverse side effects. Pentamidine, a diamidine patient, which makes it unaffordable by the affected developed in 1937, is used for early-stage T. b. gam- countries. To reduce costs, a shorter course of eflor- biese sleeping sickness. Suramin, a sulphanated nithine (7-day treatment) was compared to the stan- naphthylamine developed in 1922, is used to treat dard 14-day treatment. However, the results early stage T. b. rhodesiense. Melarsoprol, a trivalent showed that the 7-day treatment is effective only in arsenical derivative developed in 1948, is used for patients who have relapsed on melarsoprol and the treatment of late-stage of both T. b. gambiense and that, for new patients, the 14-day course is superior

36 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 (Pepin et al 2000). In recent years, there have been brain barrier in humans. The functional integrity of doubts about the future availability of eflornithine. the blood-brain barrier and its role in pharmacoki- However, through the collaboration between netics and pathogenesis is pivotal to understanding Aventis, the manufacturer, WHO and Médecins the disease. Attention should be given to new sans Frontières (MSF), potential manufacturers have approaches to drug delivery, such as across the been identified, and it is likely that a suitable pro- blood-brain barrier. In 1999, two leading com- ducer will be selected to produce the drug and that pounds, both of them diamidines that gave satisfac- funds will be provided through donor contributions tory results against trypanosomes in animal models to guarantee production for the next five years. in acute infection, failed to cure the chronic infec- Nifurtimox, nitrofurasan, was used for acute tion, due to inability to cross the blood-brain barrier. Chagas disease. Although it was not registered for A number of lead compounds have suffered the human African trypanosomiasis, it has been used same fate in the past. The ideal trypanocide must be experimentally and on a compassionate basis to safe and effective, and have a simple mode of treat T. b. gambiense sleeping sickness patients. administration to allow its use under rural condi- Bayer, the manufacturer, has agreed to continue pro- tions, where health facilities are usually poor, and duction for treatment of African trypanosomiasis; above all should be affordable. The occurrence of T. however, registration of the drug for this purpose is b. rhodesiense sleeping sickness outside the tradition- an urgent issue that needs to be addressed. al focus in south-eastern Uganda (Enyaru et al 1999), justifies fears of mixing T. b. gambiense and Vector control T. b. rhodesiense due to human population move- A variety of traps and screens impregnated with ments between foci in Uganda, the DRC and insecticide have been shown to be effective in reduc- Tanzania (Kigoma), and underlines the need for a ing tsetse populations by 99% in control pro- new drug that can treat both T. b. gambiense and T. b. grammes and are suitable for rural community par- rhodesiense. A multicentre clinical trial with eflor- ticipation. In any outbreak of sleeping sickness, nithine showed that eflornithine was relatively less tsetse control in combination with diagnosis and effective in Uganda than in three other countries treatment should arrest transmission. Besides, these (Pepin et al 2000). It should be noted that Uganda is devices can also be used as preventive measures to the only country where both T. b. gambiense and T. b. reduce human-fly contact. In vector-borne diseases, rhodesiense sleeping sickness foci exist and, there- vector control plays an important role in reducing fore, this observation needs further investigation. transmission. For example, Chagas disease caused by T. cruzi has been successfully controlled by vec- SOCIAL AND ECONOMIC IMPACT tor control as a result of national government com- OF SLEEPING SICKNESS mitment to a long-term programme. In onchocercia- The social and economic impact of sleeping sickness sis endemic areas of West Africa, blindness is no is often underestimated. Some affected countries longer a public health problem as a result of vector have agriculture-based economies, and workers on control and donated drug (Mectizan) distribution. cocoa and coffee plantations are at risk of contract- The effect of deforestation and climatic changes on ing the disease; consequently the labour force is tsetse populations in West Africa may be responsi- reduced. At community and family levels, mental ble for the lull in sleeping sickness in countries like confusion, personality and behaviour changes, Ghana and Nigeria. Operational issues, such as which often characterize central nervous system motivation of communities and recurrent costs, involvement in late-stage disease, may lead to which militate against sustainability in the use of divorce and break up in homes and present an impregnated traps and screens on regular basis, unfavourable climate for bringing up children. In need to be studied and solutions found. some cases, such people become mentally dis- turbed, suicidal and violent, and constitute a danger DRUG DEVELOPMENT to themselves and to the community. Aroke et al An oral formulation of eflornithine has many (1998) reported that, in the past, T. b. gambiense advantages over the injectable form and will allow sleeping sickness in children had an influence on use on a wider scale. A pharmacokinetic study of their physical growth and attainment of sexual oral eflornithine is in progress in Côte d’Ivoire and maturity. In Central Africa, there are important it is anticipated that Phase 3 clinical trials will take problems regarding treatment, particularly the place in 2001, to evaluate its safety and efficacy, severe social consequences of long-term hospitaliza- toward eventual submission of data for registration tion. Important behavioural factors contributing to to a regulatory authority. The need for new drugs is the risk of death from sleeping sickness, such as fundamental. One of the obstacles to finding new negative attitudes towards hospital treatment, have drugs is the difficulty of transport across the blood- often led to the patient absconding and not com-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 37 pleting treatment. In studies on the impact of try- activities. TDR’s initiatives in bringing together sci- panosomiasis on land occupancy systems, on popu- entists in Scientific Working Groups, meetings and lation movements and social conditions in Burkina workshops to deliberate on specific issues, paid off Faso and Côte d’Ivoire, the extreme mobility of the on several occasions. A few examples follow. people due to migrant labour was identified as one of the major problems in case detection, case report- A Glossina trapping meeting held in Brazzaville, ing and control of the disease. There is need to con- Congo, in March 1985, brought together scientists, tinue monitoring and mapping population move- from West, Central and Southern Africa, interested ments, as well as incidence of trypanosomiasis, par- in developing Glossina trapping technology. This ticularly as the impacts of the infection are rather meeting opened the way for better interaction latent but extremely serious in the long run. Studies between the scientists and gave impetus to in Uganda demonstrated that African trypanosomi- improvement in trapping technology for tsetse con- asis had an adverse impact on the functioning of trol. Five years later, the pyramidal trap impregnat- households at Iganga, south-east Uganda. Adverse ed with insecticide used at 10 traps per km2 was impacts included increased poverty, decline in agri- effectively employed in reducing tsetse populations cultural activities often leading to famine or lack of by over 95 per cent within 3-4 months during the basic food security, disruption of children ‘s educa- epidemics of sleeping sickness in Busoga, south-east tion, and general reversal of role obligations, which Uganda, at an estimated cost of US$0.9 per head of more often than not enhanced women’s and chil- population protected (Lancien, 1991). dren’s burdens. The debilitating nature of the disease also poses more problems for women, who The rational development of control and treatment may be stigmatized and/or rejected by their spous- requires thorough knowledge of the pathology of es even after recuperation. The extent of disruption the disease. At the inception of TDR in 1975, there of household social and economic activities is great- were less than 25 autopsies of African trypanosomi- ly influenced by such factors as the household’s eco- asis reported in the literature. The need for system- nomic status, composition and level of organization atic autopsy backed by expert histopathology led (Kyomunhendo 1995, 1998). TDR, in collaboration with the University of Glasgow, to establish a network of clinical centres GLOBAL RESEARCH ON AFRICAN that were provided with kits and protocols for TRYPANOSOMES autopsy. From this effort, evidence was provided The trypanosome has many unique biological char- from both laboratory and clinical data showing that acteristics that make it one of the most studied par- reactive encephalopathy, which occurs in 3-5% of asites. It offers many opportunities for basic patients treated with melarsoprol, points to a drug- research: it is easy to cultivate and purify to yield related immune response rather than to toxicity large amounts of protein and nucleic acid, it is a (Haller et al 1986). A set of slides showing the fea- eukaryotic experimental model for research on the tures of neuropathology in African trypanosomiasis control of gene expression, etc. Though it is difficult was prepared and made available to universities for to get funds for the control of sleeping sickness, teaching purposes. large sums of money are invested annually, particularly in the North, on basic research on African try- New strategies for managing patients with central panosomes. There is probably more information on nervous system involvement in African trypanoso- the biochemistry and molecular biology of try- miasis are needed. In 1986, TDR organized a work- panosomes than on any other non-mammalian cell shop in collaboration with the Institut de Neuro- type, and a great deal is known about the differ- logie Tropicale, Limoges, France, which brought ences between trypanosomes and mammalian cells. together clinicians, neurologists, neuropathologists Yet no drugs have come out of basic research. TDR and scientists. Following the recommendations of provides an essential link between research institu- this group, a number of studies funded by TDR in tions in the North and endemic countries, through collaboration with other institutions, resulted in sig- access to a network of national field projects and nificant progress in understanding the molecular control programmes, where spin-offs from basic mechanisms underlying pathogenesis, including research can be evaluated as tools for the control brain dysfunction and neuropsychiatric symptoms and prevention of sleeping sickness. It is necessary associated with the disease. The trypanosome lym- for TDR to preserve this unique role (Kuzoe, 1993). phocyte triggering factor, TLTF, a molecule which binds to CD8+ cells and triggers the production of For institutions in the South, TDR was conspicuous- gamma interferon, which is growth factor for T. b. ly a major source of funding for research on African brucei, was reported by the Karolinska Institute, trypanosomiasis and institutional strengthening Sweden, in 1991(Olsson et al 1991). The gene for

38 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 TLTF has since been identified, cloned and ical studies. One should, however, not overlook the sequenced, and a recombinant TLTF produced support, no matter how small, that has been provid- (Bakhiet et al 1993; Vadya et al 1997; Hamadien et al ed by other institutions in the North to these centres. 1999). The exploitation of TLTF for immunotherapy needs to be followed up. Other studies elsewhere The Bill & Melinda Gates Foundation, in December have shown that proinflammatory cytokines, 2000, awarded US$15.1 million to treat African try- including tumor necrosis factor (TNF), interleukins panosomiasis and leishmaniasis to an international 1 and 6 (IL-1, IL-6), and prostaglandins, play an consortium of researchers led by Dr Richard R. important role in the pathogenesis of central nerv- Tidwell, a scientist at University of North Carolina ous system disease (Hunter et al , 1992; Alafiatayo at Chapel Hill, to develop new drugs to fight et al, 1994; Jennings et al 1997). Furthermore, this African sleeping sickness and leishmaniasis. This pathology has been shown to be prevented and welcome news is without precedence in the history ameliorated by drugs such as eflornithine (Jennings of African trypanosomiasis. Some of the scientists et al, 1997) and megazole (Enanga et al, 1998). involved in this consortium already collaborate with Consideration should be given to the use of such TDR in drug discovery research and drug develop- drugs in the management of African trypanosomia- ment. TDR should establish links and maintain col- sis. Further, preclinical research with such drugs is laboration with this consortium. The primate facili- needed. ty at the Kenya Trypanosomiasis Research Institute (KETRI), that was established with TDR’s support The availability of clinical centres in endemic coun- many years ago, is an asset that will be available for tries supported by TDR facilitated the evaluation of the evaluation of potential lead compounds in pri- the diagnostic tests MAECT, CATT and CIATT, as mates. There are few clinical research centres in well as clinical trials of eflornithine which produced endemic areas where clinical trials can be conducted data that were presented to the US FDA for registra- and, therefore, these will be in demand for drug tion of the drug in 1990. combination trials, oral eflornithine phase 3 clinical trials, and trials with any potential candidate com- Under the auspices of TDR and its collaborators, pounds. Two years ago, TDR started an initiative to considerable progress was made in research on train clinical investigators and monitors worldwide African trypanosomiasis in: diagnosis and develop- to conduct clinical trials according to the good clini- ment of diagnostic tests; epidemiology, host-para- cal practice (GCP) concept. Training will be required site-vector relationships, animal reservoirs; devel- for clinical investigators in the clinical centres that opment of tsetse traps and screens; better under- will be involved in clinical trials. TDR has a com- standing of the pathology of the disease, and drug parative advantage in institutional strengthening targeted biochemistry of trypanosomes. However, and can make a significant contribution to the work this progress has not been matched in the control of of this consortium towards the evaluation of candi- the disease, due to lack of capacity to sustain date compounds. TDR has links with the outside improved interventions as well as civil disorder in world, with special reference to product develop- some endemic countries. These factors were largely ment, which should be maintained. responsible for the current problems of African trypanosomiasis. BIOINFORMATICS During 1993/94, TDR initiated a number of parasite From 1994, when reorganization took place in TDR genome networks - for T. b. brucei, T. cruzi, Leishma- along disciplines instead of diseases, the resources nia major, Schistosoma mansoni, Brugia malayi. The allocated to African trypanosomiasis decreased, and networks are now oriented towards post genomics, continued to decrease tremendously in subsequent and bioinformatics networks are being expanded years, up to 64% in 2000. In view of TDR’s unique for data mining annotation and in-depth analysis. role in research on African trypanosomiasis, the con- The T. b. brucei network should be assessed and the sequences of this lack of funds were grave. Several necessary inputs provided to move it forward in the trained researchers left trypanosomiasis research for genome analysis of T. b. brucei. HIV/AIDS and malaria, where they could get research funds. It is not surprising that the current COORDINATION NETWORK chairman of the Task Force on African A Human African Trypanosomiasis Treatment and Trypanosomiasis now works on a project on Drug Resistance Network was formed in 1999 in HIV/AIDS. One tsetse trap expert has turned his WHO (Communicable Disease Surveillance and ingenuity to making and supplying bednets for a Response unit). It has as objectives: to assess the malaria control project. Clinical research centres are effectiveness of current treatment regimens; col- rundown and have reduced capacity to conduct clin- lect/disseminate information on refractoriness to

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 39 treatment; ensure availability and affordability of miasis in mice. Tropical Medicine and International existing drugs; provide guidelines for treatment; Health 1998, 3(9):736-41. promote research on causes of treatment failures, drugs and treatments (WHO 1999). The Go- Enyaru JCK, et al. Evidence for the occurrence of vernment of France provides funds for running the Trypanosoma brucei rhodesiense sleeping sickness out- secretariat of the network. Participants in the net- side the traditional focus in south-eastern Uganda. work come from WHO (Communicable Diseases Annals of Tropical Medicine and Parasitology, 1999, cluster, the Regional Office for Africa); MSF; the 93(8):817-22. United States Centers for Disease Control and Prevention (CDC), Atlanta; the Swiss Tropical Insti- Hamadien M, Lycke N, Bakhiet M. Induction of the tute, Basel; the Institute of Tropical Medicine, Ant- trypanosome lymphocyte triggering factor (TLTF) werp. TDR should play an active role in the network and neutralizing antibodies to the TLTF in experi- and participate in its meetings. mental trypanosomiasis. Immunology, 1999, 96(4):606-11. Acknowledgements I wish to thank Professor David H. Molyneux, Haller L, et al. Clinical and Pathological Aspects of Professor of Tropical Health Sciences, Liverpool Human African trypanosomiasis (T. b. gambiense) School of Tropical Medicine, Pembroke Place, with particular reference to reactive arsenical Liverpool, and Dr Jacques Pepin, Associate encephalopathy. American Journal of Tropical Medicine Professor, Infectious Diseases Division, Centre for and Hygiene, 1986, 35(1):94-99. International Health, University of Sherbrooke, Quebec, Canada, for their criticisms and comments Hunter CA, et al. Astrocyte activation correlates on this article. I also wish to thank Dr Paul Nunn, with cytokine production in central nervous system Dr Johannes Sommerfeld and Dr Charity Gichuki, of Trypanosoma brucei brucei-infected mice. WHO, for reviewing the paper. Laboratory Investigations, 1992, 67(5):635-42.

References ILRAD REPORTS, April 1993. Estimating the costs of Alafiatayo RA, Cookson MR, Pentreath VW. animal trypanosomiasis in Africa. Production of prostaglandins D2 and E2 by mouse fibroblasts and astrocytes in culture caused by Jennings FW, et al. The role of the polyamine Trypanosoma brucei brucei in products and endotoxin. inhibitor eflornithine in the neuropathogenesis of Parasitology Research, 1994, 80(3):223-9. experimental murine African trypanosomiasis. Neuropathology and Applied Neurobiology, 1997, Aroke AH, Asonganyi T, Mbonda E. Influence of 23(3):225-34. past history of Gambian sleeping sickness on physical growth, sexual maturity and academic perform- Jennings FW. The potentiation of arsenicals with ance of children in Fontem, Cameroon. Annals of difluoromethylornithine (DFMO): experimental Tropical Medicine and Parasitology, 1998, 92(8):829–35. studies on murine trypanosomiasis. Bulletin de la Société de Pathologie Exotique, 1988, 81:595-607. Bakhiet M, et al. A Tryapnosoma brucei brucei-derived factor that triggers CD8+ lymphocytes to interferon- Kyomuhendo B G. The role of social and economic fac- gamma secretion: purification, characterization and tors in the transmission of human African trypanosomi- protective effects in vivo by treatment with mono- asis in S.E. Uganda. Unpublished report, 1995, TDR clonal antibody against the factor. Scandinavian Project 950094. Journal of Immunology, 1993, 37(2):165-78. Kyomuhendo B G. The effects of human trypanosomia- Burri C, et al. Efficacy of new, concise schedule for sis on the functioning of households. Unpublished melarsoprol in treatment of sleeping sickness Report, 1998, TDR Project ID: 960028. caused by Trypanosoma brucei gambiense: a randomised trial. Lancet, 2000, 22, 355(9213):1419-25. Kuzoe FAS. Current Situation of African trypanosomiasis. Acta Tropica, 1993, 54:153-162. Ekwanzala M, et al. In the heart of darkness: sleeping sickness in Zaire. Lancet, 1996, 348:1427-1430. Lancien J. Lutte contre la maladie du sommeil dans le sud-est Ouganda par piégeage des glossines. Enanga B, et al. Megazol combined with suramin: a [Campaign against sleeping sickness in south-west chemotherapy regimen which reversed the CNS Uganda by trapping tsetse flies]. Annales de la Société pathology in a model of human African trypanoso- Belge de Médecine Tropicale, 1991, 71 (Suppl 1):35-47.

40 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Legros D. et al. Risk factors for treatment failure ument WHO/CDS/CSR/EDC/99.5; available on after melarsoprol for Tryponsoma brucei gambiense request from Department of Communicable trapanosomiasis in Uganda. Transactions of the Royal Diseases Surveillance and Response, World Health Society of Tropical Medicine and Hygiene, 1999, 93:439- Organization, 1211 Geneva 27, Switzerland). 442. WHO Expert Committee on Control and surveillance of Louis JP et al. Absence of epidemiological inter-rela- African trypanosomiasis. Geneva, World Health tions between HIV infection and African human try- Organization, 1998 (WHO Technical Report Series, panosomiasis in central Africa. Tropical Medicine and No. 881.) Parasitology, 1991, 42(2):155. World Health Report 2000: Health systems improving Meda HA et al. Human immunodeficiency virus performance. Geneva, World Health Organization, infection and human African trypanosomiasis: a 2000. case-control study in Cote d’Ivoire. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1995, 89(6):639-43.

Olsson T, et al. Bidirectional activating signals between Trypanosoma brucei and CD8+ T cells: a trypanosome-released factor triggers interferon gamma production that stimulates parasite growth. European Journal of Immunology, 1991 21(10):2447-54.

Pepin J et al. The impact of human immunodeficiency virus infection on the epidemiology and treatment of Trypanosoma brucei gambiense sleeping sickness in Nioki, Zaire. American Journal of Tropical Medicine and Hygiene, 1992, 47(2):133-40.

Pepin J, et al. Short-course eflornithine in Gambian trypanosomiasis: a multicentre randomized controlled trial. Bulletin of the World Health Organization, 2000, 78(11):1284-1295.

Pepin J, Meda H. The Epidemiology and control of human African trypanosomiasis. In: Advances in Parasitology, 2001. In Press.

Smith DH, Pepin J, Stich HR. Human African trypanosomiasis: an emerging public health crisis. British Medical Bulletin,1998, 54(2):341-355.

Vaidya T, et al. The gene for a T lymphocyte triggering factor from African trypanosomes. Journal of Experimental Medicine, 1997, 4:186.

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TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 41 Annex 3 THE EMERGENCE AND RE-EMERGENCE OF HUMAN TRYPANOSOMIASIS (SLEEPING SICKNESS) IN AFRICA African trypanosomiasis

42 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 I THE SITUATION IN ANGOLA∗ already stated, and the continual movement of the Theophile Josenando population, which includes carriers of the parasite Instituto de comabate e controlo de Tripanosomiase, who act as a reservoir for its dissemination. In addi- Luanda, Angola tion, socioeconomic factors including poverty are without doubt implicated in the spread and persist- Angola is a country situated at the interface between ence of sleeping sickness. Central Africa and Southern Africa and has an estimated population of 12 million. Sleeping sickness is At the symposium organized by Fundanga, the a major public health problem in the country today, Angolan Foundation for Solidarity and Deve- with more than 32 000 new cases having been lopment, and held in September 1998 on the prem- detected during the last five years. In contrast, in ises of the Angolan National Assembly in Luanda, 1974, when surveillance was more active and better sleeping sickness was clearly identified as a major organized, there were only three new cases. public health problem. As a result, the Angolan Government committed itself financially to taking Tsetse flies, or Glossina, the vectors of the disease, are action against the disease, with annual financing of present in 14 of the country’s 18 provinces. Two mil- about $1 million in local currency (Kwanza) and $1 lion out of the 4.5 million people living in the seven million in convertible currency (dollars) since 1998 provinces affected - Zaire, Uige, Kwanza Norte, for activities of the National Programme for control Malanje, Bengo, Kwanza Sul and the peripheral of human trypanosomiasis. areas of Luanda - are exposed to the risk of direct infection. The number of patients has been rising The National Programme has been gradually continuously for more than ten years. The number of strengthened in its leadership role, and its efforts new cases identified in 1997 was 8275, the highest have borne fruit with the transformation of the figure ever reported in the history of the disease in National Programme into the Institute for the Angola. The figures reported reflect the case detec- Control of Human and Animal Trypanosomiasis tion and treatment activities introduced over the last (Instituto de Combate e Controlo das Trypano- few years. Paradoxically, the rapid increase in the somiases, or ICCT) by Government decree 2/00 of number of patients identified is witness to the efforts 14 January 2000. The newly created Institute enjoys made to manage the disease by the national health autonomy in management and its position has services with the help of non-governmental organi- changed in the structural chart of the Ministry of zations (NGOs). In the period 1996-2000, 32 445 Health. This autonomy first found expression in the patients were treated (Table 1). delegation of management of salaries of personnel working at ICCT headquarters and at the Viana Table 1. The number of patients treated Reference Centre. in the years 1996-2000 Control of trypanosomiasis addresses one of five 1996 1997 1998 1999 2000 priority endemic diseases (AIDS, trypanosomiasis, tuberculosis, leprosy, malaria) for the Ministry of 6786 8275 7373 5351 4546 Health in 2001. In Angola, the inclusion of ICCT national staff in the activities of NGOs is effective, enabling it to be present at most places where Since African trypanosomiasis is invariably fatal patients are managed. when left untreated, the increase in number of treated patients means a decrease in mortality. So the 32 This year, the Ministry of Health assigned five 331 patients treated durng the period 1996-2000 physicians to the ICCT and is presently trying to escaped from certain death. The treatment centres obtain greater participation by the State in the are in areas of military confrontation (war front) and financing of the Programme. There is also effective this might explain the fluctuations in number of financial participation by the autonomous provin- patients treated each year. In addition, this has led to ces, especially the province of Zaire, which ensures low surveillance coverage and inadequate screening supplies of specific drugs for the disease. of the population at risk, suggesting that the true number of patients is very high. Since the refurbishment of the screening and treatment centre and the Viana Reference Centre, some The main reasons for the persistence of sleeping staff have been transferred from the headquarters of sickness in Angola are inaccessibility of most of the ICCT to the Viana Reference Centre, including one territory affected by trypanosomiasis, the scant sur- physician in charge, two clinicians, one head of lab- veillance coverage of endemic areas, for reasons oratory, and one clinical assistant. This doubling of * The orginal manuscript is in French TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 43 staff has enabled us to open a reference treatment RECOMMENDATIONS FOR ACTION centre, supported by the research projects of the • Combat poverty at all levels. This is one of the Swiss Tropical Institute. We can therefore say that, in major objective, poverty being one of the factors addition to the refurbishment and the equipment of that aggravate the socioeconomic status of the the laboratories, functional practice and expertise population, including those suffering from try- have been developed. panosomiasis.

A large number of NGOs operate in Angola. Most of • Adopt a strategy of permanent, regular, epidemi- them were originally working independently of ological surveillance, consisting of active diagno- each other, applying different methodologies to the sis of new cases of the disease, with a view to control of African trypanosomiasis, but numerous early detection, treatment and effective follow-up territorial disputes arose and it became necessary of sufferers. for the Ministry of Health to take over direction of trypanosomiasis control. As a result, coordination of • Mobilize resources to combat the disease, such as the national and international NGOs through ICCT drugs, reagents, equipment and traps. was initiated. Many meetings were held for consultation and coordination (on average, one every three • Strengthen vector control actions, using the most months), while project visits by ICCT staff also appropriate techniques to combat tsetse flies. helped to enhance the legitimacy and credibility of the Institute, facilitating the coordination of inter- • Draw up an information, education and commu- ventions. Currently, there is no more rivalry and the nication (IEC) plan for the population involving role of the ICCT is understood by all, such that the political leaders, leaders of civil society and health number of visits to partner treatment centres has officials in order to take concrete steps to control substantially increased. The NGOs have accepted this disease. standardization of the methods for case detection, treatment and data collection, while a growing • Draw up a plan of work for animal trypanosomi- number of technical and medical staff belonging to asis and update Glossina mapping of the country. ICCT now work with NGOs, where they are replac- ing expatriate staff. • Strengthen cooperation between the ICCT and its national and international partners. The NGOs that have worked, and are still working, in Angola are shown in Table 2. • Prepare coherent and feasible projects that are guaranteed to receive national and international Table 2. NGO presence in endemic areas between funding. 1996 and 2000 • Look for ways and means to motivate ICCT work- Organization Location 1996 1997 1998 1999 2000 ers by improving working and social conditions. MSF-B N’dalatanado + + + + MSF-F Maquela do Zombo + Quiculungo + Caritas M’banza-Congo + + + Uige Quitexe + + + + + Lucala + + + Negage + + + APN Dondo + + + + + Casuala + + + + + MMC Quiçama + + + +

FUNDANGA Caxito + +

ADRA Cacuso

44 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 II THE SITUATION IN TANZANIA The worst affected area is along the Malagarasi Valley, where the vegetation is characterized by big Stafford Kibona, trees overhanging open grassland. The population National Institute of Medical Research, Tabora, Tanzania in this large area is, for the most part, concentrated in villages on the Kigoma–Kibondo trunk road. GENERAL OVERVIEW Sleeping sickness infections are contracted mainly In the United Republic of Tanzania, sleeping sick- by those going into the bush to hunt, fish, collect ness, also known as human African trypanosomiasis honey, beeswax, etc. There are few cases of perido- (HAT), is one of the major public health problems. mestic infection. Sleeping sickness was first recorded in 1922 in Maswa district, south of Lake Victoria (Kilama et al Each village has a population of over 2000. Some of 1981). It then spread throughout mainland Tanzania the villages are remote from the main road, for and is currently endemic in eight regions, namely example Kagera and Mvinza in the north-east and Arusha, Lindi, Ruvuma, Kagera, Kigoma, Tabora, Kitanga and Heru-Ushingu in the extreme north- Mbeya, Rukwa. The annual average number of west. cases for the last decade was 264. There are isolated foci, most of which have been producing cases for Villages Affected by Sleeping Sickness in many years. Heavy foci exist in Kigoma Region Kasulu District (Table 1). Thirty-two villages are exposed to the risk of infection in the endemic district of Kasulu. Of these, the Table 1. Number of sleeping sickness incidences diag- following are regarded as highest risk areas: Heru, nosed from district hospitals in six regions of Tanzania Kitagata, Kitanga, Makere, Mugombe, Mvugwe, for the past five years. Mwali, Nyachenda, Nyakitonto, Nyamidaho, Nyarugusu, Shingu. Others include Kagera, Region District 1996 1997 1998 1999 2000 Kaguruka, Kitema, Mvinza, Rungwe, Mpya, Titye. KIGOMA Kibondo 212 286 206 410 376 Overall, the estimated population at risk of infection ARUSHA Kasulu 155 198 172 156 191 is 230 000. Each village has a health clinic. There is Babati 12 19 15 12 34 one health centre for every six villages. Monduli 19 6 2 9 Hanang5 3 8 2 Villages in Kibondo District TABORA Urambo 1 7 2 9 RUKWA Nkansi 4 1 7 8 6 Sleeping sickness occurs in the following villages in Mpanda 5 1 - - - this district: Bitare, Biturana, Busunzu, Kanembwa, MBEYA Chunya 6 4 - - - Kazira mihunda, Kifura, Kilemba, Kingoro, TOTAL 400 531 421 588 627 Kitahana, Kumbanga, Kumhasha, Kumshindwi, Malagarasi, Mkabuye, Mvugwe, Nduta, Nyankwi, Although sleeping sickness was present in eight Nyaruyoba, Rusohoko. Each village has a dispensa- regions during the past ten years, the disease was ry. Each health centre serves six or seven villages. more concentrated in Kasulu and Kibondo districts of Kigoma Region, which accounted for about 90% Refugee Camps in Kigoma of the cases. These figures are underestimates due to There are about 280 000 refugees, mainly from the lack of accurate diagnosis and under-reporting. Eastern part of the Democratic Republic of Congo (DRC), settled in camps in Kigoma region. Sleeping KIGOMA SITUATION sickness cases have already been detected in these An outbreak of sleeping sickness is currently being camps and there is concern that an overlap of gam- experienced in Kigoma Region of Western Tanzania biense and rhodesiense sleeping sickness exists in along Lake Tanganyika. The disease represents a the region. continuing threat to the health and morale of many communities in the area. All the districts of Kigoma CAUSES OF EMERGENCE AND Region are affected by sleeping sickness, with RE-EMERGENCE Kibondo and Kasulu districts producing a great pro- The current situation of sleeping sickness in portion of the total cases over the past few years. Tanzania, especially the outbreak in Kigoma region, The whole of Kigoma Region is on the Western Fly is caused by the following factors: Belt, a portion of a large forest which extends, with • Poor surveillance due to inadequate funding and few breaks, over an area of 10 368 sq. km. and lies staff. between 31°E and 24.7°W, 3.5°N and 5.2°S. Here, • Inadequate and erratic supply of specific try- game is fairly abundant, and the people regard panocidal drugs. sleeping sickness as an old disease. • Poorly equipped field laboratories for diagnosis.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 45 • Opening up of new unauthorized settlements and IDENTITY OF THE HUMAN INFECTIVE farms in the endemic areas of sleeping sickness. In TRYPANOSOME IN TANZANIA the past, the people in Nyakitonto area, for exam- The occurrence of the chronic syndrome of sleeping ple, had wished to live in the valley (Kitome area), sickness, together with the marked variation in effi- which is wooded and well watered but also heavi- cacy of chemotherapeutic treatments, in Tanzania, ly infested with tsetse fly. Permissions to do so had may be cited as indications that the trypanosomes been consistently denied. However, a few families constitute a heterogeneous complex of organisms had occupied the area by 1983 and, as was perhaps including perhaps a mixture of T. b. rhodesiense and inevitable, many infections occurred thereafter. T. b. gambiense. • Lack of monitoring and health education campaigns. Old records show that the Malagarasi focus • Increased forest activities including cultivation (Kigoma region), for example, in north-western outside the villages or on the buffer zone. Some of Tanzania, is an old gambiense sleeping sickness the planned villages were, unfortunately, located focus with the last case of gambiense infection hav- in a rather dry and infertile area causing the vil- ing been reported in 1958 (Kihamia et al. 1991). The lagers to open new farmlands outside the villages. area is now considered endemic for rhodesiense • Apparent bush regeneration with resultant tsetse sleeping sickness. In Tanzania there are considerable encroachment. differences in the clinical types of human try- • The highly probable introduction of a virulent panosomiasis, with the severity of disease varying strain of T. b. rhodesiense. according to geographical location and becoming less virulent the further one goes south. While this The serious outbreaks of sleeping sickness in the may be attributed to the heterogeneity of T. b. rho- district are a disappointing setback in the disease desiense strains (Komba et al. 1997), there is concern control efforts of the country, and indicate a neces- that it may be due to the occurrence of the two sity for review of sleeping sickness control meas- species of trypanosome. Furthermore, the presence ures in the area. A study in Kigoma region sug- of large numbers of refugees from the DRC, a coun- gests the following factors are important in the try known to be endemic to gambiense sleeping current outbreak: sickness, increases this possibility. • Farming activities carried on outside the protected area. Further studies are required to: • Inter-village visits through tsetse infested bushes • Investigate why the disease is localized in specif- in search of the basic necessities of life. ic foci despite the tsetse fly and reservoir animals • Increased forest activities – honey and beeswax being present in large areas of the country. collection, fishing, hunting, etc. • Peridomestic activities e.g. firewood gathering, • Investigate the distribution of T. b. rhodesiense fetching water, cutting poles for building. strains in Tanzania. • Visits into the wildlife areas where no human settlement is allowed. • Investigate the possibility of an overlap of rhodesiense and gambiense sleeping sickness, especial- The problem that now confronts Tanzania is that ly in Kigoma focus, which has an influx of sleeping sickness is still endemic and there is lax- refugees from the DRC. ity of control measures. Compared to other endemic diseases, the number of human deaths References from sleeping sickness appears insignificant, but Kilama WL, Mtera KNM, Paul RK. Epidemiology of even temporary exacerbation of the disease fright- human trypanosomiasis in Tanzania. In: Proceedings ens the local people. of the 17th Meeting of the International Scientific Council of Trypanosomiasis Research and Control, 1981, While it is impossible to predict the future, the Publication No. 112, pg. 187. possibility of a larger outbreak must be considered. With the evidence currently available, it Kihamia CM et al. Trypanosomiasis. In: Health and would be a reasonable precaution to step up con- diseases in Tanzania. 1991, Harper Collins Academic, trol measures. UK.

Komba EK, et al. Genetic diversity among Trypanosoma brucei rhodesiense isolates from Tanzania. Parasitology, 1997, 115:571-579.

46 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 III THE SITUATION IN UGANDA animals in Zululand by David Bruce two centuries AND SUDAN ago (WHO, 1995). Later, morphologically identical trypanosomes were identified in the blood of a D.B. Mbulamberi, European from The Gambia, West Africa (Dutton, Ministry of Health, P.O. Box 7272, Kampala, Uganda. 1902), and transmission by riverine tsetse (Glossina palpalis) was confirmed. Subsequent studies SUMMARY revealed the extensive, often focal, distribution of Sleeping sickness is a disease of the rural poor the disease, the substantial endemicity and the which tends to emerge and re-emerge in epidemics chronic progressive nature of human infection in in some 36 sub-Saharan African countries, where West and Central Africa. close to 50 million people are at risk of contracting the disease. Together, these 36 countries report only The disease was called Gambian trypanosomiasis about 25 000 new cases of the disease to WHO annu- and the parasite, T. gambiense (later T. brucei T. brucei ally. This is an obvious underestimate attributed to gambiense). In 1908, a severe rapidly fatal trypanoso- poor reporting, difficulty in diagnosing the disease, mal infection was identified in the Luangwa valley, and poor accessibility of the affected areas. The true Zambia. Further investigation confirmed its clinical figure is currently estimated to exceed 300 000 new severity, the distinct epidemiology with transmis- cases annually. sion via savannah tsetse, and the zoonotic nature of infection from game animals harbouring T. brucei. In the recent past, Uganda and Sudan have not been This led to the description of Rhodesian trypanoso- spared epidemics of this disease. A devasting epi- miasis due to T. rhodesiense (T. b. rhodesiense). Other demic of T. b. rhodesiense sleeping sickness has members of the T. brucei T. brucei group, that are occurred in south-eastern Uganda and one of T. b. non-infective to humans and occur in game and gambiense in the West Nile region of the country domestic animals, were designated T. brucei (subse- (Uganda). In Southern Sudan, along the border with quently T. b. brucei). Uganda, there is another epidemic of T. b. gambiense sleeping sickness. The General Epidemiology of the Disease Trypanosomes, the parasites which cause the dis- The causes of the emergence and re-emergence of ease, occur in the blood of man and animals as the epidemics of this disease are varied, but can be con- trypomastigote. veniently grouped into political, economic, behavioural factors, and the effects of climate and vegeta- All members of the T. brucei group are morphologi- tion on tsetse fly distribution. cally identical. The parasites have evolved mechanisms for evading host immune responses through To limit the impact on human lives in these two variation of their surface antigen glycoproteins. The countries, external support will be required to zoonotic nature of T. b. rhodesiense was initially implement strategies for disease and vector control. established by inoculation of ‘volunteers’ with para- On the one hand, donor agencies, NGOs and mis- sites from a bushbuck (Heish et al, 1958), and later sion organizations could play an important role in from domestic cattle. supporting these control efforts. On the other hand, national authorities will need to control and coordi- Subsequently, the blood incubation infectivity test nate these efforts with assistance from WHO and the (BIIT) was developed to assess the human infective international community. potential of parasites from a range of wild and domestic animals. More recently, a number of This paper presents a general introduction to the molecular techniques, especially isoenzyme analy- disease (sleeping sickness) and a brief account of sis (Stevens and Godfrey, 1992) and restriction frag- factual epidemic outbreaks in Uganda and the ment length polymorphism (RFLP), have been used Sudan. The paper then proceeds to discuss, in gen- as markers for parasite strains to explore the molec- eral terms, possible factors for the emergence and ular epidemiology of this complex group of para- re-emergence of epidemics of the disease. sites. These techniques allow T. b. gambiense to be distinguished from T. b. rhodesiense.

INTRODUCTION In West and Central Africa, sleeping sickness is transmitted by riverine species of tsetse fly (palpalis Historical Aspects of the Disease group), which require sustained levels of humidity Trypanosoma brucei, and the role of the tsetse fly and prefer dense riverine habitats. These tsetse flies (Glossina spp.) as its vector, was identified in game feed preferentially on man, especially where man-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 47 fly contact is high, such as at water collection and tination test. Lancien in Uganda (1991) confirmed bathing points, river crossings and sacred groves. that insecticide impregnated traps can be used, with For the riverine species of tsetse fly, man provides community participation, to control sleeping sick- the reservoir of infection, although both wild and ness epidemics. domestic animals may play a minor role in particular foci. Throughout most of the range, T. b. rhode- Until recently, the treatment of sleeping sickness siense transmission is effected by savannah species relied essentially on three drugs, namely pentami- of tsetse (morsitans group). This group of tsetse flies dine, suramin and melarsoprol. Pentamidine, a survives in drier, more open areas of woodland, diamidine introduced in 1937, is currently available savannah and acacia thickets, and prefers to feed on as pentamidine isethionate and is effective against game animals and domestic stock. Human infection early infections of T. b. gambiense. Suramin, which occurs sporadically in individuals coming into con- was introduced in 1922, is effective against early tact with the zoonotic cycle, for example poachers, infections of both T. b. gambiense and T. b. rhode- hunters, honey gatherers, firewood collectors and siense. Melarsoprol, a trivalent arsenical introduced in tourists. A wide spectrum of animals, notably game 1949, was the only drug available, until 1990, for the animals and domestic cattle, provide a reservoir of treatment of late infections of both T. b. gambiense infection. However, in East Africa, the epidemiology and T. b. rhodesiense. All these drugs have adverse is different in that T. b. rhodesiense is transmitted by side effects, melarsoprol causing reactive encepha- a riverine species of tsetse, namely G. fuscipes lopathy in 5—10% of patients treated, with a fatal fuscipes, and domestic cattle are the main reservoir. outcome in 1 -5%. Resistance of T. b. gambiense to pen- This situation creates a lot of potential for epidemic tamidine, and of both T. b. gambiense and T. b. rhode- outbreaks of the disease. siense to melarsoprol, occurs. Nifurtimox, a 5—nitrofuran, is currently being used, either singly or in com- Clinical Manifestation of the Disease bination with other drugs, to treat late-stage gambi- In most endemic areas, T. b. gambiense causes a pro- ense infections on a compassionate basis. tracted, often initially unrecognized, illness with episodes of fever, headache and malaise, accompa- Eflornithine (DFMO), a potent inhibitor of nied by progressive lymphadenopathy and followed polyamine synthesis, was developed for the treat- later by the development of a progressive, fatal, ment of sleeping sickness through collaboration meningoencephalitis. This contrasts with the acute, between Marion Merrel Dow Inc., USA, and the severe, febrile disease observed with T. b. rhodesiense, UNDP/World Bank/WHO Special programme for with rapid progression to meningoencephalitis. Research and Training in Tropical Diseases (TDR). There is relentless deterioration to a stuporous state, However, while this drug provides the best alterna- with cachexia, wasting and progressive malnutrition, tive treatment to melarsoprol for gambiense sleeping deepening coma and death, within a few months in sickness, alone it is ineffective against T. b. rhodesiense the case of T. b. rhodesiense and extending for months infection. Therefore, no alternative treatment for or even years in the case of T. b. gambiense. late-stage rhodesiense infection is yet available.

AVAILABLE OPPORTUNITIES FOR THE The availability of all these drugs is currently high- CONTROL OF SLEEPING SICKNESS ly uncertain, with the various manufacturing firms The principle of control and prevention of sleeping either threatening to stop, or having already sickness relies on an integrated strategy of continu- stopped, their production. It is evident that the ous surveillance, involving diagnosis and treatment treatment of sleeping sickness is still unsatisfactory. of the population at risk, and vector control where The ideal trypanocide should be safe and effective. applicable (de Raadt, 1986). A number of tools for It must have a simple mode of administration to diagnosis and vector control have been developed allow its use under rural conditions where health through research during the past decade and are, facilities are usually of a poor standard, and, above indeed, field applicable by national health services. all, it should be affordable. These include the card agglutination test for trypanosomiasis (CATT) (Magnus et al, 1978) for serodiagnosis, and the miniature anion-exchange cen- THE PAST AND PRESENT SLEEPING trifugation technique (MAECT) (Lumsden et al, SICKNESS SITUATION IN UGANDA 1979) for parasitological diagnosis. The antigen AND SUDAN ELISA, developed by Nantulya (1989) and evaluated for detection of gambiense (Nantulya et al, 1992) Uganda and rhodesiense (Komba et al, 1992) sleeping sick- The sleeping sickness epidemic which devastated ness, was subsequently modified into a latex agglu- the shores of Lake Victoria at the beginning of the

48 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 last century is a famous event in the annals of tropi- and EATRO, the department probably in the best cal medicine. It is famous because an estimated one position to help contain this epidemic during its quarter to one third of a million people lost their outset, lost valuable logistics to a partner state and lives (Langlands, 1967). The same epidemic brought became helpless. The Ministry of Health posted controversy as to whether Dr Castellani or Colonel microscopists to the area and later opened up treat- Bruce first identified the trypanosome as the cause ment centres in the area. However, the epidemic of the epidemic. continued to increase in magnitude from 52 cases in 1976 to over 8000 cases in 1980 (Fig. 1). The cause of the epidemic was attributed to T. gambiense introduced to this part of the country by a Figure 1. Annual incidence of sleeping sickness due to party accompanying the explorer Lugard from the REPORTED T. b. rhodesiense in Eastern Uganda, 1976-March 2000 CASES Congo basin on relief of the Emin Pasha expedition 9000 8000 in 1894 (Christy, 1903). However, it is more accurate 7000 to say that the cause lay in the general increase in 6000 5000 social, commercial and military mobility which 4000 Cases up to March developed throughout tropical Africa in the late 3000 nineteenth and early twentieth centuries. 2000 1000

0 1976 77 78 79 1980 81 82 83 84 85 86 87 88 89 1990 91 92 93 94 95 96 97 98 1999 (March) Another outbreak involving about 2500 persons YEAR occurred in the same area from Jinja eastwards to the border with Kenya between 1939 and 1945 In the West Nile region, a new outbreak of T. b. gam- (Mackichan, 1944-45). The most striking feature of biense sleeping sickness occurred along the Dacha this epidemic was the virulence and rapidity of the river. During the first year of the outbreak, a total of fatal course of the disease observed on animal inoc- 12 cases were recorded. In the following year (1958), ulation. Thus, it is believed, the epidemic was this new focus produced 7 cases and it seemed as if caused by T. rhodesiense. The first cases detected the outbreak had ceased. However, the 1959 inci- were among migrant workers employed on Kakira dence of 30 cases, the highest annual figure since the sugar estates. Since these immigrants came from big epidemics had been finally checked 12 years areas of reasonable proximity to the infected areas of before, made it evident that the outbreak was by no Tanganyika (now Tanzania), this epidemic was means under control. thought to have been introduced by them. However, this outbreak was finally brought under Since that outbreak, cases continued to be reported control and the situation remained largely stable from within the infected area, though not in epi- thereafter until the early 1980s, when the current demic numbers. In 1971, infection spilled north of outbreak in the region started. This current outbreak the usual focus and involved up to 169 persons. is associated with the war of liberation against Idi Amin in 1979, when most local residents in the Following the control of the small epidemic of 1971, region fled into exile in Southern Sudan where there surveillance programmes were not instituted was a ravaging epidemic of T. b. gambiense sleeping because of the prevailing political and economic sickness, then as now. When these local residents atmosphere in the country at that time. There was returned to the West Nile region in the early 1980s, indiscriminate and haphazard movement of people some of them had the infection, which they intro- and livestock across the traditional trypanosomiasis duced into the area. The epidemiological trend of barrier zone. Besides, smuggling of commodities, the disease in the region (West Nile), over the years, including cattle, between Kenya and Uganda, across is shown in Fig. 2. the zone, became a means of livelihood. It was therefore difficult for the Ministry of Health teams to Figure 2. enforce surveillance measures. The Tsetse Control Annual incidence of sleeping sickness due to REPORTED T. b. gambiense in North Western Uganda, 1981-1999 Department could not carry out control programmes CASES 2500 due to lack of insecticide, transport and human resources. Thus, there was total breakdown of control 2000 measures and hence, by 1976, the stage was set for 1500 another epidemic outbreak of the disease in the area. 1000

500 This new outbreak started in Luuka County of 0 Iganga district in August 1976. Unfortunately, in 1981 82 83 84 85 86 87 88 89 1990 91 92 93 94 95 96 97 98 99 June 1977, the East African Community collapsed YEAR

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 49 Sudan uncontrolled movement of people into areas that Sleeping sickness due to T. b. gambiense has been may have been previously abandoned because of known to occur in the Southern Sudan since the epidemics, thereby promoting circulation of the par- early 20th century. Epidemics of the disease have asite in the population and the risk of contact occurred mainly in the Southern and South-western between people and the tsetse flies. Civil distur- parts of the country bordering Uganda, the bance and war will, in the final analysis, lead to a Democratic Republic of the Congo (DRC), and the breakdown in vital social services including med- Central African Republic. In addition, cases of the ical and vector surveillance programmes. This is disease have been reported along the Ethiopian bor- obviously the most important factor in the case of der, in Raga since 1909, in Yei since 1910, in Kajokaji the current epidemics in both Uganda and since 1914, in Nimule since 1915, in Tambura since Southern Sudan, as is the case in most other sub- 1918, and in Yambio since 1924. Saharan African countries, e.g. Angola, Mozambi- que, the DRC. Almost all the aforementioned foci are still active. Duku (1979) attributed these epidemic outbreaks to: Declining Economies the presence of active foci in neighbouring coun- Declining economies, as is the case in most sub- tries, particularly those where tribal settlements Saharan African countries, will dictate reduced straddled the borders; widespread distribution of financing of disease control programmes. This, in the vectors; and political upheavals, instability and turn, will affect field control activities as well as the civil disturbance. implementation of advances so far made in diagnosis, treatment and vector control. This is mainly The current flare-up of the disease started after the because these new advances are not available on the signing of the Addis Ababa peace agreement in local market and therefore require importing into 1972. The relative peace which followed the signing the country, for which foreign currency is required. of this agreement made it possible for some control In addition, reduced financing is likely to lead to the measures to be instituted with external assistance temptation of progressively dismantling vertical from WHO and the Government of the Kingdom of disease control programmes in preference for inte- Belgium between 1974 and 1978, hence the avail- grated, community-based programmes, thus lead- ability of the information shown in Fig. 3. Otherwise ing to loss of focus. information on the current epidemic in the Southern Sudan is not easily available. Behavioural Factors One of the factors to be considered here is the low priority rating accorded to sleeping sickness on the Figure 3. Self-reporting cases of sleeping sickness in Yambio district, part of both donors and national governments. This S. Sudan, 1974-1978 SELF- is despite the negative impact of the disease on REPORTING CASES development. One possible explanation is that 1600

1400 sleeping sickness control programmes do not have 1200 much appeal for international aid donors due to 1000 various factors including: the regional distribution 800

600 of the disease and mainly rural nature of the prob-

400 lem; the relatively small number of new cases 200 reported annually compared to other diseases; the 0 1974 1975 1976 1977 1978 requirement for long-term input to control pro-

YEAR grammes for sustainability in the absence of the prospect of eradication. Paradoxically, when epi- FACTORS RESPONSIBLE FOR THE demics of the disease occur, financial support is EMERGENCE AND RE-EMERGENCE OF made easily available in amounts which are usually SLEEPING SICKNESS disproportionately higher than those required for the regular preventive measures (Kuzoe, 1993). Factors responsible for the emergence and re-emergence of sleeping sickness are varied and diverse. Another pertinent behavioural factor, in this respect, Mbulamberi (1989) gave an outline of these factors. is the population density of the tsetse flies and their A brief account, a modified version of these factors, feeding behaviour. A tsetse fly feeding on a number is given below. of animals, and possibly also on man, may become infected with many different strains of try- Civil Disturbance and War panosome. Most of these strains will be non-patho- Civil disturbance and war cause extensive and often genic for man and, even if a man-infective strain is

50 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 acquired by the fly, the tendency will be for it to be recruited for large-scale construction work (tropical so diluted by the non-pathogenic strains that it will aggregation of labour) and pilgrims attending major not be passed on in sufficient number to cause infec- religious festivals. tion in man. A new population in an area may spark off an epi- Another important behavioural factor is increased demic outbreak of sleeping sickness as a result of man-fly contact. This phenomenon occurs most imported cases among them, which may be suffi- commonly during the hot, dry season with the ciently large to increase the reservoir of infection result that transmission is enhanced. Indeed, a peri- available to the insect vector and so, in a quantita- od of drought almost invariably means an increase tive manner, promote transmission. An imported in the number of infections because the few sources strain may also show quantitative differences such of water are shared by man, tsetse flies and game as enhanced virulence or ability to spread, or may animals, in close association; there is also more be one to which the indigenous population has not hunting and more searching for wild forest products been previously exposed and to which no resistance at times when crops are bad. This phenomenon is has been acquired. This phenomenon can also oper- particularly applicable to T. rhodesiense infections ate vice versa. Further, as with some other diseases, (Willet, 1965, and many other workers). the periodicity of epidemics of sleeping sickness may be associated with the growing up of a new The presence of domestic and wild animal reservoir generation of people with no previous experience of hosts is another important factor. The pig in the case the disease. of T. gambiense, and cattle in the case of T. rhodesiense, have been incriminated as domestic animal reser- The occurrence of sub-acute cases of the disease is voir hosts, while the kob and hartebeest in the case yet another important factor. The presence of an of T. gambiense and the bushbuck in the case of T. undetected and perhaps unsuspected reservoir of rhodesiense have been incriminated as wild game infection in the form of human “healthy carriers” of reservoir hosts. The bushbuck is particularly impor- the disease, which has been reported by several tant because it tends to live in thickets near human workers (Buyst, 1977; Rickman, 1974; Woodruff habitation, which puts it in close contact with man. et al, 1982), has important epidemiological implications. The appearance of different forms of the parasite is another important factor. The appearance of such Under conditions in which man-biting tsetse are parasites may be due either to the parasites being common and where people congregate, the ambu- introduced from outside the area or to genetic lant human carrier assumes a powerful potential for changes in the parasite. There is at least a suspicion, the onward transmission and spread of sleeping based on field observations, that zymodemes of try- sickness. The occurrence of asymptomatic carriers panosome introduced into fresh localities may of rhodesiense sleeping sickness is certainly low. exhibit an enhanced ability to spread through the However, sleeping sickness cases with non-specific community. Scott (1961) reported two instances in symptoms (fever, headache) who remain ambulant which the introduction of infected persons from an for several weeks are common, and they too may be established epidemic area resulted in outbreaks of important reservoirs of infection where man-fly the disease in endemic localities far removed from contact is intense (Wurapa et al, 1984). This threat is the original focus of infection. There are other simi- also present among many early cases of the gambi- lar observations suggesting that severe local out- ense disease, in which the initial stages are general- breaks which quickly follow the introduction of ly relatively mild and the victim may continue to infected persons to fresh localities are, in some way, work for many months or even years before he is connected with enhanced ability of the zymodeme eventually driven, by increasing illness, to seek to spread. Indeed, the possible existence of epidem- treatment or to retire to his home. During this time, ic trypanosome zymodemes has been advanced by he is a constant source of infection to tsetse so that some workers. the very nature of the illness provides great opportunities for its spread. In both the Ugandan and Another factor of importance are the changes in Southern Sudan situation, the question of delayed population movements and population growth. It is diagnosis and treatment is a big factor. generally supposed that population movements are liable to precipitate epidemics. Refugees displaced Another critical factor in this category is human as a result of war, famine, earthquakes and other behaviour and activities in the fly’s habitat. Often, similar occurrences are notoriously prone to disease man becomes infected during travel, hunting, fish- in epidemic form, as are immigrant labour forces ing, collection of honey or when working in the bush

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 51 which the fly inhabits. Fishing and “honey-hunting” vive for protracted periods. This enhances the are particularly hazardous occupations. While fish- potential for these flies to transmit the disease, of ing in riverine pools surrounded by thickets, people course depending on their infection rates. may be in close contact with tsetse flies for many days at a time, a situation in which the association References between humans, bushbuck and tsetse fly is likely to Christy C. The Epidemiology and Etiology of the be significant and conducive to transmission and sleeping sickness epidemic in the Equatorial East spread of the disease. Wyatt et al. (1985), working in Africa with clinical observations. Report of the north-east Zambia, found fishing to be more com- Sleeping Sickness Commission of the Royal Society, mon among cases of sleeping sickness than controls. 1903, 3:3-32. Fishing represented a hazard both while walking to the stream and while engaged in the activity itself. Buyst H. The epidemiology of sleeping sickness in the historical Luangwa Valley. Annales de la Société Climate, Vegetation and Tsetse Fly Belge de Médecine Tropicale, 1977, 57(4-5):349-359. Distribution Factors Climate appears to be of more than ordinary impor- De Raadt P. Integrated sleeping sickness control. In: tance. At higher temperatures, there is an increased Proceedings of the CEC International Symposium, Ispra, salivary gland infection rate in the tsetse fly, and, in Italy, 1986, 4:147-151. addition to this direct effect, there are many ways in which climate influences a closer association Duku MO. Human trypanosomiasis in the Southern between man and fly. Sudan: Present situation and control measures in: International Scientific Council for Trypanosomiasis In the current epidemic of sleeping sickness in south- Research and Control 16th meeting, Yaounde Cameroon, eastern Uganda, heavy rains coupled with the abun- 1979, 139-145. dant growth of Lantana camara thickets provided G. f. fuscipes with suitable conditions well outside its Dutton JE. Preliminary note upon a trypanosome usual riverine habitat, so it was able to live and breed occurring in the blood of man. Thomas Yates in the vegetation surrounding homesteads. Laboratory Report, 1902, 4:455.

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Wijers DBJ. The importance of the age of Glossina palpalis at the time of the infective feed with Trypanosoma gambiense. In: International Scientific Committee for Trypanosomaisis Research and Control. 7th meeting, Bruxelles, 1958. London, Commission for Technical Cooperation in Africa South of the Sahara, 1960, 319-320.

Willet KC. Some observations on the recent epidemiology of sleeping sickness in Nyanza region, Kenya, and its relation to the general epidemiology of Gambian and Rhodesian sleeping sickness in Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1965, 59:374-386.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 53 Annex 4 EPIDEMIOLOGY, DISEASE SURVEILLANCE AND CONTROL, AND VECTOR CONTROL African trypanosomiasis

54 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 I EPIDEMIOLOGY AND According to the World Health Organization (1998), CONTROL OF HUMAN AFRICAN there remain within the “tsetse belt” more than 200 TRYPANOSOMIASIS* active foci, located between latitudes 15° North and 15° South. Within this area, 60 million individuals liv- Honoré A. Méda1 and Jacques Pépin 2 ing in 36 countries are exposed to the infection. Due 1 Project SIDA2, Benin (ACDI-Canada) B.P. 900 Tri to shortages of financial and human resources, less Postal, Cotonou, Republique du Benin. than 4 million benefit from an adequate surveillance 2 University of Sherbrook, Infectious Diseases Division, and control programme; all endemic countries are Centre for International Health, 3001, 12th Avenue, characterized by shortages of the financial and Sherbrook, Quebec, Canada. human resources necessary to implement or sustain a comprehensive control programme. Reports from INTRODUCTION national control programmes can only give a rough Human African trypanosomiasis (HAT) is caused by idea of the epidemiological situation, because of the hemoflagellates of the Trypanosoma genus, difficult security situation and the decay of commu- Trypanozoon subgenus and brucei species, which nication systems in many parts of high-incidence classically includes three subspecies: Trypanosoma countries. Year-to-year incidence for the 1977-1994 brucei brucei, T. b. gambiense and T. b. rhodesiense. period in all endemic countries can be found in a These subspecies are morphologically identical but recent WHO report (WHO, 1998), where Figure 1 differ in their ability to infect various hosts. T. b. bru- shows the geographical distribution of the cumula- cei is essentially a parasite of domestic and game tive number of new cases reported between 1977 and animals and is not pathogenic to humans because it 1997 for the endemic countries. T. b. gambiense try- is lysed by a haptoglobin-like molecule (Smith et al, panosomiasis is now a major public health problem 1995). Only T. b. rhodesiense and T. b. gambiense are in Central Africa, specially in the Democratic considered to be human pathogens. There are two Republic of Congo (DRC), Angola and Southern clinical variants: an acute syndrome attributed to T. Sudan, where the ongoing civil war hampers control b. rhodesiense and a chronic one caused by T. b. gam- efforts to such an extent that national statistics give biense. Both diseases result from complex interac- only a very incomplete view of the problem. In DRC, tions between the parasite and its tsetse fly where relatively better information is available, the (Glossina) vector and vertebrate hosts. total number of people at risk is estimated, by the national control programme, to be 12 500 000. The During the last couple of decades, considerable number of new cases reported each year has now progress has been made toward the improvement of reached levels comparable to those seen in the early epidemiological knowledge. This has led to the 1930s, despite substantial underdiagnosis due to development of new tools suitable for control. A inadequate coverage of endemic regions; this situa- recent paper by Pépin et and Méda (2001) provides details tion may result in the death of as many adults as of the advances in the epidemiology and control of AIDS (Ekwanzala et al, 1996). Underdiagnosis is also HAT. However, these advances have not been suffi- exacerbated by the poor sensitivity of diagnostic ciently used in the field for what they were intended. methods. The number of cases reported annually The aim of this paper is to try to summarize what we increased dramatically from about 10 000 in 1980 to know of the epidemiology and control and to identi- more than 27 000 in 1998. In the most endemic fy some of the most important gaps that need to be regions (e.g. Equateur and Bandundu), many com- urgently tackled by the scientists and programme munities have been found to have a prevalence of managers involved in research and control activities. over 10% during recent case-finding surveys. In 1994, an extra-ordinary prevalence of 72% in a small village CURRENT EPIDEMIOLOGICAL of the Bandundu region was reported. SITUATION AND DISEASE BURDEN HAT is the only vector-borne parasitic disease whose Angola is the country with the second highest inci- geographical distribution is limited to the African dence of HAT, respectively 8275 and 6610 new cases continent. T. b. gambiense is seen in West and Central were reported in 1997 and 1998 by the national con- Africa, and T. b. rhodesiense in East and Southern trol programme. Variations in the annual number of Africa. Uganda is the only country where both sub- reported cases must be interpreted with caution due species are found: T. b. gambiense in the north-west to the impact of the civil war on case-finding. The and T. b. rhodesiense in the south-east. This distribu- disease is endemic in the north-west provinces. The tion has probably remained constant over time. prevalence rates reported vary between 1.3% and

* The contents of this paper are drawn largely from Pépin J. Méda AH. The Parasitology, 2001, 49:71-132, by permission of the publisher Academic epidemiology and control of human African trypanosomiasis. Advances in Press.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 55 9.7%. Uganda is the only country where both T. b. 30 days on average; it varies, according to species gambiense and T. b. rhodesiense are found, the former and the ambient temperature, between one and in the north-west of the country, near the Sudanese eight weeks. When Glossina takes a blood-meal from border, and the latter in the south-east, without over- an infected host, it ingests bloodstream trypo- lap. Between 1000 and 2000 T. b. gambiense cases were mastigote forms in its salivary glands. Trypanoso- reported annually in 1990-1994, but this has at least mes then move to the fly’s midgut where, over a few stabilized with 978 cases reported in 1998, more than days, they transform into the procyclic stage. The half of them from Arua district. A major epidemic of variant surface glycoprotein (VSG), the dominant Rhodesian HAT devastated SE Uganda from the constituent of the surface of bloodstream try- mid-1970s, with a cumulative total of about 40 000 panosomes, is replaced by an invariant surface pro- new cases over 15 years (Smith et al, 1998; Hide, tein. After two to three weeks of maturation and 1999). There has been some improvement recently, multiplication, trypanosomes migrate to the sali- after many years of efforts by national authorities vary gland, where other transformations lead to with substantial external support. Only 271 cases their development into metacyclic trypanosomes, were reported in 1998 from the SE of the country which require VSG to be infective for a susceptible (Busoga). In Sudan, where reliable statistics are not human or animal host during the next blood-meal. available, the foci of T. b. gambiense HAT are located These forms are the metacyclic trypomastigotes, the in the southern part of the Equatoria region, west of only stage which is infective to vertebrates. Once the Nile, within 100 kilometres of the borders with infected, a tsetse fly remains so for the rest of its life Central African Republic (CAR), the DRC and span of between three and four months. While tak- Uganda. Extrapolations suggest that there must be at ing subsequent blood-meals, the fly is capable of least a few thousand cases per year. In other coun- inoculating another vertebrate host with the meta- tries of Central Africa, HAT is more or less an emerg- cyclic trypanosomes. Within the human host, the ing public health problem. Elsewhere in East and parasite multiplies at the site of inoculation, where a Southern Africa, the incidence of T. b. rhodesiense try- chancre might develop. Inoculation chancres are panosomiasis remains low. In West Africa, the dis- rarely recognized on African skin. From this site, the ease has regressed or disappeared from several parasite gets into the bloodstream and lymph nodes countries as ecological changes reduced the intensity and multiplies through binary scissiparity with of man-fly contact. Only Côte d’Ivoire and Guinea three morphological forms: the short and stumpy, still report a significant number of cases. Only a few the intermediate, and the long and slender forms. dozen cases are seen each year in Burkina Faso and Mali, corresponding both to importation of cases and After infection of a human host, there is a switch local transmission. The situation is less well known from the expression of metacyclic VSG to blood- in other West African countries. stream VSG. To evade the host’s immune response, trypanosomes can successively express different It is difficult to estimate the overall burden of HAT. VSG, but only one at a time. This antigenic variation There are about 100 000 new cases per year, with is a unique feature of trypanosomes (Barry, 1997); it between one third and one half of cases remaining has direct consequences for the epidemiology of the undetected and untreated. Rhodesian trypanosomi- disease. Up to a thousand different VSGs are genet- asis represents less than 5% of the overall burden of ically encoded, and it is thought that the VSG cur- the disease. Recent estimates (WHO, 2000) are rently expressed protects invariant constituents rather similar, with 2.05 million disability adjusted from the host’s immune response. The mechanisms life years (DALYs) lost, and 66 000 deaths, in 1999 through which trypanosomes switch to expressing a due to HAT. As a comparison, the number of mil- different VSG are complex, but it allows the parasite lions of DALYs lost is estimated at 45.0 for malaria, to escape from antibodies directed against the pre- 4.9 for lymphatic filariasis, 2.0 for leishmaniasis, 1.9 vious VSG. This complex process explains the inter- for schistosomiasis, 1.1 for onchocerciasis, 0.7 for mittent parasitaemia and the very long, largely Chagas disease. asymptomatic, incubation period. Thus, there is an alternation between periods of higher parasitaemia following expression of a new VSG, during which EPIDEMIOLOGY OF T. B. GAMBIENSE the human host might be more infectious, and peri- HUMAN AFRICAN TRYPANOSOMIASIS ods of lower or undetectable parasitaemia, during which infectivity must be lower. Variations in the The Life Cycle of T. brucei virulence of T. b. gambiense strains were noted early The complex life cycle undergone by T. brucei in its by Van Hoof (1947). More recently, biochemical, tsetse fly vector and human host is described in var- molecular and immunological methods have been ious standard textbooks. The whole cycle lasts about developed for the identification of trypanosomes by

56 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 examining their genetic material (Gibson, 1994). savannah of Central Africa. The savannah is the These methods include two-dimensional gel elec- exclusive habitat of G. morsitans in various regions trophoresis and isoenzyme, chromosome and DNA of Africa. Its geographical distribution is limited to analysis using polymerase chain reaction (PCR) gallery forests bordering streams, where optimal techniques, which have substantially contributed to survival conditions are found. In a given geograph- a better understanding of the parasite’s taxonomy ical area, the distribution of tsetse flies varies and the epidemiology of the disease, especially in depending on the species and is mainly determined studies on animal reservoirs of human trypano- by the climate, presence of water, vegetation and somes. Cellular and molecular characteristics of availability of sources of blood-meals (humans and human trypanosomes have been reviewed else- animals). Studies conducted in the forest zone of where (El-Sayed and Donelson, 1997; Barry 1997). West Africa have shown that G. p. palpalis is a very mobile vector found in different biotopes and that its distribution is closely linked to human occupa- The Vectors tion patterns (Baldry, 1980; Challier and Gouteux, 1980; Gouteux and Laveissière, 1982). Highest Vector species and sub-species apparent densities per trap (ADT) per day were The tsetse fly is a vector of trypanosomes that infect observed on the edges of villages where the swine man as well as wild (antelopes, giraffes, etc.) and population provides an abundant, easily accessible domestic (pigs, cattle, sheep, goats, dogs, horses, food source. High densities have also been found etc.) animals. It belongs to the genus Glossina, which near sources of potable water, in coffee and cocoa includes about 30 known species divided into three plantations, especially those located on the edge of groups: the Glossina sub-genus which includes forests or gallery forests, and along paths separating species of the morsitans group; the Nemorhina sub- plantations from the remaining forest used by genus (palpalis group) and the Austenina sub-genus humans and some wild animals, especially the (fusca group). A software has been developed for the bushbuck, which are food sources for Glossina. identification of glossinids by species and subspecies and the determination of their epidemiolog- The tsetse fly is only active for a short time when ical importance (Brunhes, 1994). Several factors looking for blood-meal (35 minutes on average per related to the vectors determine the transmission of day). It spends most of the time resting to digest or trypanosomes to humans: vector biology and ecolo- gestate. The amount of activity of each species gy, vectorial capacity, man-fly contacts, longevity, determines its chances of encountering a host on dispersal, feeding behaviours, etc. which a blood-meal may be obtained and varies depending on climatic factors (temperature, humid- Distribution and ecology of Glossina ity, amount of light, wind, rain), olfactory and visu- A large variety of traps have been tested for tsetse al stimuli (smelling and seeing a potential feeding sampling and control, a dozen of which were host), and on intrinsic factors (physiological age, reviewed by Leak (1999). The favourite technique nutritional status, gravidity). currently used to study Glossina habitats, densities and ecodistribution is the biconical trap developed Vectorial capacity, competence and by Challier and Laveissière (1973). Turner (1980) susceptibility of Glossina proposed the “marking-release-recapture” tech- The vectorial capacity of Glossina is determined by nique using a radioactive compound (e.g. Fe59) and its ability to infect itself while feeding on a verte- scintillometer to study its bioecological characteris- brate host, and to subsequently develop an infection tics such as Glossina habitats, behaviour and dynam- and transmit the trypanosome to another verte- ics, population size, densities, dispersal, survival brate host (Challier, 1982). According to these crite- rates, resting sites. ria, only the palpalis and morsitans groups contain species and sub-species that are vectors of T. b. gam- The palpalis group contains two excellent vector biense. It has been demonstrated that not all flies in a species of T. b. gambiense and of animal trypanoso- given area have the same capacity to transmit the miases in West and Central Africa: G. palpalis palpalis parasite. It is therefore important to determine whe- in forest areas and G. p. gambiensis in savannah ther or not there are local conditions that may areas. The former inhabit the forest areas and the reduce or increase this capacity. The tsetse fly’s abil- moist savannah, whereas the latter is found in the ity to infect itself while feeding on a parasitized host semi-arid savannah. G. tachinoides and G. fuscipes depends on several poorly understood factors. The fuscipes are related to the palpalis group and are vec- number of trypanosomes ingested by the fly during tors of sleeping sickness in the savannahs of West its blood-meal could be one such factor. However, it and Central Africa while G. pallidipes is found in the has been demonstrated that a single trypanosome

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 57 can infect G. m. morsitans (Maudlin and Welburn, distinguish between meals of human and animal ori- 1989; Baker, 1991). The concept that a blood-meal gin. Glossinids of the palpalis group are notable for taken from an individual with low-level para- their eclecticism and opportunism: they feed indis- sitaemia is less infectious for the vector than one criminately on many species and are therefore very taken from a host with high-level parasitaemia dangerous to man. In the forest zone of Côte d’Ivoire, needs to be investigated. Under natural conditions, Gouteux et al (1982) found that at least 75% of the the infection rate among tsetse flies is quite low. On blood-meals were from suidae around the villages. In average, less than 1% of tsetse flies are infected with plantations in the same zone, however, Laveissière T. brucei ssp (Molyneux, 1980b). Teneral flies are et al (1985) found that 46% of the blood-meals were more susceptible to infection by trypanosomes. from man. This observation was confirmed by recent results of Sané et al (2000). As reviewed by Leak (1999), it has been suggested that lectins present in the haemolymph and midgut A comparison of the results of analyses of blood- of the fly are responsible for increasing the resist- meals collected in the five main foci in Côte d’Ivoire ance of the tsetse fly, with age, to infection with showed that the proportion of human blood-meals T. brucei. Lectins are absent or blocked in unfed ten- varies significantly from one focus to the other, eral flies, thus permitting infection, while in older although the socio-geographic conditions in these flies lectin production seems to be stimulated by foci appear to be identical: a human origin corre- blood-meals, preventing subsequent infections. The sponded to 42% of blood-meals of G. p. palpalis at trypanosome also has effects on Glossina. Jenni et al Vavoua, 73% at Daniafla, 55% at Zoukougbeu, 91% at (1980) have shown that the infected insect has a ten- Gagnoa and 27% at Sinfra. Humans, therefore, seem dency to bite more often and feed more voraciously, to be the favourite host, in differing degrees, of which can have a substantial impact on its transmis- G. p. palpalis in plantation zones of Côte d’Ivoire. This sion potential. According to Maudlin et al (1998), the preference for man was greater in the foci with low risk of death increases with age, but much more transmission rates (Daniafla, Gagnoa) than in the quickly in infected than non-infected tsetse flies. more active foci (Sinfra, Vavoua and Zoukougbeu). Infected flies are much more susceptible to insecti- Domestic animals appear to play an important role in cides than non-infected flies (Nitcheman, 1990). feeding tsetse flies at Vavoua and Zoukougbeu, the most active foci, whereas, in the low-incidence foci, Feeding behaviour and trophic preferences the percentage of animal blood-meals is insignificant. The first concern of the teneral Glossina is to find a At Zoukougbeu, apart from on humans, G. p. palpalis host on which a blood-meal may be taken. The feeds freely on domestic swine (30% of meals), young adult needs a blood-meal to complete its whereas at Vavoua it prefers the bushbuck which maturation. This first meal takes place between 24 provides an equal percentage of its food as man and 72 hours or even later, and depends on intrinsic (42%). The diversity of the feeding regimen of G. (diurnal rythm, sex, gravidity, species, etc.) and p. palpalis might explain to some extent the varia- environmental (climatic conditions, visual, mechan- tions in levels of incidence of human disease. In foci ical and olfactory stimuli) factors (Colvin and where transmission is more intense, G. p. palpalis Gibson, 1992). The tsetse’s feeding ground may be feeds on both man and animals and proper case-find- restricted by the relatively limited dispersion of ing and treatment of infected humans is not sufficient potential sources of blood-meals. In forest areas, to control the disease. With a diversified regimen G. p. palpalis confines itself to the outskirts of vil- alternating between man and animals as sources of lages where swine hide, along paths through plan- blood-meals, tsetse flies are able not only to transmit tations, near sources of potable water, and around trypanosomes to humans, but also to maintain the farm camps and other places used by man putative animal reservoir. In contrast, in low trans- (Laveissière and Hervouët, 1981). mission foci, the tsetse fly depends on man, transmission to and from animals is rare, and case-finding Most studies on the trophic preferences of Glossina prevents the accumulation of human cases. This West were conducted in West Africa and based on analyses African paradigm should probably not be systemati- of blood-meals collected by dissecting the insect. cally extrapolated to high-incidence foci of Central Methods with different degrees of sensitivity have Africa, where the role of animals is less clear. If one been reviewed by Leak (1999), including the precip- of its usual mammal hosts is not available, G. itin test, the agglutination inhibition test, the comple- tachinoides replaces it with reptiles as observed in the ment fixation test, direct and indirect ELISA assays, gallery forest of the savannah: snakes or lizards tests based on the latex agglutination technique, and, account for between 54% and 67% of the blood-meals more recently, one based on electrophoresis of super- of G. tachinoides, while only 8% of its meals are from oxide dismutase (Diallo et al, 1997) which can only man (Laveissière and Boreham, 1976). The feeding

58 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 preferences of this vector also vary seasonally The number of trypanosomes inoculated into the because of changes in the availability of hosts. In the mammal host during a blood-meal is probably one hot season, 30%-55% of its meals come from mammal of the factors that determine the probability of trans- hosts (mainly humans and bushbucks) whereas in mission. It has been estimated that, to infect man, the cold season, the only animals available are rep- the inoculum must contain between 300 and 500 try- tiles, and these provide over 50% of its meals panosomes (Challier, 1982). Transmission is also (Laveissière and Boreham, 1976). influenced by factors intrinsic to the vector (physiological age, nutritional status, gravidity, etc.) and by Transmission cycles of T. b. gambiense climatic and other environmental conditions. The In the forest zone of Côte d’Ivoire, man-fly contacts low infection rate naturally observed among tsetse occur in almost all biotopes but especially in planta- flies limits the devastating epidemic potential of tions and peridomestic areas where it is easier for African trypanosomiasis, and explains the apparent the fly to find hosts (Challier and Gouteux, 1980; paradox between the abundance of the G. p. palpalis Laveissière et al, 1985). Some authors attribute the and G. morsitans vectors, and the relative rarity of persistence of residual foci of HAT to the existence human disease. There is no direct correlation of other cycles adjacent or parallel to the common between the densities of G. p. palpalis, the major vec- man-fly-man cycle (Molyneux, 1980a; WHO, 1998), tor living in close contact with man, and the inci- i.e. cycles where the parasite travels between dence of sleeping sickness. domestic and/or wild animals (swine, sheep, goats, bushbuck, etc.) and man. They identify two such In endemic foci, the nature, frequency and intensity transmission cycles: a) a domestic cycle where the of man-fly contacts are the major determinants of Glossina transmits the parasite between human the risk of African trypanosomiasis. In the forest hosts and domestic animals; b) a sylvatic cycle zone of Côte d’Ivoire, there is virtually no ecological where the vector transmits the parasite between zone where humans are safe from being bitten by wild animals, sometimes with humans or domestic G. p. palpalis. Man-fly contacts can occur in all botan- animals entering this cycle, resulting in sporadic ical zones and are affected not only by the vegetal cases or even epidemic outbreaks. The domestic environment but also, and especially, by human cycle hypothesis is supported by similarities hosts. Multidisciplinary studies have shown that observed between parasites isolated in humans, ani- human activities and behaviours have an important mals and the vectors (Gibson et al, 1978; Mehlitz impact on the epidemiology of sleeping sickness, et al, 1982), whereas there is little evidence that wild through an increase in the frequency and intensity animals are infected with T. b. gambiense. of man fly-contact (Laveissière et al, 1985, 1986a, 1986b; Hervouët and Laveissière, 1987; Méda et al, Tsetse flies and the epidemiology of African 1993). For example, coffee-growing, which requires trypanosomiasis the planter to spend more time in the plantation The transmission of infectious trypanosomes to than if growing cocoa or food crops, is associated humans depends on many factors: the density of with a high risk of infection due to the increased Glossina populations, Glossina longevity, the vector’s contact time with the vector. This risk is heightened susceptibility to infection, Glossina infestation rates by residing in farm camps and by procuring water and the factors that influence these, and human from natural water sources located near edges of behaviours and activities in the biotopes of the flies plantations and gallery forests. Many other activi- that determine the frequency of man-fly contacts. As ties, such as collecting firewood, washing and fish- described in the previous sections, the vector’s biol- ing, bring human hosts into contact with tsetse flies. ogy, ecology and feeding behaviour have direct con- The availability of other sources of blood-meals (e.g. sequences on transmission of the parasite. Thus, pigs) reduces the chances of transmission. through its biological cycle, the Glossina allows the maturation, development, multiplication, transmis- The Human Reservoir sion and dissemination of the parasite. The feeding Humans are the main reservoir of T. b. gambiense. behaviour of various species of Glossina determines Four factors potentially influence man’s potential in their epidemiological contribution to the transmis- transmitting T. b. gambiense: the duration of infec- sion of T. b. gambiense to the humans, and to some tion, the degree of parasitaemia, the number and extent the animals, that constitute the parasite’s distribution of individuals who are infected, and the reservoir. The female G. p. palpalis, because of its intensity of contact with the vectors. more aggressive feeding behaviour and longer life span, plays an essential role in the transmission of The duration of infection in humans the parasite. Given that the tsetse fly is a relatively ineffective vector, the very long duration of infection (from a

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 59 few months to several years) during which the take most blood-meals on non-human sources and human host can maintain normal activities, and transmission to humans is unlikely. On the other provide blood-meals for the vectors, must be the hand, high human population density results in key determinant behind the endemic features and modifications of the habitat that reduce the tsetse epidemic potential of Gambian HAT (Baker, 1974). population. Thus, an intermediate population den- Fortunately, this long duration of infection offers a sity is optimal for Gambian trypanosomiasis to golden opportunity for intervention. Control pro- prosper. A recent study in Côte d’Ivoire showed a grammes focusing on the identification and treat- strong correlation between the epidemiological risk ment of asymptomatically infected humans, and and settlement density (Laveissière and Méda, thus on a shortening of the duration of infection, are 1999). Whether or not there are secular or seasonal extremely effective if an overwhelming majority of variations in the frequency with which humans the population shows up during case-finding sur- become infected with T. b. gambiense is unknown but veys and if sensitive diagnostic methods are used. plausible through changes in tsetse densities, man- fly contact, presence of alternative sources of blood- The distribution of infection in human popula- meals, etc. tions Prevalence of trypanosome-infected individuals in Familial aggregation human populations varies tremendously according Familial clustering of trypanosomiasis has been rec- to socio-demographic factors. Trypanosomiasis is ognized since the beginning of the century. Compa- not found equally in males and females. The appar- red to children of mothers without a past history of ent preponderance of cases among females would HAT, the risk of a child having had trypanosomiasis not correspond to substantial differences in inci- was four times higher if the mother had had the dis- dence or prevalence if reliable denominators were ease, while it was two times higher in brothers and available (which is rarely so). Females often partici- sisters of a case than in their half-brothers and half- pate more regularly in case-finding sessions, which sisters. Such clustering could be due to either genet- can also lead to a higher number of cases (Ason- ic susceptibility or to shared exposure to the vector. ganyi and Ade, 1994). Variations in incidence and Several arguments reviewed elsewhere (Khonde prevalence between age and ethnic group have also et al, 1997) suggest that the latter is the most plausi- been noted. This probably relates to differences in ble explanation. Shared exposure could result from occupational exposure and other determinants of simultaneous contact with an infective tsetse whose man-fly contact rather than to genetically deter- blood-meal on a first individual is interrupted and mined susceptibility (Laveissière et al, 1986a; Her- resumed on a nearby relative, or from members of a vouët and Laveissière, 1987; Méda et al, 1993). Other same family sharing an ecological microcosm and risk factors have been described, which are probably being similarly but not simultaneously exposed to all markers of exposure to infective tsetse flies: lack the vector bites (Gouteux et al, 1989). of formal education, absence of pigs (an alternative source of blood-meals for tsetse) in the habitat, etc. The influence of human behaviour (Méda et al, 1993; Méda et al, 1995). Human behaviour plays an important role in the epidemiology of Gambian trypanosomiasis. Health Occupation is also related to exposure and to inci- seeking behaviour might delay the recognition of an dence. For instance, in Côte d’Ivoire, HAT is more epidemic and enhance transmission of the parasite frequent in coffee and cocoa plantation workers or if symptomatic individuals wait for months before people who fetch water than in other inhabitants reaching a health facility where trypanosomes can (Laveissière et al, 1986a and b; Méda et al, 1993). be detected, maybe because they first attributed the More than 80% of cases occur in people who not disease to other, supra-natural, causes and sought only work but also live in small plantation settle- traditional treatment. Participation in case-finding ments; a case-control study showed that such peo- surveys varies from place to place and over time, ple were five times more likely to develop try- and is a key determinant in the success or failure of panosomiasis than their counterparts who resided such programmes. in villages (Méda et al, 1993). In some foci of the DRC, a higher prevalence was found in fishermen, Migrations have contributed to trypanosomiasis while elsewhere, farmers were more likely to get epidemics, as they favour the circulation of try- infected (Henry et al, 1982; Mentens et al, 1988). panosomes from high-incidence to low-incidence areas where the population is more susceptible Human population density influences the risk of (Prothero, 1963). Although this remains controver- epidemics in gambiense sleeping sickness (Scott, sial, the explosive epidemics seen in Uganda and 1970). If population density is very light, tsetse flies the Congo a century ago have been attributed by

60 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 many authors to the large-scale circulation of (Van Hoof, 1947; Schutt and Mehlitz, 1981). These workers organized by the colonizers to suit their infections generally resulted in low parasitaemia needs (Leak, 1999). A recent example of the impact that lasted less than a year. The study of naturally of migrations is the epidemic in NW Uganda, which occurring infections in animals was facilitated by resulted from the exodus of Ugandan refugees to the development of appropriate laboratory meth- Sudan and Zaire where they got infected, followed ods: the blood incubation infectivity test (BIIT), a few years later by the migration to Uganda of isoenzyme electrophoresis, and DNA analysis. infected Sudanese refugees and the return of Domestic animals were found to be infected with Ugandans back home (Paquet et al, 1995). parasites enzymatically identical to T. b. gambiense. Pigs have generated more interest because they are Immunity a frequent source of blood-meal for tsetse flies, and It was generally thought that no immunity followed have been found infected with T. b. gambiense in a first diagnosis of HAT, because of the parasite’s Liberia (a country with little human disease), Côte antigenic variation and its repertoire of VSG which d’Ivoire, Congo and the DRC (Gibson et al, 1978; includes hundreds of different antigens. However, Schutt and Mehlitz, 1981; Mehlitz et al, 1982; observational and experimental studies in animal Noireau et al, 1989; Truc et al, 1991). In Côte d’Ivoire, models have shown these animals to be more resist- 52 sympatric T. brucei strains were characterised by ant to homologous trypanosomes when re-chal- isoenzyme electrophoresis: among 12 zymodemes lenged after adequate treatment of a previous infec- revealed, the most frequent was found both in tion. This protection resulted from exposure to humans and pigs (Penchenier et al, 1997). In Congo, metacyclic trypanosomes rather than to blood- sheep were also found to be infected with T. b. gam- stream forms (Nantulya et al, 1984; Akol and biense, and the prevalence of Trypanozoon infection in Murray, 1985; Vos et al, 1988). The existence of pro- domestic animals was estimated to be 0.5% tective immunity in humans was investigated in a (Noireau et al, 1989; Truc et al, 1991). A dog was very-high incidence community of the DRC where found infected with T. b. gambiense in Liberia 38% of adults had a past history of trypanosomiasis (Zillmann et al, 1984). A more recent study (Khonde et al, 1995). The results suggest that a first using PCR showed the simultaneous presence episode of trypanosomiasis confers to adults about of T. b. gambiense in humans and animals (a dog and 85% protection against subsequent reinfection. a pig) from the Lower Congo province of the DRC (Schares and Mehlitz, 1996). However, the epidemi- The impact of HIV ological significance of the animal reservoir is Little is known about the interactions between the unknown. Whether or not animals may become a human immunodeficiency virus (HIV) and Gam- threat for reintroduction or persistence of the para- bian trypanosomiasis. Three studies performed in site in foci where near elimination of HAT has been Central Africa (Louis et al, 1991; Pépin et al, 1992) achieved remains an important research question and Côte d’Ivoire (Méda et al, 1995) suggest that (Molyneux, 1980a). HIV infection has so far had little impact on the epidemiology of Gambian trypanosomiasis, to some EPIDEMIOLOGY OF T. B. RHODESIENSE extent because the prevalence of HIV remains rela- TRYPANOSOMIASIS tively low in rural communities where HAT is HAT caused by T. b. gambiense and T. b. rhodesiense endemic. No data are available for T. b. rhodesiense differs in its epidemiological features. We will only areas. Given that HIV infection is getting more stress these diffences. T. b. rhodesiense is a zoonosis prevalent in rural areas, it is worthwhile undertak- sporadically transmitted to humans when they ven- ing a well designed nested case control study to fur- ture into bush infested by tsetse fly vectors whose ther investigate these interactions. It has been blood-meals are normally taken on game animals. observed that HIV co-infected HAT patients Humans enter this sylvatic cycle as an incidental respond less well to eflornithine treatment than host. Except during epidemics, human-fly-human seronegatives (Milord et al, 1992), but this is unlike- or domestic animal-fly-human transmission is ly to have any impact on transmission. thought to be rare. The epidemics usually involve G. f. fuscipes, a peridomestic fly, and transmission The Animal Reservoir occurs around the villages. The disease, character- The existence of an animal reservoir of T. b. gambi- ized by an acute or subacute malaria-like syndrome ense has been investigated for a long time with high parasitaemias, progresses over weeks or (Makumyaviri et al, 1989; Leak, 1999). Earlier stud- months rather than months or years. The vectors ies showed that many species of domestic animal differ from those of T. b. gambiense. The parasite is could be experimentally infected with T. b. gambi- transmitted mostly by three species of the morsitans ense: pigs, dogs, goats, sheep and even chickens group: Glossina morsitans morsitans and G. m. cen-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 61 tralis in East Africa, G. pallidipes in East and The limiting factor is the relatively low sensitivity of Southern Africa and G. swynnertoni in Kenya and the standard parasitological techniques. A more fre- Tanzania. G. fucipes fucipes, which belongs to the pal- quent use of finer serological and parasitological palis group, is a vector of human and animal try- techniques could lead to a rapid and sustainable panosomiasis in Central and East Africa. reduction of the human reservoir. In order to reduce man’s exposure to infectious bites, the tsetse flies The existence of animal reservoirs was established must be destroyed. This must be carried out with in the 1950s. Cattle are the major reservoir (Hide the active involvement of the community, who can et al, 1996), and played a major role in an epidemic take on trap laying, spraying, bush clearing, etc. in south-east Uganda, with 23% of cattle carrying There are a variety of vector control techniques, the human infective strains. Other domestic (dog, choice of which depends on financial and human sheep, maybe pig) and various game (warthog, resources, the epidemiological situation, and the bushbuck, hartebeest, impala, lion, zebra, hyena) programme duration. Within the African context, animals can harbour the parasite in their blood; vector control must be carried out with cheap, cost- some of these animals are well adapted to the para- effective and easy-to-use methods. Control integrat- site and can remain infected for more than two years ed into primary health care and targeted on groups without overt disease. Conversely, some other at risk has been found feasible, cost-effective and species (e.g. dog) rapidly succumb from the infec- cheaper, provided village health workers (VHWs) tion. In contrast to T. b. gambiense trypanosomiasis are motivated and the beneficiary population partic- which results from peridomestic infections in ipates fully. Community involvement, both in case Central Africa, rhodesiense trypanosomiasis is, in finding and vector control, through the implication endemic situations, acquired far from the village, of VHWs, has been successfully tested on a large where the animal reservoir lives. It is mainly an scale in various epidemiological contexts. occupational disease of adult men: hunters, fishermen, firewood collectors and honey gatherers have Case Detection been found to be at higher risk. Tourists visiting Case detection has been the cornerstone of HAT con- game parks are also exposed to the infection. trol. For many years, specialized case-finding mobile However, in epidemic situations, when transmis- teams have relied essentially on the presence of sion becomes peridomestic, all groups, including swollen cervical lymphs nodes. Lymph node fluid, males and females are at similar risk. Indeed, it has when present, is examined for evidence of try- been observed that an increase in the number of panosomes. In some very active foci, the mobile cases in children and women is an indication that an teams carry out wet film and giemsa-stained thick outbreak is developing (Apted, 1970). As it is in gam- smear examinations. Because of the low sensitivity of biense sleeping sickness, familial aggregation has these methods, better serological and parasitological been noted (Okia et al, 1994). In contrast to Gambian tools have been developed over the last two decades. trypanosomiasis, there is a marked seasonality of disease occurrence, with a higher incidence during Serological methods the warmer season (Smith et al, 1998). Population Various serological assays were developed to help movements and political upheavals played an case-finding teams identify a small number of anti- important role in development of recent epidemic body carriers on whom to concentrate efforts for try- in south-east Uganda (Mbulamberi, 1989a; Smith panosome detection using parasitological methods. et al, 1998). Changes in agricultural practices also In the 1970s, the indirect fluorescent antibody test led to more favourable conditions for the develop- (IFAT) was deemed the most reliable technique for ment of G. fucipes, and resulted in the recent perido- epidemiological surveillance of T. b. gambiense try- mestic epidemic, which was brought into control by panosomiasis. However, it required relatively expen- the surveillance and early diagnosis through sleep- sive equipment and qualified staff, and its imple- ing sickness orderlies and vector control (Smith mentation was possible only in laboratories. Delays et al, 1998; Okoth, 1999) in obtaining results were such that some seropositive suspects could not be located again to undergo the CONTROL OF HUMAN AFRICAN parasitological confirmation test. From the 1980s TRYPANOSOMIASIS onwards, the IFAT was supplanted by the card Sleeping sickness control relies on two principles: agglutination test for gambiense trypanosomiasis reduction of the parasite reservoir through case (CATT) (Magnus et al, 1978), the advent of which detection and treatment, and reduction of man- considerably enhanced detection of cases of Gam- fly contact through vector control. In the case of bian trypanosomiasis. The CATT is a latex aggluti- T. b. gambiense, reduction of the human reservoir can nation test relying on the detection of antibodies be achieved through case-finding and treatment. using the variable antigen type (VAT) LiTat 1.3 anti-

62 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 gen, which is expressed by several T. b. gambiense some in biological fluids. This confirmation can be stocks. This is the only serological assay currently obtained through the examination of wet blood or used by control programmes. It can be performed in giemsa-stained blood smears, or by direct examina- the field, without electricity and without specialized tion of the lymph node aspirate when a typical lym- staff, and the results are available within 10 minutes. phadenopathy is palpable (Cattand and de Raadt, It is cheap, at approximately 0.40 USD per test, and 1991; Miézan et al, 1994). These classical methods is generally performed on whole blood. Its sensitivi- have a rather low sensitivity in gambiense try- ty varies between 92% and 100% when assessed in panosomiasis. Demonstrating the presence of try- patients parasitologically confirmed in hospital or panosomes in blood and cerebrospinal fluid (CSF) other settings, and its specificity is thought to be 94- has been considerably facilitated by the develop- 97% (Noireau et al, 1988; Miézan et al, 1991). The ment of concentration methods. The micro-haemat- CATT proved highly specific during testing in non- ocrit centrifugation technique (Woo’s test) is much endemic areas (Bafort et al, 1986). Some strains of more effective in the rhodesiense disease (and in vet- T. b. gambiense do not express the LiTat 1.3 antigen, erinary medicine) than in T. b. gambiense HAT. The but their distribution seems fairly limited (Dukes miniature anion-exchange centrifugation technique et al, 1992). The positive predictive value of the (mAECT) is deemed at present to be the most sensi- CATT depends on the prevalence of disease in the tive parasitological method for the detection of population. This prevalence ranges between 1 and blood parasites (Miézan et al, 1994). Because of the 5% in most endemic foci and the positive predictive cost (about US$2) and its relative complexity, it is value generally varies between 14% and 40% used selectively to test CATT-positive suspects (Miézan et al, 1991). The CATT can be performed on among whom the diagnosis could not be confirmed filter paper, using smaller volumes of reagents, thus by classical methods. Two new parasitological tech- reducing costs (Miézan et al, 1991). However, the niques have recently been added to the diagnostic reliability of this micro-CATT is less satisfactory arsenal: the quantitative buffy coat technique (QBC) under field conditions than in a research setting. The (Bailey and Smith, 1992) and the kit for in vitro iso- CATT can also be used on diluted serum rather than lation of trypanosomes (KIVI) (Aerts et al, 1992). whole blood, resulting in higher specificity at the Neither the QBC, which requires relatively expen- expense of lower sensitivity (WHO, 1998). sive equipment, nor the KIVI, which is a parasite isolation method rather than a screening test, has The card indirect agglutination trypanosomiasis test proved to be superior to the mAECT (Truc et al, (CIATT) is an indirect assay that was newly pro- 1994). posed for the detection of circulating trypanosome antigens in patients’ blood (Nantulya, 1997). The In the CSF, the most sensitive technique is that of parasite antigen detected is an internal, invariant double centrifugation (Cattand et al, 1988; Miézan molecule that is common to both T. b. gambiense and et al, 1994). A variation of the latter, the single cen- T. b. rhodesiense. This test can be used to detect cur- trifugation of cerebrospinal fluid in a sealed Pasteur rent infection in both forms of the disease. It has pipette, has been recently developed (Miézan et al, been found to be highly sensitive in endemic areas 2000). It makes detection of trypanosomes in the (Asonganyi et al, 1998). The test was shown to be CSF simpler, quicker and more sensitive, and is spe- easy to use in field conditions and does not need cially suitable for passive diagnosis in suspects who chain maintenance for the storage of reagents. A consult in health facilities with symptoms sugges- recent evaluation in non-endemic areas revealed a tive of sleeping sickness. The combination of all specificity ranging from 61% to 98% depending on these seroparasitological methods ensures increased the proximity of the endemic zone (Meda et al, sub- sensitivity, but falls short of perfectly reliable para- mitted for publication). Specificity is improved by sitological diagnosis in all seropositive persons, titration of seropositive specimens. Apart from their hence the need to pursue efforts to develop more diagnostic potential, the CATT and CIATT have also sensitive parasitological assays and more specific been found to have potential for use in patient fol- serodiagnostic techniques. low-up to determine chemotherapeutic cure. Further operational studies aimed at testing the use- Treatment and Drug Resistance fulness of the CIATT in clinical settings and for field Pentamidine remains the standard treatment for use by national control programmes should be car- early-stage patients and melarsoprol for late-stage ried out. cases. The treatment for HAT is selected by first establishing the stage of infection. The diagnosis of Parasitological methods late-stage trypanosomiasis is based on at least one of Confirmation of diagnosis among seropositive indi- the following criteria (WHO, 1998): CSF white cell viduals depends on demonstrating the trypano- count (WCC) > 5/mm3 or CSF proteins >37 mg/100

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 63 ml (as measured by dye-binding protein assay), or bioecological and epidemiological studies. Before on both criteria, with or without the presence of try- the advent of insecticides, vector control depended panosomes in CSF. Miézan et al (1998) reported that primarily on elimination of the wooded vegetation the CSF WCC is, by itself, as sensitive for diagnosis which constitutes the habitat of Glossina. Nowadays, of central nervous system involvement as is the insecticides are applied to various types of traps and combination of the above criteria. Therefore, the screens to destroy the vector. A number of improved WCC was recommended in patients with confirmed biconical, monoconical and pyramidal traps, infection, especially in poorly equipped facilities. inspired by the Challier-Laveissière biconical trap Advances have been made in the treatment of HAT. (Challier and Laveissière, 1973), have been tested for Between 5% and 10% of late-stage patients treated the control of G. p. palpalis, G. morsitans and with melarsoprol, an arsenical derivative, succumb G. f. quanzensis in West and Central Africa. Lancien to its undesirable effects. Until recently, melarsoprol designed the monoconical trap (Lancien, 1981), was the only available treatment for late-stage which was followed by the pyramidal trap patients. A new drug, eflornithine, has been devel- (Gouteux and Lancien, 1986), and later by the oped (Pépin and Milord, 1994). Results obtained Vavoua trap (Laveissière and Grébaut, 1990). To with eflornithine are excellent, but its future avail- enhance trap efficacy, especially against G. morsi- ability remains doubtful. tans, olfactory attractive baits are attached to them (carbonic gas, acetone, urine phenols and host skin Drug resistance has been a relatively uncommon secretions) (Leak, 1999). These act at a greater dis- phenomenon in Gambian trypanosomiasis, despite tance than purely visual baits. suramin, pentamidine and melarsoprol having been used for five decades (Pépin and Milord, 1994), and, Pilot studies conducted in various epidemiological as a consequence, has not had any impact on the settings have shown that trapping is effective. epidemiology of the disease. Suramin is little used Efficacy is measured in terms of the apparent densi- in the treatment of Gambian trypanosomiasis. ty of flies captured per trap per day (ADT), and Suramin and Pentamidine are given throughout varies according to the type of trap, the species or Africa to patients with early-stage of T. b. rhodesiense sub-species of Glossina, the environmental and cli- and T. b. gambiense disease respectively. So far, the matic conditions, etc. (Laveissière, 1988). In the for- treatment failure rate remains fairly low. The situa- est areas of Côte d’Ivoire, it was demonstrated that tion is quite different for melarsoprol. There are at the black/blue/black screen is about twice as effi- least two foci where melarsoprol resistance is more cient as the simple blue one (Laveissière et al, 1987). frequent than elsewhere; clearly this is an issue that In the West African savannah, the ADT of G. tachi- will need better investigation and monitoring over noides and G. p. gambiensis populations was reduced the next few years. One is the Mbanza Kongo focus by 88-92% using the blue screens (Mérot et al, 1984). of northern Angola, close to the border with Lower- The same screens reduced the ADT of G. tachinoides Congo: a 40% failure rate was reported 25 years ago by 98% in only 15 days (Laveissière and Couret, (Ruppol and Burke, 1977) and similar failure rates 1981). In contrast, in the forest areas of Congo, the have been observed in recent years. Limited epi- screens did not yield satisfactory results. The ADT demiological data suggest that there has been little was more drastically reduced (99%) after five spread of resistant strains over time. In the Arua dis- months in the forest areas of Côte d’Ivoire using trict of northern Uganda, a 27% failure rate has biconical traps (Laveissière and Hervouët, 1981). recently been reported among new cases treated Later on, in the same areas, about 16 000 blue with melarsoprol (Legros et al, 1999); a 10-fold screens sited in coffee and cocoa plantations lower failure rate has been documented in the adja- reduced the ADT of G. p. palpalis by 90% in one cent focus of Adjumani. In the DRC, where melarso- week, and by 98% at the end of five months prol failures are not specially frequent, statistically (Laveissière et al, 1986c). significant differences in failure rates were found. It has been also observed that HIV co-infected HAT Traps and screens have thus replaced insecticide patients respond less well to eflornithine treatment spraying. These vector control methods have gener- than seronegatives (Milord et al, 1992), but this is ated much interest: they are effective, simple, envi- unlikely to have any impact on transmission as ronmentally friendly and suitable for use by the eflornithine is little used and relapsing cases of HAT communities themselves. Pilot projects in Burkina are often not parasitaemic. Faso (Mérot et al, 1984), Côte d’Ivoire (Laveissière et al, 1994b), Uganda (Lancien, 1991) and Congo Vector Control (Gouteux and Sinda, 1990) have demonstrated the The tsetse fly is one of the rare insects for which sev- feasibility and efficiency of traps and screens used eral control methods have been developed, based on with the participation of rural communities under

64 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 the supervision of specialized teams. The number of equipment maintenance, especially during the traps and/or screens required for a particular loca- farming season, and to lower efficacy due to the tion depends on the type of vegetation in the tsetse insecticide being washed away by rains and the habitats, and on the presence or number of edges, screens being concealed by weeds which grow water sources, paths and encampments which quickly during the rainy season. Two years later, the determine the frequency and intensity of man-fly impact of the control programme on the incidence of contacts. The communities are in a position to pro- human disease was nevertheless obvious: no new vide information on locations where the villagers patient had been detected during the case-finding are most often bitten. The life span of a trap depends survey carried out in the study area where the over- on the quality of materials and of maintenance, and all prevalence was initially higher than 1%. Globally, on the environmental conditions. In experiments this experiment had shown that about a year’s vec- conducted in West Africa, it was estimated that tor control was needed to substantially reduce trans- about 10% and 20% of the traps or screens needed to mission. Similar results were recorded in Congo be renewed each year in the forest and savannah (Gouteux and Sinda, 1990) and Uganda (Lancien, zones respectively (Laveissière, 1988). It is not pos- 1991). Clearly, efficient case-finding must be con- sible to give the precise number of traps or screens ducted simultaneously with vector control, other- to be installed per hectare, since each microcosm wise the persistence of the human reservoir will will have its own features. The number of traps or lead to a rapid resurgence of disease when vector screens to be installed does not depend on the den- control is pursued less vigorously. The cost of a sity of the human population to be covered. In the screen and a trap (Vavoua type) was estimated to be Vavoua pilot study, traps and screens were installed US$3 and US$6 respectively (Laveissière and Méda, as follows: one screen every 100 metres, one or two 1992). The cost of one hectare protected was esti- screens per water-point or encampment, one screen mated at US$1 for the first year, and much less for at each working place in the plantation, one trap the second year. Costs vary depending on the type every 300 metres in forest areas and one every 100 of trap or screen used and on whether or not the metres around villages (Laveissière et al, 1994). equipment is insecticide-impregnated.

Traps are preferable to screens if re-impregnation Genetic control by the release of sterile males is and surveillance cannot be carried out by the popu- unsuitable for control in epidemic situations; it lation. Traps need to be re-impregnated once a year, poses a series of technical, financial and logistic at the end of the rainy season. Screens have to be re- problems which do not facilitate its implementation impregnated once during the first year of the cam- in endemic countries. paign, and twice a year later on. Traps should be installed as far beyond the endemic area as possible, GAPS IN KNOWLEDGE AND so as to provide an effective barrier. In the gallery RESEARCH NEEDS forest of savannah areas, open and sunny places fre- Over the last two decades, new and much more quently visited by people, such as washing and effective tools for HAT control have been developed bathing places, water collection points, bridges and compared to those that existed some forty years ago banks of rivers, are the preferable sites. In forest when the disease was almost eliminated. This areas, the tsetse flies must be intercepted at the progress contrasts with the present epidemiological interfaces between different ecological patterns, i.e. situation. The disease remains a serious emerging the edges that are considered as epidemiologically public health problem in Central, East and West dangerous areas (Laveissière et al, 1986b), such as Africa. WHO, industrial firms, bilateral and multi- areas around villages, paths separating wooded lateral organizations have decided to mobilize ade- areas and other types of ecological patterns, espe- quate resources to combat it. Many gaps in knowl- cially cocoa or coffee plantations, etc. edge relevant to control of the disease need to be urgently filled. Some of these gaps have been iden- Vector control with community involvement, as part tified as priority areas by the Working Group on of a comprehensive sleeping sickness control pro- Operational Research on African Trypanosomiasis gramme, was organized in the forest areas of (Geneva, 1997) and the International Colloquium on Vavoua (Côte d’Ivoire) where Laveissière et al Operational research priorities on African try- (1985) recorded a 90% reduction of ADT in the vil- panosomiasis (Antwerp 1998). Scientists and pro- lages and farms a week after setting up the traps gramme managers are invited to pay much more and screens, and over 99% after three months. These attention to the multiple questions related to the results remained stable for the first six months. development of new tools for control or better use of Subsequently, ADTs have increased. This phenome- existing ones. Most perspectives in HAT research non was linked to the gradual abandonment of and control proposed by Kuzoe (1991) still need to

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 65 be considered. In the short and intermediate terms, was evaluated. This test has been found to be high- some of the research priorities include the following ly sensitive and sufficiently specific for both types of fields. HAT. Its suitability and predictive value in clinical management need to be assessed in different epi- Improvement of Epidemiological demiological settings. The test is easy to use in field Knowledge and Disease Surveillance and conditions and does not need cold chain mainte- Control nance for storage; therefore, it is worthwhile evalu- Little is known of the basic epidemiology of HAT ating its suitability for screening in primary health disease in many endemic countries, especially care programmes. The last meeting of the Task Force Tanzania, Southern Sudan, Angola, Chad, Central on Operational Research on African Trypano- African Republic and Guinea. It has been speculat- somiasis elaborated guidelines for designing the fol- ed that T. rhodesiense sleeping sickness occurs among lowing studies on the CATT and CIATT: tourists in East Africa; if this is true, it does mean • Assessment of chemotherapeutic cure of sleeping that maybe the disease is rising to an epidemic level sickness using the CIATT in T. b. gambiense and among local populations. Improvement in knowl- T. b. rhodesiense HAT. edge of the host-parasite and vector-parasite rela- • Comparison of the CIATT and CATT in the diag- tionships is of great interest for the development of nosis of Trypanosoma brucei gambiense sleeping new tools and products for control, including new sickness in a clinical setting. drugs for treatment. The zoonotic nature of T. rhode- • Applicability of the CIATT in the diagnosis of siense was confirmed about forty years ago. The sit- Trypanosoma brucei rhodesiense sleeping sickness in uation is completely different for T. gambiense; it has a clinical setting. been shown that domestic animals such as pigs, • Operationality of the CIATT versus CATT in a dogs, sheep and cattle can, or do, carry parasites field survey of T. b. gambiense sleeping sickness. whose isoenzyme features match those of parasites infecting humans. An important question arises as The question of the treatment of CATT-positive but to the epidemiological importance of these animal aparasitaemic individuals is under study in the DRC reservoirs in T. b. gambiense trypanosomiasis. At and NE Uganda. The significance of the results of present, the exact role of these reservoirs is these studies need to be assessed for impact on the unknown; it is difficult to establish the relative epidemiology of T. b. gambiense infections in humans. importance of transmission between animals and humans. Some studies suggest that such transmis- The usefulness of remote sensing as a tool for sup- sion could play a significant role by maintaining a porting vector control and surveillance needs to be minimum level of transmission when the level of explored further (Kuzoe, 1991). Is it a reliable tool endemicity is low. If this transmission is epidemio- for the identification of high risk areas for human logically significant, what are the factors that influ- trypanosomiasis? Is it an adequate tool for surveil- ence the infective contacts between animals and lance of the disease? Some questions, specifically humans? Is there a sylvatic cycle which contributes related to surveillance and treatment and requiring to the persistence of Gambian trypanosomiasis? investigation, are given in the handbook by Leak How can this cycle be interrupted? Several methods (1999), including: Is there a level of vector density have been proposed for blood-meal analysis. What that ensures a low endemic level of trypanosomia- is the most reliable, easy to use in field conditions, sis? Is trypanosome prevalence, as determined by and inexpensive, technique for the determination of passive surveillance, a reliable indication that more blood-meal origin of the vectors? resources are needed for active surveillance? What should be the interval between visits of mobile Surveillance and Management of teams in the context of active surveillance? Sleeping Sickness The card agglutination test for trypanosomiasis PCR has recently been introduced as a new tool for (CATT) is a simple, easy to use, sensitive and rela- the identification of trypanosomes; however its use- tively effective tool for the diagnosis of T. b. gambi- fulness in the diagnosis and management of the dis- ense trypanosomiasis, which has been available for ease in humans, and its application in studies on two decades. However, there is no equivalent test animal reservoirs, remain unsatisfactory. The tech- for T. b. rhodesiense sleeping sickness; it has been nology needs to be refined, adapted for field use, found that T. b. gambiense in Central Africa does not and transferred to field workers who need to be express the antigens used in the CATT. Therefore trained in its application. there is need to improve the reagents presently used. More recently, another simple test, the card Geographical information system (GIS) and map- indirect agglutination trypanosomiasis test (CIATT), ping technology offers a cost-effective and rational

66 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 tool for monitoring and control of disease, and is mining the epidemiology of resistance to the drug, now available for spatial analysis of data collected in are lacking. Nifurtimox is a synthetic nitrofuran endemic foci. This tool has been successfully applied which was primarily used for treatment of Chagas in the control of animal trypanosomiasis in savannah disease due to T. cruzi. In HAT, it has only been areas. In the context of limited resources, GIS is more used in the former Zaire and in Sudan, mostly in and more perceived as an efficient tool for the iden- patients with infection refractory to melarsoprol, in tification and mapping of high risk areas where con- combination with other trypanocidal drugs. Fur- trol efforts should be directed. However, its applica- ther studies using an improved protocol should be tion in HAT has been very limited so far. Therefore, carried out in T. b. gambiense areas where malarso- there is a need to provide opportunities for multidis- prol resistance is expanding (the DRC, Angola, ciplinary research teams, including geographers, to Sudan) in order to identify effective combinations further use GIS in pilot control programmes. of drugs. Controlled clinical trials on prednisone versus placebo, to assess reduction of incidence of Another gap mentioned by some young researchers melarsoprol-associated encephalopathy, might also is that there is no functional network for scientists be interesting. No data are available on the efficacy working in the field of trypanosomiasis at regional of new drugs (nifurtimox, steroids) in T. b. rhode- or sub-regional levels. Such a network would link siense sleeping sickness. senior and young scientists together, allow efficient use of human resources and strengthening of the Research on Tsetse Flies and Vector Control scientific capabilities of young researchers. The biotopes most suitable for tsetse flies are known for almost every biogeographical area, except man- Research on Existing and New Drugs, grove swamps. There is a need to collect basic epi- and Drug Resistance demiological information on types of sites in order to The ideal drug is one that is safe, effective, afford- determine which areas present the greatest risk to able, and can be taken orally. Scientists and indus- humans in West and Central Africa. A better under- trialists are invited to collaborate in the develop- standing of the variability of vectorial capacity of the ment of new effective compounds for the treatment tsetse fly, including age, population structure, and of HAT. Pharmacokinetic studies have shown that feeding patterns, might lead to improvement of con- it is worthwhile evaluating pentamidine adminis- trol; populations that are genetically different might tered for tree days versus seven days in the first have different vectorial capacities in different epi- stage of the disease. Studies in laboratory animals, demiological contexts. What are the sociological fac- using several combinations of existing drugs such tors that affect man-fly contact and are responsible as pentamidine and eflornithine, suramin sodium for the outbreak of epidemics? A recent meeting held and melarsoprol, or melarsoprol and nifurtimox, in Harare recommended setting up a quality control have shown them to be synergistic. However, few system for new insecticides to be used for tsetse con- data on the effects of drug combinations on either trol. What is the role of population mobility in the type of African trypanosomiasis are available. In emergence and maintenance of epidemics of sleep- view of the increasing development of resistance to ing sickness in various epidemiological settings? melarsoprol monotherapy in some areas, studies on the available drugs, given separately or in combi- Data collected from catching tsetse flies with traps nation (eflornithine/melarsoprol, nifurtimox/me- are classically analysed using latin squares. Howe- larsoprol, etc.), should be undertaken in humans, ver, this method is questionable; there is a need to based on the latest pharmacokinetic information. identify more adequate statistical techniques for the Relapses following treatment with melarsoprol, analysis of these particular types of data. with or without pentamidine, occur on a large scale in the DRC and Angola. Studies on factors associat- Social and Economic Impact of ed with treatment failure with melarsoprol should Sleeping Sickness be designed to identify preventive measures. Trials Little information is available in the literature on the on alternative regimens of melarsoprol (e.g. 14 days social and economic impact of HAT. Therefore, there versus 10 days) might improve its efficacy and tol- is a need for social and economic studies to evaluate erance. Many hypothetical reasons have been given the sustainability and long-term cost-effectiveness of for treatment failure, such as possible reinfection, integrated control programmes (Kuzoe, 1991). Some misclassification of the disease, lack of compliance evidence suggests that sleeping sickness can reduce with treatment, and individual variation in the physical attributes and socio-economic potential at pharmacokinetics of the drug. Studies on the mag- individual, family and community levels; but physi- nitude of the phenomenon, and on the risk factors cians, demographers, social scientists and econo- and biological and environmental conditions deter- mists have so far been unable to quantify and quali-

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72 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 II VECTOR CONTROL IN ense epidemics were brought under control in the RELATION TO HUMAN AFRICAN francophone region of West Africa. This francopho- TRYPANOSOMIASIS ne approach was also adopted during the 1930s to control epidemics in Nigeria and Ghana with the I. Maudlin, E. M. Fèvre, P. G. Coleman, newer arsenical tryparsamide. The different colo- Centre for Tropical Veterinary Medicine, The University nial administrations appear to have became locked of Edinburgh, Easter Bush Veterinary College, into ‘their’ strategies of either treating the cases or Midlothian, EH25 9RG, UK dealing with the vector (de Raadt and Jannin, 1999). It is, nonetheless, clear that both approaches were CONTROLLING VECTOR-BORNE effective. DISEASES Controlling vector-borne diseases represents a spe- In the case of sleeping sickness, as with other vector- cial challenge to societies in developing countries. borne diseases, the first line of defence in the face of Apart from the sheer size of the public health prob- an epidemic would appear to be tsetse control – lems posed by diseases such as malaria and sleeping why then, was this approach not universally adopt- sickness, vector-borne diseases are especially diffi- ed across colonial Africa? And why were the alter- cult to control. One of the main reasons for this is native strategies adopted in francophone Africa suc- that vector-borne diseases are quantitatively differ- cessful? It is instructive to examine the reasoning ent from directly transmitted diseases in that the behind the decisions governing interventions for sleeping sickness control, not only to explain the basic reproductive number (R0 - the number of new infections which arise from a single current infec- events of history but also to inform policy-makers tion, introduced into a population of susceptibles, faced with the resurgence of sleeping sickness during the period of its infectiousness) for vector- (World Health Organization 2001). borne diseases is often an order of magnitude greater than for directly transmitted diseases. This TWO DISEASES – TWO APPROACHES means that, generally, vector-borne diseases are TO CONTROL more difficult to control and may bounce back from In retrospect, we tend to view this division between control faster than directly transmitted diseases. francophone and anglophone, in approaching what However, it follows from our theoretical under- was apparently the same problem, as merely anoth- standing that targeting control activities at the vec- er example of the failure of communications tor has potentially the greatest impact on disease between two competitive colonial powers. This may, transmission (see below). however, be simplistic. Assuming that there was some logic to this dichotomy of approach, it is prof- CONTROLLING SLEEPING SICKNESS itable to first consider how this position arose on the It is over a century since it was first recognized that two sides of the continent, as this may influence tsetse flies were responsible for transmitting try- how we might best proceed to control sleeping sick- panosomes from wild animals to domestic livestock ness in the future. (Bruce, 1895). When it subsequently became clear that tsetse were also responsible for the transmis- The reasons for the differing approaches to disease sion of human sleeping sickness (Bruce and control have their origins in the biology of two quite Nabarro, 1903), control of these insects assumed distinct diseases. These differences may be expres- massive importance for the colonial powers because sed within a theoretical framework of vector-borne of the threat the disease posed to the economic disease transmission, as discussed below. development of Africa south of the Sahara. Subsequently the largely anglophone countries of AN EPIDEMIOLOGICAL FRAMEWORK East, Central and Southern Africa aimed a large part TO EVALUATE SLEEPING SICKNESS of their research and control efforts at elimination of CONTROL STRATEGIES the vector combined with passive case detection. By The basic reproductive number, R0, is central to contrast in West Africa, and particularly among the understanding how the parasite spreads through, francophone countries, the more direct approach of and is maintained, in a population. A threshold con- controlling the disease in humans became estab- dition of R0>1 must be achieved if a parasitic lished practice. This followed from the success of species “is capable of invading, and establishing Jamot, who pioneered large-scale case detection and itself within, a host population” (Anderson and treatment with the arsenical drug Atoxyl (de Raadt May 1992). Ideally, disease control will force R0<1, and Jannin 1999). By combining large-scale arsenical and so eventually lead to the eradication of the par- chemotherapy with institutional interventions such asite from the population. Determination of this as regulating the movement of people, T. b. gambi- threshold condition for transmission is, therefore,

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 73 important for the design and implementation of Two well recognized differences between sleeping control strategies. (If the threshold condition cannot sickness caused by T. b. gambiense and T. b. rhodesiense be met, then control activities should aim to main- are the duration of infection and the role of the animal tain R0 at as low a value as possible.) reservoir. T. b. rhodesiense infections are characteristi- cally acute, with death often occurring within a few The study of vector-borne disease epidemiology months of infection (Odiit et al, 1997; Apted, 1970; and the design of control programmes has benefited Wellde et al, 1989). The role of a non-human animal from the development of a basic mathematical theo- reservoir in T. b. rhodesiense transmission has long ry by Macdonald (based on Ronald Ross’ earlier been recognized (Heisch et al, 1958; Onyango et al, work), which captures the essentials of vector trans- 1966). Indeed, movement of cattle from the sleeping mission (Macdonald, 1957). From this theory, the sickness endemic areas of south-east Uganda has been reproductive number of a vector borne infection, R0, implicated in the origins of a T. rhodesiense outbreak in is defined as: an area previously free from the disease (Fèvre et al, 2001). By contrast, T. b. gambiense infections tend to be chronic, with the time between onset of symptoms, development of late-stage disease and subsequent where m is the ratio of vectors to humans; a is the death often occurring over several years (Apted, feeding rate of vectors, such that the average time 1970). Also, humans are generally considered to be the between feeds is 1/a; c is the probability that a sus- main host for T. b. gambiense infections with the animal ceptible vector acquires infection after feeding on an reservoir less important than in T. b. rhodesiense. infectious human; b is the probability that a suscep- However, the role of the animal reservoir in gambiense tible human acquires infection after being bitten by infections is still unresolved. Rogers (1988) argued an infected vector; r is the rate at which humans that domestic animals may be essential in the mainte- recover from infection, with the average duration of nance of T. b. gambiense as R0 in humans alone may infection being 1/r; T is the time taken for the para- fall below unity, leading to suggestions that the exis- site to mature in the vector; and u is the vector mor- tence of a non-human animal reservoir in T. b. gambi- tality, such that 1/u is the average vector life ense infections may have contributed to the failure of expectancy. A fuller explanation of how this expres- human population surveillance and treatment cam- sion is derived, both mathematically and intuitively, paigns to eradicate sleeping sickness in certain set- is given by Anderson and May (1992). tings (Morris, 1946; Rogers, 1988). However, it has also been argued that the existence of an animal pop- The epidemiological impact of a particular control ulation on which tsetse flies preferentially feed may intervention may be investigated by assigning its result in humans receiving infectious bites at a lower effects to one or more of the parameters in the rate than if flies only feed on humans (Goutex, expression for R0. For example, vector control will Laveissièrre and Breham, 1982). kill vectors, and so increase u and decrease m; while active case detection and treatment would increase r. These differences in the natural history of the dis- Thus the expression for R0 provides insights into eases may be incorporated into the expression for vector-borne disease epidemiology and a frame- R0, and their impact on the relative effectiveness work in which to evaluate the relative impact of dif- (expressed in terms of reducing R0) of strategies to ferent interventions. An important qualitative con- control the two types of sleeping sickness demon- clusion from the simple expression for R0 is that strated (Welburn et al, 2001). It was shown that killing adult vectors is more effective than the early detection and treatment of cases profoundly reduces treatment of cases. The time a case is infected enters the transmission of T. b. gambiense but has relatively into the expression for R0 linearly via the parameter little impact on the transmission of T. b. rhodesiense. r. In contrast, adult vector mortality enters in a non- This is because the duration of T. b. rhodesiense is rel- linear fashion via the term exp(-uT). atively short, and the animal reservoir is a relatively important component of R0. This general model has subsequently been developed to specifically model the African trypanosomi- In the light of these theoretical and biological con- ases, in both animals and humans, by Rogers (1988). siderations, the different approaches adopted by the The model provides a theoretical basis to investigate colonial authorities of the time are seen to be quite how differences in the natural history of T. b. gambi- logical. ense and T. b. rhodesiense will effect the epidemiology of sleeping sickness caused by the two pathogens, CONTROL IN THE 21ST CENTURY and how these differences will impact on the relative While, theoretically, vector control will proportion- effectiveness of the same control interventions. ally have the greatest impact on disease transmis-

74 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 sion, it may not always be the approach of choice in ered, as in reality these resources are always limited. the field when other variables are taken into Indeed, in countries affected by sleeping sickness, account: the many resources necessary for implementation of • Treatment of infected people has to be carried out certain control strategies are often non-existent or for humanitarian reasons, and funding may usu- severely constrained. Control policies should be ally being found for such emergency interven- based on the optimal utilization of the available tions. resources. To help determine this optimal utiliza- • In the war zones of Africa, sustaining a tsetse con- tion, cost-effectiveness analysis is an important tool trol operation is very difficult and logistically (Murray et al, 2000; Shaw et al, 2001). Such analysis more demanding than case finding. Tsetse control must be based on a sound understanding of the nat- operations in these circumstances may be merely ural history of the disease, to best predict the likely token, having little impact when carried out by epidemiological effects of the same resources invest- poorly equipped and under-trained personnel. ed into alternative interventions. Given limited resources and societal instability (e.g. as in the current T. b. gambiense outbreaks in References Southern Sudan, Congo and Angola), it may not Anderson RM, May RM. Infectious diseases of humans: be appropriate to divert resources from case find- dynamics and control. Oxford University Press, 1992. ing towards tsetse control. • Targeted treatment of the animal reservoir, in the Apted FIC. Clinical manifestations and diagnosis of case of T. b. rhodesiense, is a potentially effective, sleeping sickness, In: Mulligan HW, Potts WH, inexpensive and sustainable control option Kershaw WE, eds. The African trypanosomiases. (Welburn et al, 2001). George Allen and Unwin, 1970:661-683.

Tsetse control operations, whether top-down or Brightwell B et al. Reality vs. rhetoric - a survey and enacted by the community, will always suffer from evaluation of tsetse control in East Africa. problems of sustainability, which have been consid- Agriculture and human values, 2001, pp 1-17. ered in great depth elsewhere (Brightwell et al, 2001). While trapping programmes run by the com- Bruce D. Preliminary report on the tsetse fly disease or munity are appealing to donors, they may simply be nagana in Zululand. Durban, Bennett and Davis, ineffective in the face of an epidemic. Under such 1895. circumstances, alternative actions (including aerial spraying) may have to undertaken, which may Bruce D, Nabarro D, Greig EDW. Further report on require very large inputs from the donor communi- sleeping sickness in Uganda. Reports of the Royal ty. Moreover, sleeping sickness control has unfortu- Society Sleeping Sickness Commission Reports, 1903, nately become a supply driven endeavour. As more 4:2-87. and more smart technology has become available for the control of tsetse, so the need has been felt to Fèvre EM et al. The origins of a new Trypanosoma apply it to the control of sleeping sickness epidemics brucei rhodesiense sleeping sickness outbreak in east- – but this may not always be appropriate. ern Uganda. Lancet, 2001, 358:625-628.

By contrast, treatment of sleeping sickness cases Gouteux JP, Laveissière C, Boreham PFL. Écologie and the domestic animal reservoir form part of the des glossines en secteur pré-forestier de Côte normal activities of the medical and veterinary d’Ivoire. 3. Les prévérences trophiques de Glossina services. When these services break down, for palpalis s.l. Cahiers de l’Office de la Recherche whatever reason, it is usual for aid agencies, the pri- Scientifique et Technique Outre-Mer, série entomologie vate sector and even communities themselves to médical et parasitologie, 1982, 20 :3-18. step in to the breach. Therefore, these activities are likely to be more sustainable in the medium and Heisch RB, McMahon JP, Manson-Bahr PEC. The long-term. isolation of Trypanosoma rhodesiense from a bushbuck. British Medical Journal, 1958, 2:1203-1204. CONCLUSION AND FUTURE RESEARCH Macdonald G. The epidemiology and control of malaria. From a theoretical perspective, killing tsetse flies is London, 1957, Oxford University Press. a preferred strategy. However, epidemiological effects are only one side of the story. Financial and Morris KRS. The control of trypanosomiasis by logistical resources needed to implement and sus- entomological means. Bulletin of Entomological tain control interventions also have to be consid- Research, 1946, 37:201-250.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 75 Murray CJL et al. Development of WHO guidelines on generalized cost-effectiveness analysis. Health Economics, 2000, 9:235-251.

Odiit M, Kansiime F, Enyaru JCK. Duration of symptoms and case fatality of sleeping sickness caused by Trypanosoma brucei rhodesiense in Tororo, Uganda. East African Medical Journal, 1997, 74:792-795.

Onyango RJ, van Hoeve K, de Raadt P. The epidemiology of Trypanosoma rhodesiense sleeping sickness in Alego location, central Nyanza, Kenya. I. Evidence that cattle may act as reservoir hosts of trypanosomes infective to man. Transactions of the Royal Society of Tropical Medicine and Hygiene , 1966, 60:175- 182.

Rogers DJ. A general model for the African trypanosomiases. Parasitology, 1988, 97:193-212. de Raadt P, Jannin J. International co-operation: past and present. In: Dumas M, Bouteille B, Buget A, eds. Progress in human African trypanosmiasis, sleeping sickness. Paris, Springer, 1999: 315-333.

Shaw APM et al. Abasis for financial decision-making on strategies for the control of human African trypanosomiasis. Scientific Working Group Meeting on African Trypanosomiasis, Geneva, World Health Organization, 4-8 June 2001.

Welburn SC et al. Sleeping sickness: a tale of two diseases. Trends in Parasitology, 2001, 17:19-24.

Wellde BT et al. Presenting features of Rhodesian sleeping sickness patients in the Lambwe Valley, Kenya. Annals of Tropical Medicine and Parasitology, 1989, 83:73-89.

World Health Organization. African trypanosomiasis or sleeping sickness. Fact Sheet No. 259, March 2001 (www.who.int/inf-fs/en/fact259.html).

76 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 III A BASIS FOR FINANCIAL DE- have only been studied sporadically. Thus, whilst CISION-MAKING ON STRATEGIES most people working on the disease have a very FOR THE CONTROL OF HUMAN clear idea of what the most cost-effective strategies AFRICAN TRYPANOSOMIASIS are in their locality, given their resources and price structure, little has been done to bring together the APM Shaw,1 P Cattand,2 PG Coleman,3 M John4 information gained in order to help decision-makers 1AP Consultants, Upper Cottage, Abbotts Ann, design strategies and prioritize in new areas or Hampshire, SP11 7BA, UK. under new circumstances. 2Association against Trypanosomiasis in Africa, 1 rue de l’Hôtel Dieu, F-74200 Thonon, France. This paper is the first step in an ongoing exercise to: 3CTVM, University of Edinburgh, Easter Bush, Roslin, • identify the main factors influencing the cost- Midlothian, EH25 9RG, UK. effectiveness of the different approaches to con- 4Le Kerrio, 56230, Berric, France. trolling the disease. • review the current literature on this subject. INTRODUCTION • identify potential research areas and information This discussion paper seeks to identify the main needs. issues involved in designing cost-effective strategies for the control of human African trypanosomiasis For 1999, the burden of HAT was estimated at 66 000 (HAT) and to highlight areas where more informa- deaths and 2 million DALYs lost (World Health tion needs to be compiled or new research initiated. Organization, 2000). Although only 45 000 new cases were reported (World Health Organization, Some form of control of the disease has been ongo- 2001), the likely number of people affected is proba- ing in most parts of Africa since the beginning of the bly ten times greater, thus approaching half a mil- last century. Thus many schemes have been funded lion. Of the 60 million people thought to be ‘at risk’, and budgeted for. However, the economics of the only 3–4 million are covered by some form of dis- different approaches and their cost-effectiveness ease surveillance.

Figure 1. Control options

Control of Disease ReservoirsPoint of Control of the vector contact

HumanPoint of Animal contact

TREATMENT TSETSE CONTROL of patients found - 1st/2nd stage Traps, targets, screens, rhodesiense / gambiense aerial spraying, insecticide treatment of cattle TREATMENT OF LIVESTOCK

DETECTION cattle and, in the past, some wildlife control finding patients: passive/fixed-post surveillance, active surveillance

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 77 The control of HAT is based on combinations of the years in the case of gambiense sleeping sickness, and four different approaches illustrated in Figure 1. It have made several attempts to have their symptoms involves: treated and obtain a correct diagnosis. Usually this • treating those human patients diagnosed with the will have involved several trips to their rural health disease. centre, possibly also to a nearby hospital or treat- • trying to improve diagnosis, by some level of sur- ment centre, a visit to a local healer, and being treat- veillance, to find patients and ensure that they are ed for malaria and other diseases before being diag- treated earlier, hopefully while still in the first nosed as having sleeping sickness. Both during trips stage of the disease. to the health centre and while the patient is hospi- • proceeding to more active forms of case-detec- talized, it will usually be necessary for a relative to tion, aimed not only at finding and diagnosing accompany and look after the patient. As the disease patients but also at reducing the size of the human progresses, the patient in search of a diagnosis will reservoir of the disease. This applies to the gambi- have become more and more of a burden on his fam- ense form of the disease. ily, requiring care and being unable to undertake • trying to reduce the chance of people picking up normal activities. the infection from domestic animals or wildlife, as in Uganda, where cattle are routinely treated A very rough estimate of the possible cost to around new outbreaks to control the rhodesiense patients trying to obtain a diagnosis was made by form of the disease (personal communication, I Landell Mills (2000) for the rhodesiense situation in Maudlin). Uganda. This came to US$25 per patient, including • controlling tsetse fly populations so as to reduce estimates for the cost of transport to rural health transmission of both forms of the disease. centres and hospitals, treatments for malaria, painkillers, and the time taken by relatives to Finally, as indicated in Fig. 1 by the two boxes accompany and care for the person. For those rhode- marked ‘point of contact’, control of HAT involves: siense patients who are never correctly diagnosed • avoiding areas likely to lead to infection - a strat- but who do receive some treatments, this cost would egy that people have used since time immemori- rise to a minimum of US$50, including consultations al. This strategy applies, above all, to avoiding with a local healer, further trips to treatment centres, contact with the tsetse fly and is an approach that possibly a short stay in hospital, and care by rela- has mainly been employed by livestock keepers to tives. prevent their cattle becoming infected and either dying or becoming less productive. As people For those correctly diagnosed, the costs of treat- become aware of the dangers of working in cer- ment, as estimated by WHO in 1998, are given in tain tsetse-infested thickets, they avoid them. In Table 1. These figures are based on the then current the past it was thought that people could avoid drug costs, treatment regimes in use, and estimates contracting the rhodesiense form of the disease of the cost of hospitalization. simply by avoiding areas containing wildlife. In the early stages of an epidemic of sleeping sick- Table 1 Costs of treatment

ness, the disease tends to be found among people Item First-stage disease Second-stage disease

whose occupations put them at risk by bringing Pentamidine Suramin Melarsoprol Eflornithine them into contact with infected flies, particularly Estimate of total cost 107 114 253 675 at certain times of the day (e.g. when collecting Cost excluding water, washing clothes, entering game reserves - drug cost 87 79 190 367 Drug cost as % as in the case of beekeepers, hunters, park of total cost 19 31 25 46 rangers). Thus, avoidance, whilst not specifically Source: WHO, 1998 discussed here, is a control strategy of sorts, However, these costings will need to be re-evaluat- although in practice it has mainly been used by ed in the light of current plans to make drugs avail- cattle herders to protect their stock. able free of cost from WHO for a specified number of years. The costs of transport and administration FINDING AND TREATING PATIENTS will remain, but, for the relevant drugs, the cost paid As cited above, WHO estimates are that only about by recipient countries or programmes will consist 10% of sleeping sickness patients are correctly diag- only of transport and drug administration. As the nosed and receive treatment. This proportion may table indicates, treatment costs could be reduced by be larger in endemic foci where active surveillance significant percentages, but nevertheless, in eco- is undertaken, but it can also be a great deal smaller. nomic as against purely financial terms, the use of Typically, patients who are detected passively have these drugs will still represent a resource cost which suffered from symptoms for some time, possibly should be taken into account. Furthermore, drug

78 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 availability on these terms will alter with time. used innovative techniques of filter paper sampling Taken together therefore, this implies that the aver- by trained community health workers who either age cost for patients (a high proportion of whom visit the community or are based at rural health cen- will already be in the second stage of the disease) tres: passively diagnosed and correctly treated would be • Fixed-post or passive surveillance. Using this in the order of US$50–300 each. strategy, patients who present with symptoms that are difficult to diagnose, or who don’t In order to analyse this aspect further, there is a need respond to treatments e.g. for malaria, are eventu- to: ally referred to a treatment centre and tested for a • analyse case histories of individuals being treated variety of diseases, including trypanosomiasis, so for the disease in order to determine by what that those with the disease are eventually diag- process they obtained a diagnosis, how long it nosed. The initial screening test is performed on took, and how much it cost them and the health wet blood. services. • Filter paper sampling at rural health centres. • collate information on currently used treatment Under this strategy, community health workers regimes and the costs of hospitalization at try- (CHWs) based at rural health centres receive panosomiasis treatment centres. some training in collecting samples on filter paper and routinely test all new patients presenting ECONOMICS OF CONTROLLING themselves at the health centre for whatever rea- THE HUMAN RESERVOIR: son. GAMBIENSE FORM OF THE DISEASE • Filter paper sampling by community health Turning next to active surveillance, which has been workers. CHWs trained in collecting filter paper the mainstay of programmes to combat the gambi- samples spend 20% of their time collecting sam- ense form of the disease, calculated costs are given ples and following up seropositive individuals - in the World Health Organization (WHO) Expert as based on experience in Uganda (John, 1995) Committee report of 1998 (see series of tables in and Côte d’Ivoire (Laveissière et al, 1995). Annex 9 of that report). The discussion below is • Monovalent mobile teams. These are the classic based on these calculations, as originally present- surveillance teams. The card agglutination test for ed by Shaw et al (1995) and following the same trypanosomiasis (CATT) is performed on whole approach as those prepared for the previous WHO blood, and all the parasitological tests, except Expert Committee on HAT (Shaw and Cattand, lumbar punctures, are conducted in the field. 1985). In order to produce a coherent set of cost- Monovalent teams work only on trypanosomia- ings, it was necessary to use a real situation as a sis. basis while making some adjustments to produce a • Polyvalent mobile teams. These operate in the scenario in line with generally accepted norms, same way as monovalent teams except that only a and the figures and prices used were based main- third of their work consists of screening for try- ly on work conducted in the Moyo District of panosomiasis. Uganda (John, 1995). The figures used in 1985 were based on WHO’s work in the Daloa area of Côte In order to standardize the results, calculations were d’Ivoire. based on areas with a population of 100 000 people, containing 10 rural health centres, and where 20 WHO’s calculations of 1998 were used to create a community health workers were operating. The spreadsheet (Microsoft Excel®) model, so allowing numbers screened by each strategy were assumed to results to be calculated for any starting prevalence. be as given in Table 2. The population covered, number of units involved in surveillance, sampling intensity, sensitivity and Table 2 Numbers screened for trypanosomiasis using specificity of screening and diagnostic tests, as well different surveillance strategies in an area with a pop- as all prices and other costs, can also be varied. The ulation of 100 000 results of repeated runs of this model enable the rel- 10 rural 20 community One One Surveillance Fixed-post ative cost-effectiveness of different sampling strate- surveillance health health monovalent polyvalent strategy centres workers mobile team mobile team gies at different prevalences to be analysed. These Number are presented and discussed in the series of graphs screened per 300 3000 24 000 36 000 20 000 annum below. The cost calculations covered: The original analyses were based on five alternative • initial screening using the CATT test. surveillance strategies, including the classic mobile • parasitological confirmation using gland punc- teams, fixed-post surveillance, and the less widely tures, the capillary tube centrifugation technique

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 79 (CTC) test, the miniature anion centrifugation Firstly, the relative performance of the different sur- technique (m-AECT), and lumbar punctures. veillance strategies was analysed in terms of the cost • training of staff in specialized techniques needed of finding gambiense patients. The results, in terms of for diagnosing trypanosomiasis. US$ per trypanosomiasis patient found, are given in • administrative overheads. Figure 2. Obviously the cost of finding a patient • depreciation (annual cost) of capital items (vehi- declines very rapidly as the prevalence increases, cles, laboratory equipment, etc.). since a higher proportion of those screened are • travel allowances and a share of salaries of all staff infected. This clearly does not reflect any increase in in proportion to the amount of time they spend on efficiency, simply that more and more of those trypanosomiasis control. screened are infected, so the costs of the operation • running costs for vehicles, specialized equipment average out over a larger number of individuals. and other recurrent costs for each surveillance Fig. 2 is given in three sections so that the differen- strategy. tials between the costs of each sampling strategy can be seen more clearly.

Figure 2a. Figure 2b. Cost of detection per Cost of detection per patient patient at low prevalences: 0.01% to 1% at medium prevalences: 1% to 5%

US$ PER US$ PER PATIENT PATIENT FOUND FOUND 3000 160

140 2500

120

Rural 2000 Health centre 100 Community health worker

Fixed post 1500 surveillance 80

Monovalent mobile team 60 Polyvalent 1000 mobile team

500 20

0 0 00.5 1 012345

PREVALENCE (%) PREVALENCE (%)

At very low prevalences (of 1% or less, see Fig. 2a), person, while surveillance using CHWs costs just such as those encountered in past years in areas under US$100 per person, and using mobile teams where surveillance was reasonably regular and the or rural health centre costs between US$120 and disease was considered to be under control, the US$140. costs (at a 0.05% prevalence) are well over US$2000 per patient identified using rural health centres or At medium level prevalences (from 1% to 5%, see mobile teams, and drop to just under US$2000 using Fig. 2b), the costs per patient found continue to fall, CHWs. Passive or fixed-post detection costs only US and the differentials between strategies narrow fur- $50 per patient identified since there are virtually no ther, with the cost of passive detection being US$14, overheads. When the prevalence reaches 1%, the of detection by CHWs being US$22, and of the other cost of passive detection falls to just over US$20 per three options being about US$30.

80 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 At high level prevalences (over 10%, see Fig. 2c), the Given the way in which the figures were calculated, cost of patients found passively falls to around by independently building up the cost of each strat- US$10, as it does for all surveillance strategies at a egy using local norms and prices, it is surprising prevalence of 20%. When the prevalence reaches how similar the costs for finding patients using dif- 50%, the cost of detection becomes very low, around ferent strategies are. Setting aside passive detection, US$5 for all surveillance strategies, except passive of the four active detection strategies, CHWs using detection where it remains at about US$10. filter paper is consistently the most cost-effective.

Figure 2c. Cost of detectionFigure 2c. per patient at high prevalences:Cost of detection per 5% patient and over at high prevalences: 1% to 5%

US$ PER PATIENT FOUND 35

25 Rural Health centre

Community 20 health worker

Fixed post surveillance 15 Monovalent mobile team

Polyvalent mobile team 10

0 01020304050 6070

PREVALENCE (%)

The purpose-built mobile team concerned only with what proportion of the population could be sam- trypanosomiasis surveillance is consistently the pled in a year using the different approaches and most costly. reflecting the assumptions made about the possible workload that each form of active surveillance can The almost total convergence of costs at high preva- tackle. The figure shows the situation in the hypo- lences is due to the fact that, at higher prevalences, thetical area with a human population of 100 000, a more than half of all costs are diagnostic costs for fixed number (10) of rural health centres, and a fixed initial screening and parasitological examinations number (20) of CHWs who can assign a significant (ranging between US$3.50 and US$2.50 per person). proportion of their time to active case detection for At these prevalences, most individuals are sero-pos- sleeping sickness. In such a situation, only the itive and have to be re-examined, so the running inputs by the mobile teams can be varied, for exam- costs and overheads associated with each strategy ple increased as illustrated, from spending half their are spread over a large number of patients. time in the area to having two teams working there full time. Based on experience, it was also assumed In order to further examine the relative effectiveness that mobile teams were able to undertake further of the different surveillance strategies, Fig. 3 shows examinations on a higher proportion of CATT-posi-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 81 tive patients than was possible under the surveil- Under these conditions, as illustrated in Fig. 3, find- lance strategies based on CHWs. The filter paper ing and treating a majority of the patients in the based sampling strategies involve a delay before the population can only be done using one or more results are known, after which individuals testing monovalent mobile teams. The other active surveil- positive are recalled and taken to a treatment centre lance strategies could be effective in detecting the for further testing. In contrast, mobile teams can presence of the disease, or in gradually eroding the undertake many of the parasitological tests them- size of the human reservoir, provided that the inci- selves, and transport the suspected patients to a dence is not high. treatment centre for final confirmation of disease status.

Fig. 3 Percentage of population sampled and percentage of all trypanosomiasis patients found according to surveillance strategy used % people

80,00

70,00

60,00

50,00

% 40,00

30,00

20,00

10,00

0,00

's t % people

P % patients a m m

a e m a

s d

e 0 a e

2 m e

e F

a t

e l

T e

10 Health Centres

ly n

n l

o o

2 2

Fig. 4 Total trypanosomiasis specific costs for Turning from the costs per individual found with the case-finding by surveillance strategy disease to the total investment required for finding trypanosomiasis patients, Fig. 4 illustrates these for the five different surveillance strategies for preva- 60 000 lences of 1% and 10%. As would be expected, the costs largely reflect the proportion of population 50 000 screened by each strategy (Fig. 3 and Table 2). 40 000 Although the costs would vary from country to country, and have probably increased somewhat US $ 30 000 since these figures were published in 1998, they give 20 000 an idea of the orders of magnitude involved, ranging 10% Prevalence from US$50 000 to US $60 000 for a monovalent 1% Prevalence 10 000 mobile team, to a minimal investment for fixed post/passive detection. 0

orkers obile Teamobile Team ealth W

unity H m olyvalent M 10 Rural Health CentresFixed Postonovalent Surveillance M om 1 M 1 P 20 C

82 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 In order to better understand how the situation nate the total costs at very low prevalences. But once evolves at high prevalences, details of the calcula- a prevalence of 1% is reached, all other costs are tion for intervention by a mobile team are shown in dwarfed by the cost of treating patients. Given the Fig. 5. Here, the cost of treating the identified uncertainty about how to value drug costs, these patients has been added to produce the total costs, have been included at their recent commercial cost which have been broken down into four categories. but separated from other treatment costs (adminis- The costs of surveillance (logistics, share of salaries, tration of drugs and hospital care). Thus the graphs etc.), which are given as ‘sampling strategy’, and the can be read so as to exclude the cost of drugs. A costs of diagnostic tests, which cover both initial notional figure for transport, or for their economic screening and parasitological confirmation, domi- cost, could be included in subsequent analyses.

Fig. 5a Breakdown of costs of detection and treatment of patients at low to medium prevalences (one mobile team sampling 36 000 people)

400,000

300,000 CostCost of of Drugs Drugs US$ 200,000 OtherOther treatment treatment cost cost Diagnostic Tests 100,000 Diagnostic Tests Sampling Strategy 0 Sampling Strategy 0.1 0.5 1 2 3 4 5 % Prevalence

Fig. 5b Breakdown of costs of detection and treatment of patients at high prevalences (one mobile team sampling 36 000 people)

4 500 000 4 000 000 3 500 000 3 000 000 2 500 000 CostCost of of Drugs Drugs US$ 2 000 000 OtherOther treatment treatment cost cost 1 500 000 DiagnosticDiagnostic Tests Tests Sampling Strategy 1 000 000 Sampling Strategy 500 000 0 10 20 30 40 50 60 70 % Prevalence

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 83 The costs of controlling the disease in the area pos- A preliminary and very approximate economic tulated, with one monovalent mobile team screen- analysis of the control of rhodesiense disease in ing 36% of the population in a year, thus range from south-eastern Uganda was undertaken as part of a just over US$50 000 where the prevalence is 0.1%, to DFID-commissioned review of its research work over US$4 million where the prevalence is 70%. (Landell Mills, 2000). Although based on extremely approximate assumptions, this analysis highlighted This analysis, based on conditions in Uganda and the potentially very favourable situation where it is extrapolation of the situation encountered there, possible to control the disease by treating cattle. thus examines the ways in which the costs of con- Drug treatments cost US$1.75 to US$2 per dose, and trolling the human reservoir vary with the sampling it is thought that between 5% and 20% of cattle carry strategy, sampling intensity, and prevalence. The cattle pathogenic trypanosomes (personal commu- spreadsheet model produced provides a basis for nication, Paul Coleman). Based on work done on the extending this analysis to cover other surveillance impact of trypanosomiasis on livestock production, protocols, price sets, test sensitivities, etc. It is likely and current milk and cattle prices in Uganda, it was that the costings for the extremely high prevalences estimated that treating 1000 cattle around a disease need revising upwards, since in the model, labour focus could yield a benefit of between US$500 to and time requirements were not fully adjusted to a US$3000, as compared to a cost of US$750 to situation where virtually all individuals test positive US$2000. Furthermore, if this expenditure on cattle to the screening test and need parasitological confir- treatment was successful in reducing the incidence mation. in humans, the financial benefit in terms of sleeping sickness treatment cost avoided would probably To update and validate this analysis in different cir- more than justify the expenditure. cumstances, what is required is: • collation and examination of data from budgets In this preliminary analysis of the economics of dis- and actual expenditures from a range of surveil- ease control over the past decade in south-eastern lance activities in different countries. Uganda, four categories of costs were considered: • to find out what protocols are used in different research, vector control, medical surveillance and field situations and what results are obtained, cattle treatment (Landell Mills, 2000). The benefits particularly in terms of what sequence of tests are were calculated by considering three likely alterna- used to obtain parasitological confirmation and tive scenarios for what the disease incidence might how many patients are detected by each test at have been if there had been no control activities. The different prevalences. monetary benefits consisted of costs saved for treating patients, benefits to cattle production, and ECONOMICS OF CONTROLLING THE patients’ costs incurred while seeking treatment (see ANIMAL RESERVOIR: RHODESIENSE section Finding and treating patients above). The non- FORM OF THE DISEASE monetary benefits were calculated in terms of Turning to the rhodesiense form of the disease, its DALYs averted. The monetary benefits produced more acute course means that patients usually pres- benefit-cost ratios ranging from 1.08 to 1.98. Because ent with symptoms shortly after infection. the project produced a net financial gain, the cost Nevertheless, diagnosis is often slow and inaccurate. per DALY was actually negative, ranging from - Control of this form of the disease has relied less on US$1.50 to -US$7.50. These results should be treated active surveillance and more on vector control. with caution, as they depend on very rough assumptions. However, they do point to the likeli- Recent research results have added a powerful and hood that this control approach, where it is feasible, cost-effective tool to the armoury of control methods could be extremely cost-effective. for this form of the disease. The proof that cattle have now become the main reservoir of the disease Research into the involvement of cattle as a reser- (Hide et al, 1996) in south-eastern Uganda has, as its voir of T. b. rhodesiense is ongoing. From the eco- corollary, shown that treating cattle would control nomic point of view, one key issue is, what propor- the reservoir, and, if at suitable level, would stop tion of the cattle population will need to be treated, transmission to humans. Although T. b. rhodesiense is and with what frequency, in order to generate finan- not pathogenic to cattle, the drugs used to control it cial benefits which cover the costs of controlling the also kill T. vivax and T. congolense, which are patho- disease in humans? Another important variable is genic to cattle and are prevalent among the cattle in the ratio of reported to unreported cases of the dis- the area. Thus, treating cattle generates an econom- ease in humans. Obtaining this information ic benefit which is independent of the control of the depends on the results of epidemiological studies. disease in humans. The economic gains to cattle production need to be

84 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 more accurately estimated, using data on the preva- The cost of pour-on formulations currently ranges lence of cattle pathogenic trypanosomes and their around US$1.50 to US$2 for an adult bovine. impact on livestock productivity. Insecticide treatment of cattle reduces fly density, but whether or not this would be of use in pre- VECTOR CONTROL venting HAT depends entirely on the local epi- At this stage of the work, it has not been possible to demiology of the disease. review existing information about the costs of vector control in detail. Recent years have seen few A useful series of comparative costings for tsetse initiatives that use vector control to deal with control methods for one country, along with details human trypanosomiasis. Costs are available from of how they were calculated, can be found in Barrett recent work in Uganda, where pyramidal traps (1997) for the case of Zimbabwe. Current research were very effective in reducing tsetse density and and information needs include updating costings disease incidence; these costs (personal communi- for the various strategies, and collating information cation R. Floto) amounted to ECU 781 000, exclud- on the impact that vector control operations under- ing technical assistance - at today’s prices, about taken in the past have had on the incidence of sleep- US $1.1 million. ing sickness. The extent to which community involvement and inputs, especially of labour, can be The costs of vector control will tend to be very spe- sustained over long periods of time also needs to be cific to the situation, varying with terrain (especial- revisited. ly for target and spraying operations), type of organization (especially for targets, traps or It is difficult, especially for gambiense disease, to screens), and type of settlements and rural economy separate the effects of controlling the human reser- (being affected by the availability of labour for voir from those of vector control, since the two are maintaining traps and targets, and the presence of usually undertaken at the same time. The cost- cattle where these are to be treated with insecti- effectiveness of vector control versus case-finding cides). Estimates of the cost of vector control opera- and treatment was modelled by Shaw (1989). At this tions, such as those cited below, almost invariably time, it appeared that case-finding and treatment include only the marginal costs, i.e. the extra costs was the more cost-effective at lower incidences and involved in field work, but neglect the considerable when dealing exclusively with a human reservoir. overheads, which can double or triple the costs per The conclusion, then as now, was that epidemiolog- sq. km. Vector control operations include: ical models and economic models need to be inte- • aerial spraying, using fixed wing aircraft and the grated in order to be of use in decision-making. sequential aerosol technique, which typically involves spraying the area in a cycle of five at DALYS AND THE COST-EFFECTIVENESS carefully timed intervals. Apart from the sterile OF SLEEPING SICKNESS CONTROL insect technique, aerial spraying tends to be the Finally, in order to examine the wider economics of most expensive control method. It has the great controlling sleeping sickness in relation to the con- advantage of achieving a very rapid reduction in trol of other diseases, an estimate of the cost per the fly population. Costs are likely to be well DALY averted is needed. So far there have been very upwards of US$500 per sq. km. few comprehensive attempts to calculate the actual • traps, screens and/or targets. The cost of these and potential DALYs lost due to either form of operations is far more variable than for aerial sleeping sickness. spraying, depending on the number deployed per sq. km. or per linear km. and on the way in which Recent work in Uganda has produced calculations they are deployed and serviced. The costs for tar- of DALYs for rhodesiense (Odiit et al, 2000a and get operations probably range between US$300 2000b). This work will be integrated into more and US$400 per sq. km., and low-cost trap- or comprehensive economic analyses. The authors screen-based operations could cost as little as point out that the age distribution of trypanosomi- US$100 to US$150 per sq. km. asis patients very closely follows that of the active • treating cattle with insecticides (not to be con- adult population, so that the disease tends to hit fused with treating cattle with trypanocides, as the most economically productive group of society discussed above). Here, various ‘pour-on’ formu- hardest, affecting family livelihoods and communi- lations are used, but again the costs are very diffi- ty prosperity very much. These data confirm obser- cult to estimate since the pour-on formulations vations made throughout Africa for both forms of also protect against tick-borne diseases, and the the disease. Odiit et al estimate that, at the time of number of cattle to be treated per sq. km. is also a diagnosis, patients will have been suffering from variable as is the number of treatments per year. symptoms of the disease for an average of 61 days

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 85 and will then require hospitalization for an average to look at the economics of alternative treatments for of 34 days. For patients correctly diagnosed and second-stage gambiense patients was based on the treated, the case fatality rate is 5.3%, but for unre- age-at-death distribution calculated for rhodesiense ported cases, the outcome is assumed to be patients in Uganda (Politi et al, 1995). These authors inevitably fatal. Based on the age distribution of also concluded that the standard treatment for sec- patients, Odiit et al estimate that the number of ond-stage patients represented a very attractive cost DALYs lost for unreported, and thus untreated, per DALY averted, ranking with the most cost-effec- patients is just over 20 years (personal communica- tive interventions such as childhood immunization tion Dr Paul Coleman). This figure was used to and blood-screening for HIV. underpin the economic analysis discussed in the section on Economics of controlling the animal reser- Figure 6 takes this discussion a bit further by mod- voir above. In this case, because controlling the ani- elling how the situation could be analysed if more mal reservoir reduced the rate of transmission to data were available. The cost of treatment and humans, and it was assumed that for every report- detection of patients using a mobile team at differ- ed case there was one unreported case, the full ent prevalences, as shown in Figure 2, is divided by twenty years applied to half of the cases averted. a conservative estimate of the number of DALYs This DALY figure thus also means that the cost- averted. The ‘zero’ baseline figure, which gives the effectiveness of controlling rhodesiense sleeping highest cost, is based on each patient treated, i.e. sickness compares very favourably with that of each premature death prevented, representing 15 other high priority health control activities, such as DALYs averted. This figure was selected as a con- malaria (Goodman et al, 2000), the expanded pro- servative estimate, taking into account the long gramme on immunization (EPI), and human asymptomatic period for gambiense disease, and cal- immunodeficiency virus (HIV). ibrated to be rather lower than the figure for rhodesiense. On this estimate, once the prevalence has For gambiense disease, no published DALY figures reached 1%, the cost per person found and treated based on detailed field records are available. falls to US$330, and thus the cost per DALY averted However, ongoing work in Southern Sudan indi- falls below the ‘good value for money’ threshold of cates that a similar situation probably exists (per- US$25. At higher prevalences, the cost per DALY sonal communication Dr Anne Moore). An attempt averted stabilizes at between US$10 and US$12.

Figure 6. US$ per DALY averted at different prevalences (Surveillance for gambienseFigure 6. using a monovalent US$ per DALYmobile averted at team)different prevalences (Surveillance for gambiense using a monovalent mobile team)

200

175

150 0

125 0.5

US$ 1 100 1.5

75 Multiplier applied to DALYs averted

0 0.05 0.5 2 4 1020 30 40 50 60 70

PREVALENCE

86 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 In order to add another dimension to the analysis, three further lines have been drawn showing what the effect would be if the number of DALYs averted per patient found and treated was increased by 0.5, 1 or 1.5. This analysis needs to be developed when more data have been analysed or collected to show: • what the actual figure for DALYs averted per patient treated is likely to be. • how the prevalence evolves from year to year in gambiense foci. The latter brings the discussion back to epidemiology, since, at low prevalences, this multiplier can be seen to be a measure of Ro, the basic reproductive rate of the disease.

There is thus a clear agenda for analysing existing data on the progress of epidemics and for collecting new data in order to add to the knowledge of the epidemiology of the disease. The low proportion of all cases actually recorded has meant that knowledge of the year-to-year changes in prevalence is often patchy or anecdotal. Nevertheless, for foci which have been the subject of more intensive control work over a number of years, data do exist. In this context, epidemiological models (e.g. Rogers, 1988) have an important role to play.

CONCLUSIONS This paper has tried to cover the main issues involved in the economics of controlling both gambiense and rhodesiense sleeping sickness. Some aspects, notably vector control, have only been superficially treated. Nevertheless, it is hoped that the paper provides a sufficient basis for discussion on what research and information gathering is needed to help plan funding and resource allocation and gauge what returns to expect from these invest- ments.

Returning to the information requirements that were noted at the end of each section, these fall into one of two categories: economic and epidemiological. Some of the issues and needs identified have been included in the table below, which provides a simple framework for categorizing the information required.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 87 Table 3 Framework for identifying information requirements and sources

Epidemiological Economic

• Analyse changes in preva- • Look at finances of past con- lence over time, relating to trol programmes, budgets, control strategies (including expenditure, overheads. vector control) being used. • Compare prices and costs of • Collate diagnostic protocols surveillance between coun- used in different tries and in different epi- countries/situations. demiological situations. • Collate information on sensi- • Update costings on vector Analyse existing data tivity and specificity of dif- control. ferent tests in the field. • Cost out different surveillance strategies, and refine existing spreadsheet model or develop new ones. • Cost out different treatment protocols, determine appropriate cost for drugs, estimate hospitalization costs.

• Determine the ratio of • Determine the costs of the reported to unreported disease to the local economy. cases. Initiate new data collection • Determine changes in prevalence over time and with respect to control work undertaken.

From the point of view of those allocating funds of controlling rhodesiense. Any errors remain the within the health sector, the recent emergence of responsibility of the first author,who would also like DALY calculations for sleeping sickness has made it to thank Ian Maudlin, Anne Moore, Jean Jannin and very clear that control of this disease represents an Martin Odiit for their helpful comments and extremely cost-effective investment. This is linked to encouragement. two factors. The first is the inevitably fatal outcome of the disease, which has been discussed above. The sec- References ond is the focal nature of the disease, which means Barrett JC. Economic Issues in Trypanosomiasis that although the population at risk is large, the dis- Control. Bulletin 75, Natural Resources Institute, ease is nevertheless location specific, so that control Chatham Maritime, UK, 1997. operations can target circumscribed geographical areas where the disease is known to be present. Goodman C, Coleman P, Mills A. Economic Analysis of Malaria Control in Sub-Saharan Africa. Acknowledgements Strategic research series. Geneva, Global Forum for This paper reflects: the inputs and ideas provided Health Research, WHO, 2000. over many years by Pierre Cattand, the joint work on the cost of surveillance undertaken with Michèle Hide G et al. The origins, dynamics and generation John, and Paul Coleman’s many suggestions and of Trypanosoma brucei rhodesiense epidemics in East generous inputs into the analysis of the economics Africa. Parasitology Today, 1996, 12: 50-55.

88 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 John M. Utilisation of testryp catt applied to samples of World Health Organization. The world health report. dried blood for sleeping sickness screening (T. b. gambi- WHO, Geneva, WHO, 2000. ense trypanosomiasis) of the population in villages and health units, Moyo District, Uganda 1993-1994. Dis- World Health Organization. African trypanosomiasis sertation for the MSc in Public Health in Tropical or sleeping sickness. Geneva, WHO, 2001, Fact Sheet Countries, London School of Hygiene and Tropical No. 269 (March 2001). Medicine, University of London, UK, Sept 1995.

Laveissière C et al. Intégration de la surveillance de la maladie du sommeil aux soins de santé primaires dans le foyer de Sinfra (Côte d’Ivoire). O.C.C.G.E., Institut Pierre Richet, Bouaké, Côte d’Ivoire, 1995.

Landell Mills. Evaluation of selected livestock research themes. Report prepared for DFID by Landell Mills Ltd, researched and written by APM Shaw and L Sibanda, 2000.

Odiit M et al. Incorporating the burden of human sleeping sickness in an economic impact assessment of trypanosomiasis. ISVEE, Colorado, 2000(a).

Odiit M et al. The burden of sleeping sickness in Southeastern Uganda – geographical variations. Paper presented to the British Society of Parasitology, Oxford, 2000(b).

Politi C et al. Cost-effectiveness analysis of alternative treatments of African gambiense trypanosomiasis in Uganda. Health Economics, 1995, 4:272-287.

Rogers DJ. A general model for the African trypanosomiases. Parasitology, 1988, 97:193-212.

Shaw APM, Cattand P. The costs of different approaches towards the control of human African trypanosomiasis. Paper Prepared for the WHO Expert Committee on the Epidemiology and Control of African Try- panosomiasis, Ref. TRY/EC/WP/85.17, 1985.

Shaw APM. Comparative analysis of the costs and benefits of alternative disease control strategies: vector control versus human case finding and treatment. Annales de la Société Belge de Médecine Tropicale, 1989, 69 (Suppl. 1):237-253.

Shaw APM, John M, Cattand P. The financial implications of different approaches to the detection and treatment of human African trypanosomiasis. Paper Prepared for the WHO Expert Committee on the Epidemiology and Control of African Trypanoso- miasis, Ref. TRY/EC/WP/95.14, 1995.

WHO Expert Committee on control and surveillance of African trypanosomiasis. Geneva, World Health Organization, 1998 (Technical Report Series 881).

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 89 Annex 5 DRUG DEVELOPMENT, PRECLINICAL AND CLINICAL STUDIES AND DRUG RESISTANCE African trypanosomiasis

90 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 I DRUG DEVELOPMENT FOR developed in the late 1970s (at the Merrell Dow AFRICAN TRYPANOSOMIASIS research laboratory) as an antitumour agent, it proved to be minimally active in that context but was Cyrus Bacchi effective against laboratory infections of T. b. brucei, Pace University, Haskins Laboratories, 41 Park Row, and later against T. b. gambiense in clinical trials. It New York, NY 10038, USA cured late-stage and melarsoprol-resistant CNS infections, but was not active against T. b. rhodesiense infection. Its side effects are generally mild and reversible INTRODUCTION upon withdrawal of the drug (Van Nieuwenhove There is an urgent and obvious need for novel, effec- 1992; Pepin and Milord 1994; Burri et al, in press). It tive, non-toxic drugs for treatment of human was approved by the US Food and Drug Admini- African trypanosomiasis (HAT). This is evident stration (FDA) in November 1990. Three problems from: arise with its use: expense (about US$500 per • Dramatic recent increases in incidence of the dis- patient), availability, and dosing (four times a day, ease due to civil unrest, warfare, and general intravenously, for 14 days). Aventis (current holder of breakdown of health monitoring and delivery the patent) now has a production agreement with infrastructure in large areas of Africa. Bristol-Myers Squibb for the synthesis of DFMO to be • An increase in resistance to standard trypanocidal used in a depilatory cream (Vaniqa). agents. • Toxicity of standard agents. In a recent development, WHO and Aventis have entered into an agreement in which Aventis will Of the standard agents available, pentamidine is supply 60 000 vials of eflornithine each year for five used for early-stage Trypanosoma brucei gambiense years. WHO will continue to explore avenues to infections but is not generally recommended for maintain production after the five-year period. early-stage T. b. rhodesiense. Pentamidine has been in WHO is conducting clinical studies to determine the use since 1940, is available from Aventis, and is also efficacy and safety of oral dose eflornithine, which used for treatment of Pneumocytis carinii in AIDS. would allow its use on a wide scale (FAS Kuzoe, Resistance is due in part to inability to transport the personal communication). agent. Suramin, a polysulphonated naphthylurea, has been in use since 1920 and is presently used for New agent in Phase I clinical trials: early-stage T. b. rhodesiense. It does not penetrate the DB 075/289 blood-brain barrier and is not used for the central A number of 2,5-bis(4 amidinophenyl)furan caba- nervous system (CNS) disease. It has the longest mates are reported to have activity agains half-life of any routinely used trypanocide due to Pneumocysitis carinii (Rahmathhulla, et al 1999). One binding to serum proteins and there is no consistent of these compounds (DB 075/289) has nearly com- appearance of resistance. Melarsoprol (Mel B, pleted Phase I testing (toxicity, pharmacokinetics), Arsobal) is the first choice for treatment of late-stage aimed at development against Pneumocystis carinii CNS infection. Developed in 1949, it has saved mil- pneumonia (PCP) and African trypanosomiasis. In lions of lives but suffers from toxicity problems case of a favourable outcome, first field trials (Phase (encephalopathic syndrome) in about 10% of IIA: proof of principle in patients) are to be con- patients, which are life-threatening in about 5% of ducted starting in May 2001. Studies in try- patients. Recently, there has been an increase in the panosome infected monkeys are ongoing and are number of patients who are refractory to treatment, thus far promising (C. Burri, Pesonal communica- which can largely be attributed to lack of uptake by tion). DB075/289 does not pass the blood-brain bar- the parasite (Burri et al, in press). rier and is only active against the first phase of the disease. However, as an orally dosed agent, it would In a recent development, Aventis and WHO have be ideal for treatment of actue disease as an alterna- signed an agreement in which Aventis will supply tive to pentamidine and potential additional means pentamidine and melarsoprol free of charge for five for disease control in high transmission areas. The years. There are indications that Bayer will enter development of DB 075/289 is conducted by an into a similar agreement for suramin (FAS Kuzoe, international consortium lead by Dr Richard personal communication). Tidwell of the University of North Carolina, and funded by a 15.1 million grant from the Bill and APPROVED NEW AGENT Melissa Gates Foundation. DL-α-difluoromethylornithine (DFMO, Orni- dyl®‚) is an inhibitor of ornithine decarboxylase, the lead enzyme of polyamine biosynthesis. Initially

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 91 LEADS TO OTHER AGENTS of 150 mg/kg for seven days (infusion pumps). Antagonists of Polyamine Metabolism. In HETA costs about US$2/g to synthesize under labo- addition to DFMO, a number of other agents target- ratory conditions. It deserves further study and ing polyamine metabolism have shown promise. should be examined in preclinical trials. These include MDL 73811 (5’-{[(Z)-4-amino-2- butenyl]methylamino}-5’-deoxyadenosine), an enzy- Trypanothione Reductase Inhibitors. me-activated inhibitor of S-adenosylmethionine Trypanosomes produce a unique glutathione ana- (AdoMet) decarboxylase which supplies decarboxy- logue, N1,N8-bis(glutathionyl)spermidine, for use lated AdoMet, the source of aminopropyl groups for as a redox defence mechanism in combination with spermidine and spermine synthesis. This agent was a specific trypanothione reductase, which restores developed by Marion Merrell Dow in the late 1980s. oxidized trypanothione to the reduced state. The lat- It cures acute laboratory infections of T. b. brucei and ter is thus a logical drug target. Many inhibitors of T. b. rhodesiense clinical isolates (Bitonti et al, 1990). It this enzyme have been developed, and some have is active against late-stage model infections when proven highly effective in vitro against bloodstream used in combination with low dose DFMO (Bacchi form trypanosomes, yet none have been shown to et al, 1994). MDL-73811 is not toxic to mice at >10 have significant activity in model infections. Classes times the curative dose levels used. It is rapidly of compounds include phenothiazines, tricyclic transported by the parasite through the P2 adeno- compounds, diphenylsulphides, phenylpropyl and sine site (Bitonti et al, 1990; Goldberg et al, 1998). It is naphthylmethyl β-substituted polyamines. Dif- given intraperitoneally once a day at 10-25 mg/kg. ficulties with bioavailability, pharmacokinetics, and Supplies are now limited. However, the absence of metabolism of these compounds need to be toxicity and the strong activity against T. b. rhode- addressed (Werbovetz, 2000; Keiser et al, 2001). siense isolates indicate steps should be taken to obtain further supplies and initiate preclinical trials. Cysteine Protease Inhibitors. Cysteine pro- teases have been detected in most parasitic proto- CGP 40215 is a bicyclic analogue of methylglyoxal zoans and are considered important potential tar- bis(guanylhydrazone) (MGBG), an inhibitor of gets for drug development. Initial studies examined AdoMet dc. This agent also resembles the diamidines cruzain, the major cysteine protease activity in T. berenil and pentamidine. It was the most active, both cruzi, while more recently trypanopain-Tb (the in vitro and in vivo, of a series of derivatives produced major lysosomal cysteine protease of T. b. brucei) has by Ciba-Geigy in the 1990s, curing laboratory infec- been the subject of extensive study. In this context, tions of T. b. brucei, T. b. rhodesiense, T. congolense, and treatment with the cysteine protease inhibitor Cbz- T. b. gambiense - a total of 19 isolates (Brun et al, 1996; Phe-Ala-CHN2 at 250 mg/kg/day for three days Bacchi et al, 1996). Used singly, CGP 40215 was not reduced parasitemia to undetectable levels in curative to a CNS model, but was curative when used T. b. brucei infected mice (Scory et al, 1999). in combination with DFMO. Unfortunately, when However, the parasitemia returned and animals tested in vervet monkeys with a CNS infection, the relapsed after treatment had ceased. This study, cou- compound was not curative and pharmacokinetic pled with similar results obtained with T. cruzi, indi- studies indicated that it did not cross the blood-brain cate these inhibitors have potential, but will need barrier (BBB) (Keiser et al, 2001). extensive refinement to adjust the availability and pharmacokinetics (Werbovetz, 2000). Methionine Recycling. Methylthioadenosine phosphorylase (MTA-Pase) is the lead enzyme of a Nitro-imidazoles. Megazole is a 5-nitroimida- salvage pathway which regenerates adenine and zole (2-amino-5-[1-methyl-5-nitro-2-imidazolyl]- methionine from MTA, the by-product of amino- 1,3,4-thiadiazole) which was first synthesized in propyl group transfer from decarboxylated AdoMet. 1968, but not studied because of risk of mutagenic- African trypanosomes have an MTA-Pase with a ity (Keiser et al, 2001). Recent studies, however, broad substrate specificity. The MTA substrate ana- have demonstrated significant activity of megazole logue hydroxyethylthioadenosine (HETA) is cleaved against acute murine infections of T. b. brucei and T. by MTA-Pase and the ribose moiety is metabolized, b. gambiense. Megazole was also effective in elimi- possibly to a keto acid, which appears to be the nating a murine model CNS infection when used in active agent (Bacchi et al, 1999). HETA is able to cure combination with suramin. Suramin prolonged acute T. b. brucei infections and infections caused by megazole’s elimination half-life and altered other 6 of 11 T. b. rhodesiense isolates in mice. HETA was 500 pharmacokinetic parameters including the reten- times less toxic to mammalian cells in culture than to tion time (doubled) and serum profile (extended trypanosomes (Sufrin et al, 1996; Bacchi et al, 1997). peak). In most studies, single-dose oral administra- In mice, it has given no evidence of toxicity at doses tion of megazole was used (Enanga et al, 1998). In

92 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 a primate model, cerebrospinal fluid (CSF) levels studies are planned with this series (JRL Pink and in an animal dosed with a combination of mega- FAS Kuzoe, personal communication). zole and suramin were found to be 5-10% of plasma levels after a single 100 mg/kg dose - a level VSG Synthesis. African trypanosomes incorpo- about 10 times that of the MIC100 for T. b. gambi- rate myristic acid into the glycosyl phosphatidyli- ense. This animal was cured of a CNS infection nositol (GPI), which serves as an anchor for the vari- (Enanga et al, 2000). Excretion of megazole is pri- ant surface glycoproteins (VSGs) which cover the marily (80%) in the urine in the primate model, and trypanosome. Myristate is obtained by try- four metabolites were consistently found in signif- panosomes either by scavenging from the host by icant amounts (Enanga et al, 1999). Although myristate exchange, or by fatty acid remodelling. promising, further preclinical studies will be need- The critical nature of VSG in the trypanosome’s exis- ed to determine the structure of the metabolites tence in the host makes the myristoylation step an and their overall mutagenic potential before clini- attractive chemotherapeutic target (Werbovetz, cal studies can begin. 2000). Fatty acid analogues of myristate gave IC50 values <100 nM in vitro against T. b. brucei and Inhibition of Glycolysis. The predominant T. b. rhodesiense bloodform trypanosomes. These form of African trypanosomes in the blood is the analogues were inactive in vivo however, because long slender trypomastigote, which depends on gly- they were rapidly metabolized by the host colysis for energy production. Recent molecular (Werbovetz et al, 1996). The second pathway is one modelling studies on glyceraldehyde-3-phosphate in which other fatty acids are remodelled to myris- dehydrogenase (GAPDH) have detected differences tate and then preferentially incorporated into GPI in the nicotinamide adenine dinucleotide (NAD)+ and not into other lipids (Morita et al, 2000). This binding site between the trypanosome and human pathway can be inhibited by the antibiotic thiolacto- enzyme, and an overall sequence identity of only mycin, which kills trypanosomes in vitro with an µ 28.5% with the trypanosome enzyme (Suresh et al, IC50 concentration of 150 M. However, in pharma- 2000). From these observations, adenosine ana- cokinetic studies in mice, thiolactiomycin was found logues modified in the C2’ ribose and N6 adenine to be rapidly excreted. Peak serum levels were positions have been synthesized. The most active of obtained 15 minutes after intramuscular injection these analogues was an N6 derivative, N6-(1- and then rapidly declined until, at 60 minutes, they napthalenemethyl)-2’-(3-methoxygenzamido) were 1/10th that of peak levels. This compound µ adenosine, with an IC50 value of 12 M against could be given orally with similar rapid elevation T. b. brucei and inhibition of pyruvate excretion in and decline of serum levels and tissue distribution in vitro incubations. This compound did not inhibit patterns (Miyakawa et al, 1982). It would be impor- µ human GAPDH at 50 M, but had an IC50 of tant to pursue these lines of study and develop 0.28 µM against L. mexicana GAPDH (Aronov et al, effective myristate analogues which are not metabo- 1999). Although promising, no in vivo studies have lizable by the host and/or thiolactomycin analogues been reported with these compounds. which have an extended serum half-life.

SIPI 1029. This agent is a triazine derivative Amidine Prodrugs and Cis-platinum (Trybizine.HCl) which is used against T. evansi in Derivatives. A number of 2,5-bis (4 amidino- buffaloes in China. SIPI 1029 was effective both phenyl) furan carbamates are reported to have activ- in vitro and in acute mouse model infections against ity against Pneumocystis carinii, and one is undergo- T. b. brucei, T. b. gambiense and T. b. rhodesiense ing Phase I clinical trials for this target. As an orally (Bacchi et al, 1998; Kaminsky and Brun, 1998). In dosed diamidine, it would be of significant interest combination with low-dose DFMO, it cured a model to examine these agents for treatment of acute T. b. CNS infection (Bacchi et al, 1998), although used gambiense infections as an alternative to pentami- singly it was not active in this model, or in a vervet dine (Rahmathhulla et al, 1999). monkey CNS model (Keiser et al, 2001). Pharma- cokinetic studies in the latter model revealed only A group of organometallic complexes derived from low concentrations of the agent in CSF. pentamidine have been evaluated for activity in vivo against acute T. b. brucei in mouse and sheep model Over 200 analogues of SIPI 1029 were tested at the infections. Cis-platinum pentamidine-bromide, Swiss Tropical Institute against African try- –thiocyanate and -seleniocyanate were curative in a panosomes in vitro. Over 40 were tested in an acute single dose of 1-3 mg/kg. The bromide derivative mouse model and the most active compounds (5-10) was curative at 1 mg/kg and had a chemotherapeu- were examined for activity in a CNS model. None tic index of 200 compared to pentamidine, which showed activity in this model and no additional was curative at 3.5 mg/kg and had a chemothera-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 93 peutic index of 13. Further studies are planned with diamidines and melarsoprol has been the focus of a CNS model infection and with pentamidine-resist- much study (Hasne and Barrett, 2000; Barrett and ant isolates (Loiseau et al, 2001). Fairlamb, 1999). Thus P2 carries adenosine and adenine, while melarsopol and pentamidine interfere Combination Chemotherapy. A number of with their uptake. Trypanosomes resistant to me- compounds have been identified in the past seven- larsoprol and to berenil have lost P2. Megazole, a eight years which are excellent trypanocides both 5-nitromidazole active against African trypano- in vitro and in vivo, with the exception that they do somes (see above) is also transported through P2 not penetrate the BBB and hence do not cure model (Hasne and Barrett, 2000). It now appears that there CNS infections. In light of the lack of lead com- are other transporters, in addition to P2, for dia- pounds, and the fact that even current promising midines and melarsoprol (DeKoning, 2001). compounds have not undergone extensive preclinical toxicology studies, which may very well elimi- Another aspect of trypanosome nucleoside uptake nate them from consideration, it would be prudent is the demonstration of an AdoMet transporter sep- to examine drug combinations for CNS activity. arable from P1 and P2 (Goldberg et al, 1997). Which compounds however, should be the starting AdoMet transporter is not a usual component of points? In light of the many studies and clinical mammalian cell nucleoside uptake. Its appearance activity, eflornithine (DFMO) at low dose levels has in trypanosomes should be exploited in terms of proven active in curing model CNS infections in transport of currently active agents and in develop- combination with: suramin, melarsoprol, SIPI 1029, ment of novel agents with the ability to transverse a MDL 73811, HETA, CGP 40215, 9-deazainosine. wide range of transporters. For example, the MTA- Clearly, the ability of DFMO to act synergistically Pase analogue HETA is taken up by both P2 and the with so many chemically distinct agents does not AdoMet transporter (Goldberg et al, 1998). Overall, imply a common biochemical basis of action. Rather, the function and substrate specificity of try- the literature on artificially induced BBB injury indi- panosome nucleoside/drug transporters should be cates that polyamine metabolism is significantly out made a priority for study and drug development. of balance after injury and that DFMO has a role in correcting this (Croft, 1999). A recent review indi- Acknowledgements cates that a number of different types of injuries to I thank Michael Barrett, Reto Brun, Christian Burri, the brain result, after a complex cascade of events, in Felix Kuzoe, and Karl Werbovetz for supplying the enhanced synthesis, degradation and release of recent references, helpful discussions, and/or other polyamines in brain tissue. Ultimately, damage may information essential for this document. be attributable to formation of toxic reaction products as a result of the oxidative degradation of References polyamines (Seiler, 2000). DFMO may ameliorate Aronov AM et al. Structure-based design of sub- this injury, and in so doing a situation may develop micromolar, biologically active inhibitors of try- which allows passage of molecules impervious to panosomatid glyceraldehyde-3-phosphate dehydro- the BBB. Since, in most combination studies with genase. Proceedings of the National Academy of Sciences DFMO, only one or two doses of the other agent of the United States of America, 1999, 96:4273-4278. need be given, usually within in three-four days of the onset of the DFMO, there appears to be a short Bacchi CJ et al. In vivo trypanocidal activities of new S- window during which the other agent will pass the adenosylmethionine decarboxylase inhibitors. Anti- microbial Agents and Chemotherapy, 1996, 40:1448-1453. BBB. There is a real need to explore these combinations and the basis for their activity in primate mod- Bacchi CJ et al. Metabolic effects of a methylth- els using low-dose DFMO in combination with ioadenosine phosphorylase substrate analog on known agents, e.g. suramin and melarsoprol. It may African trypanosomes. Biochemical Pharmacology, be that other polyamine antagonists, e.g. MDL 73811 1999, 57:89-96. which blocks AdoMet dc and hence spermidine production, may have similar synergistic activity. Bacchi CJ et al. Combination chemotherapy of drug resistant Trypanosoma brucei rhodesiense infections in Transport of Nucleosides. Recently, a signifi- mice using DL-α-difluoromethylornithine and stan- cant effort has been made by a number of investiga- dard trypanocides. Antimicrobial Agents and Chemo- tors concerned with the mechanism of drug uptake therapy, 1994, 38:563-569. by African trypanosomes. While the normal functions of P1 and P2 transporters in bloodstream try- Bacchi CJ et al. In vivo efficacies of 5’-methylth- panosomes were determined to be nucleoside and ioadenosine analogs as trypanocides. Antimicrobial nucleobase transport, the ability of P2 to take up Agents and Chemotherapy, 1997, 41:2108-2112.

94 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Bacchi CJ et al. Antitrypanosomal activity of a new Hasne M, Barrett MP. Drug uptake via nutrient triazine derivative, SIPI 1029, in vitro and in model transporters in Trypanosoma brucei. Journal of Applied infections. Antimicrobial Agents and Chemotherapy, Microbiology, 2000, 89(4), 697-701. 1998, 42:2718-2721. Kaminsky R, Brun R. In vitro and in vivo activities of trybizine hydrochloride against various pathogenic Barrett MP, Fairlamb AH. The biochemical bases of trypanosome species. Antimicrobial Agents and Che- arsenical-diamidine cross resistance in trypanoso- motherapy, 1998, 42:2858-2862. mes. Parasitology Today, 1999, 15:136-140. Keiser J, Stich A, Burri C. New drugs for the treatment Bitonti AJ et al. Cure of Trypanosoma brucei brucei and of human African trypanosomiasis, research and Trypanosoma brucei rhodesiense infections in mice development. Trends in Parasitology, 2001, 17:42-49. with an irreversible inhibitor of S-adenosylmethionine. Antimicrobial Agents and Chemotherapy, 1990, Loiseu PM et al. Antitrypanosomal properties of cis- 34:1485-1490. platinum-pentamidine bromide, thiocyanate and seleniocyanate on Trypanosoma brucei brucei mouse and Brun R et al. In vitro trypanocidal activities of new sheep models. Journal of Chemotherapy, 2001, 13:59-65. S–adenosylmethionine decarboxylase inhibitors. Anti- Miyakawa S et al. Thiolactomycin, a new antibiotic. microbial Agents and Chemotherapy, 1996, 40:1442 1447. Journal of Antibiotics, 1982, 35:411-419.

Burri C, Stich G, Brun R. Current chemotherapy of Morita YS, Paul KS, Englund PT. Specialized fatty human African trypanosomiasis. In: Mulligan HW. acid synthesis in African trypanosomes: myristate The African trypanosomiases (in press). for GPI anchors. Science, 2000, 288:140-143.

Croft SL. Pharmacological approaches to antitry- Pépin J, Milord J. The treatment of human African trypanosomal chemotherapy. Memorias do Instituto panosomiasis. Advances in Parasitology, 1994, 33:1-47. Oswaldo Cruz, 1999, 94:215-220. Rahmathullah SM et al. Prodrugs for amidines: synthesis and anti-Pneumocystis carinii activity of carba- De Koning HP. Uptake of pentamidine in Trypa- mates of 2,5-bis(4-amidinophenyl)furan. Journal of nosoma brucei brucei is mediated by three distinct Medicinal Chemistry, 1999, 42:3994-4000. transporters: implications for cross-resistance with arsenicals. Molecular Pharmacology, 2001, 59:586-592. Scory S et al. Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine pro- Enanga B, et al. Pharmacokinetics, metabolism and teinase inhibitor z-phe-ala-CHN2. Experimental excretion of megazole, a new potent trypanocidal Parasitology, 1999, 91:327-333. drug in animals. Arzneimittelforschung /Drug Rese- arch, 1999, 49:441-47. Seiler N. Oxidation of polyamines and brain injury. Neurochemical Research, 2000, 25:471-490. Enanga B et al. Megazole combined with suramin: a chemotherapy regimen, which reversed the CNS Sufrin JR et al. Antitrypanosomal activity of purine nucleosides can be enhanced by their conversion to pathology in a model human African trypanosomia- o-acetylated derivatives. Antimicrobial Agents and sis in mice. Tropical Medicine and International Health, Chemotherapy, 1996, 40:2567-2572. 1998, 3:736-741. Suresh S et al. A potential target enzyme for trypanoci- Enanga B et al. Pharmacokinetics, metabolism and dal drugs revealed by the crystal structure of NAD- excretion of megazol in a Trypanosoma brucei gambiense dependent glycerol-3-phosphate dehydrogenase from primate model of human African trypanosomiasis. Leishmania mexicana. Structure, 2000, 8:541-552. Arzneimittelforschung /Drug Research, 2000, 50:158-162. Van Nieuwenhove S. Advances in sleeping sickness Goldberg B et al. Effects of intermediates of methio- therapy. Annales de la Société Belge de Médecine nine metabolism and nucleoside analogs on S–ade- Tropicale, 1992, (Suppl. 1):39-51. nosylmethionine transport by Trypanosoma brucei Werbovetz KA. Target-based drug discovery for brucei and a drug-resistant Trypanosoma brucei rhode- malaria, leishmaniasis, and trypanosomiasis. siense. Biochemical Pharmacology, 1998, 56:95-103. Current Medicinal Chemistry, 2000, 7:835-860.

Goldberg B et al. A unique transporter of S-adenoyl- Werbovetz KA, Bacchi CJ, Englund PT. Trypanocidal methionine in African trypanosomes. The FASEB analogues of myristate and myristoyllsophos- journal: official publication of the Federation of American phatidylcholine. Molecular and Biochemical Parasi- Societies for Experimental Biology, 1997, 11:256-260. tology, 1996, 81:115-118.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 95 II DRUG RESISTANCE IN However, reports have been mounting that many SLEEPING SICKNESS cases in the current epidemics of sleeping sickness are not responding to melarsoprol, the principal drug Michael P Barrett, used against stage II disease. This report aims to dis- Institute of Biomedical and Life Sciences, University of cuss the current status of drug resistance in human Glasgow, Glasgow G12 8QQ, UK infective trypanosomes, to consider mechanisms by which parasites become resistant to drugs, and to dis- INTRODUCTION cuss how resistance is detected and how detection No vaccines exist against sleeping sickness, and the may be improved. Suggestions as to how to deal with prospects of prophylactic immunization are poor drug resistant parasites and how to delay the onset since the parasites change their surface coat period- and spread of resistance are also made. ically in a process known as antigenic variation. Drugs remain the principal means of intervention. EPIDEMIOLOGY OF SLEEPING Five drugs are currently used against Human SICKNESS: IMPLICATIONS FOR THE African Trypanosomiasis (HAT) (Pépin and Milord, SPREAD OF RESISTANCE 1994), the drug of choice depending on which sub- A full understanding of the epidemiology and pop- species of T. brucei is involved and on whether the ulation structures of human infectious try- disease is diagnosed before or after the parasites panosomes, and their relationship with non-human have become established within the cerebrospinal infectious parasites, is crucial in understanding pos- fluid (CSF). Problems associated with the current sible routes to the spread of drug resistance. therapies for sleeping sickness include toxicity, Currently the epidemiology of the disease is not suf- resistance, and a lack of a guaranteed supply ficiently well understood to make solid conclusions. (Barrett, 2000) (although it seems likely that licensed However, recent data have started to clarify the sit- drugs will be available for at least the next five uation in the field, allowing room for some specula- years). It is unlikely that new formulations will be tion on this topic (MacLeod et al, 2001). available for at least ten years. This report deals with the problem of drug resistance. T. brucei parasites are able to undergo genetic exchange inside the tsetse fly, although this process Antimicrobial drug resistance is widespread. Many is not obligatory. It has recently been shown that antibiotics are obsolete and the antimalarial drug many infected tsetse flies in the field carry mixed chloroquine has been rendered useless in many populations of trypanosome (MacLeod et al, 1999), parts of the world due to the emergence of resist- thus genetic exchange is possible and studies on the ance. Drug resistance has also become a serious population genetics of trypanosomes have revealed problem in the treatment of animal trypanosomias- that genetic exchange does occur sufficiently fre- es (Geerts and Holmes, 1998). The epidemiology of quently to be an important determinant of genetic sleeping sickness and recommended regimens for diversity. the administration of drugs against this disease appear to be unfavourable for the development of However, it is not yet clear whether the human resistance. HAT is relatively uncommon when infectious Trypanosoma brucei gambiense does engage compared to malaria (estimates of 300 thousand in genetic exchange. Moreover, in East Africa it cases of sleeping sickness compared with 300 mil- appears that T. b. rhodesiense may have a predomi- lion of malaria). No drugs are currently used pro- nantly clonal structure (MacLeod et al, 2000), sug- phylactically against sleeping sickness and admin- gesting that it does not frequently engage in genetic istration of all drugs is parenteral and should occur exchange, although this does not mean that it can- in a clinical setting and not through self-adminis- not do so. While T. b. gambiense (type I) appears to be tration. Therefore the conditions which have nor- genetically distinct from other T. brucei sub-species, mally been implicated in the selection of drug- T. b. brucei and T. b. rhodesiense may simply be con- resistance, i.e. widespread use, significant under- sidered as host-range variants of local T. b. brucei dosing associated with self-administration and populations that become genetically isolated. Type improper prophylactic use, do not appear to be rel- II T. b. gambiense found in West and Central Africa evant to sleeping sickness. In addition, the zoonot- also appear to closely resemble T. b. brucei (MacLeod ic nature of the trypanosomes which cause human et al, 2001). disease could mean that drug selection would be relieved as parasites are transmitted to untreated Studies on trypanosome population genetics are still animals, thus diminishing the pressure to maintain in their infancy and it cannot be ruled out that resistance genes in the parasite population. human infectious parasites can engage in genetic exchange with non-human infectious organisms.

96 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Moreover, human infectious parasites are not limited • Treatment failure is increasingly being reported in host-range to humans, and many trypanosomes from epidemic loci (30% failure in northern (around 20%) isolated from cattle in Uganda and Uganda compared with 5-10% normally). Western Kenya are human infectious (Hide, 1999). In • T. b. gambiense isolates from treatment failures in West Africa it appears that T. b. gambiense may also north-west Uganda have recently been shown to have a host range beyond humans, with pigs repre- have reduced sensitivity to the drug. senting a particularly important reservoir • While plasma levels of melarsoprol appear to (Penchenier et al, 1999). A critical corollary of the remain at levels high enough to kill the parasites, essential zoonotic nature of trypanosomes is that the same may not be true in the CSF and other resistant strains selected by drug use in animals extravascular compartments. could be transferred to humans (this will be dis- • The amino-purine transporter P2, encoded by the cussed later). In addition, human infectious parasites TbAT1 gene, is at least partially responsible for could also acquire resistance from non-human infec- uptake of melamine-based arsenicals. Loss of the tious parasites as a result of genetic exchange during transporter can contribute to resistance. One mixed transmission in tsetse flies. It is important to study has shown that removal of the TbAT1 gene stress, however, that this has yet to be demonstrated renders parasites only fourfold less sensitive to in experimental or population genetic studies. melarsen oxide than wild-type trypanosomes. However, this degree of resistance might be However, it is clear that even in instances where try- enough to allow survival in the CSF (and relapses panosomes in humans are not exposed to the classi- appear to depend on CSF involvement). cal risk factors for the development of drug resist- • Many of the isolates from treatment failures have ance, the possibility exists that human infectious an altered TbAT1 gene. parasites are exposed to these classic factors in ani- • It is possible that a combination of reduced drug mals. Domestic animals do receive large doses of sensitivity (in part due to reduced uptake because various trypanocides, often administered at the dis- of changes to the P2 transporter) and variability in cretion of farmers, and frequently given over pro- accumulation of drug in the CSF can explain cur- longed periods as prophylaxis. While trypanocides rent treatment failures in Africa. used to treat humans and animals do differ, the potential to develop cross-resistance between The drug and its use human and animal trypanocides does exist. For Melarsoprol (Mel B) is a melaminophenyl-based example, diminazene is used extensively for the organic arsenical which was introduced as an anti- treatment of cattle. Cross–resistance between dimi- trypanosomiasis reagent in 1949 (Friedheim, 1949). nazene and the related diamidine pentamidine can It was Paul Ehrlich who promoted the idea that be induced in trypanosomes (Barrett and Fairlamb, arsenicals could be useful drugs for use against 1999). Cross-resistance between diminazene and sleeping sickness at the turn of the century. His com- melarsoprol can also be selected with relative ease pound, salvarsan or ‘606’, developed for use against (reviewed in Barret and Fairlamb, 1999). This is syphilis, is often considered as the prototypic because all of the drugs can enter trypanosomes via chemotherapeutic reagent. Ernst Friedheim devel- the P2 amino-purine transporter. Loss of this trans- oped the melamine-based arsenicals in the late porter can diminish uptake of these drugs into par- 1940s after the dangers of serious side effects associ- asites, which decreases sensitivity. Although the sit- ated with tryparsamide and high rates of treatment uation with respect to cross-resistance is not entire- failure decreased confidence in this product. ly straightforward, as outlined in the sections about Interestingly, melarsoprol has recently been used in resistance with respect to each of the human drugs, clinical trials against leukaemia (Soignet et al, 1999). it should be considered when looking at treatment The trials were abandoned when it became clear of animal trypanosomiases in regions where the that patients were suffering from seizures at more or human disease is endemic. less the same rate as in sleeping sickness treatment. However, different regimes might be tried. THE STATUS OF RESISTANCE TO CURRENTLY USED DRUGS IN Melarsoprol itself is amphipathic and will diffuse SLEEPING SICKNESS across cellular membranes. However, the drug is very rapidly converted to the highly hydrophilic Melarsoprol melarsen oxide in plasma (96% clearance of melarsoprol within 1 hr) (Burri et al, 1993). Melarsen Summary points on melarsoprol resistance oxide levels peak within 15 minutes and have a half- • Melarsoprol has been widely used in the field life of 3.9 hours. Relatively little melarsoprol, or its since the 1950s. metabolites, accumulate across the blood-brain bar-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 97 rier, with maximum levels being equal only to Trypanosomes exposed to arsenicals lyse very rap- around 1-2% of maximum plasma levels. The new idly. A mode of action has yet to be established. Loss pharmacokinetic information (Burri et al, 1993; of ATP due to inhibition of glycolysis could under- Keiser et al, 2000) has been very useful in establish- lie lysis caused by the drug, as bloodstream form ing a new regimen for administration of the drug trypanosomes depend solely upon glycolysis for which is likely to engender better patient compli- ATP production. However, it seems that the cells ance (Burri et al, 2000). Detailed information on lyse before ATP supplies are seriously depleted, pharmacokinetics may also be crucial in permitting leading several workers to question whether glycol- an understanding of the factors that underlie clinical ysis is a target for arsenical action (Van Schaftingen drug failure. It seems that levels of drug that reach et al, 1987). the CSF might be insufficient to kill parasites that are only a few fold less sensitive to drug than wild- Another suggested target of melarsoprol was try- type trypanosomes. panothione, a key low molecular weight thiol found in trypanosomatids but not mammalian cells. Since Of all the trypanocides, toxic effects are worst with arsenic is known to form stable interactions with melarsoprol; up to 10% of patients suffer a frequent- thiols, trypanothione was also proposed as the ly fatal reactive encephalopathy (Pépin and Milord, definitive target for these compounds (Fairlamb 1994). A high proportion of leukaemia patients suf- et al, 1989). Arsenicals, however, interact more tight- fered neurological seizures when treated with ly with other thiols including lipoic acid and, at the melarsoprol (Soignet et al, 1999), demonstrating that point of arsenical-induced lysis, only a small frac- melarsoprol is itself responsible for the reactive tion of trypanothione is conjugated with the drug encephalopathy associated with drug treatment. (Fairlamb et al, 1992). Thus it seems unlikely that trypanothione is the in situ target of these drugs. Exfoliative dermatitis has also been observed. Hypersensitivity, renal and hepatic dysfunction are Resistance also known. Myocardial damage, albuminuria and Treatment failure with melarsoprol has been report- hypertension can also occur. Headache, fever, gen- ed in the field. There has always been a cohort of eral malaise, urticaria, abdominal pains, vomiting 5–10% of treated patients who relapsed after treat- and acute diarrhoea are all less severe but common ment, although it was never clear what factors were side effects (Pépin and Milord, 1994). responsible for this. However, in northern Uganda relapse rates after melarsoprol treatment of around Mode of uptake and action 30% have been reported (Legros et al, 1999). It has been proposed that both the melaminophenyl Anecdotal reports of similar failure rates in northern arsenicals and diamidine classes of drug enter T. Angola are also in circulation. The incidence of brucei by the P2 amino-purine transporter (Carter relapse after treatment does not, at present, seem to and Fairlamb, 1993). Trypanosomes selected for be as serious in southern Sudan, although around resistance to sodium melarsen had lost the P2 trans- 16% failure rate has also been reported here. The porter (Carter and Fairlamb, 1993), and a T. equiper- recent build up of data on the pharmacokinetics of dum line selected for resistance to diminazene, the drug (Burri et al, 1993; Keiser et al, 2000), cou- which displayed some cross-resistance to arseni- pled with advances in understanding of the molec- cals, had a P2 transporter with markedly reduced ular and biochemical basis of resistance and an affinity for substrate (Barrett et al, 1995). These data analysis of field isolates from relapse cases in suggested that the P2 transporter is involved in Uganda (Matovu et al, in press), has allowed the uptake of arsenicals (Carter and Fairlamb, 1993) development of a model for the likely causes of and diamidines including diminazene (Barrett et al, treatment failure in the field. 1995) (the situation for pentamidine is more complicated, as described later). A simplistic model pro- Precise quantification of drug levels in a patient’s posing that loss of the P2 transporter was necessary plasma and CSF is difficult, although best results and sufficient to induce resistance to have been obtained using a bioassay for trypanoci- melaminophenyl arsenicals and diamidines was dal activity in fluids containing melarsoprol or its developed (Barrett and Fairlamb, 1999; Carter and active metabolites (Burri and Brun, 1992). The logis- Fairlamb, 1993; Barrett et al, 1995). In vitro, unme- tics of drug delivery in primary health care centres tabolized melarsoprol is likely to cross the mem- is difficult, so administration of the drug can differ brane by passive diffusion (Scott et al, 1997) from patient to patient in terms of both total deliv- although melarsen oxide does not. The possibility ered dose and timing between doses. Moreover, the of additional modes of uptake should not be metabolism of the drug and also its distribution excluded. within the body will vary from patient to patient

98 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 (i.e. accumulation of the drug in the CSF and other Experiments that led to the identification of a role extravascular compartments can be highly vari- for the P2 transporter in resistance (Carter and able). Fairlamb, 1993) involved laboratory derived isolates selected for high level resistance to sodium These variables can be crucial to the success of the melarsen (which, like other melaminophenyl arseni- drug. Absolute quantities vary between patients; cals, is probably rapidly metabolized to melarsen however, maximum serum levels following four oxide in vivo). However, recent data involving injections of the drug according to the “classic” pro- genetic manipulation of trypanosomes, removing tocol were 5 – 6 µg ml-1 (Burri et al, 1993). This falls the TbAT1 gene, revealed that loss of the P2 trans- to 0.22 µg ml-1 120 hours after the final injection in porter yields only around fourfold reduced sensitiv- the series. Drug detected in the CSF 24 hours after ity to melarsen oxide (Matovu et al, 2001). the final injection varied between individuals. The Additional mechanisms must therefore be at play in -1 maximum concentration was 260 ng ml but this determining high level resistance in laboratory descended to undetectable quantities in some derived lines. The modest change in sensitivity patients and average levels in the CSF are estimated relating to impairment of P2 function, however, to be in the order of 50-fold lower than those in plas- could render parasites resistant to levels of melarsen ma (Burri et al, 1993). Melarsoprol is rapidly con- oxide accumulating in the CSF and other extravas- verted to melarsen oxide (and possibly other cular compartments. It appears that the majority of metabolites) in plasma (Keiser et al, 2000). relapses reported in Uganda do involve small numbers of CSF parasites re-invading the bloodstream There is also some variability in intrinsic sensitivity post-treatment (Matovu et al, 2001). It is also note- to melarsoprol (or melarsen oxide) among different worthy that genetic removal of TbAT1 had no isolates of trypanosome. Sensitive parasites appear impact on parasite viability. to have minimal inhibitory concentration (MIC) val- -1 ues of around 1-30 ng ml (De Koning, 2001). Many of the drug resistant T. b gambiense isolates from northern Uganda have mutations in the TbAT1 The quantity of active melamine-based arsenical gene that encodes the P2 transporter (Matovu et al, reaching extravascular parasites, combined with the 2001). Many of these mutations are common with MIC of the trypanosomes, will determine whether mutations found in laboratory derived drug resist- they are killed by the drug. The MIC values of most ant isolates (Maser et al, 1999). Although some of the parasites are only marginally lower than estimated resistant isolates apparently do not have changes to concentrations of drug that are achieved in the CSF the sequence of TbAT1, it cannot be ruled out that of most patients. Data regarding melarsoprol in other genetic alterations, beyond the open reading other extravascular compartments that also harbour frame, would downregulate expression of that gene. trypanosomes are absent. The 5-10% of melarsoprol One example of a T. b gambiense line lacking the refractory cases could conceivably represent a TbAT1 gene has been reported (Matovu et al, 2001). cohort of individuals in whom extravascular accu- A multi-drug resistant T. b rhodesiense isolate from mulation of the drug is at the lower end of a nor- south-east Uganda (Matovu et al, 1997) had an iden- mally distributed curve and does not reach steriliz- tical set of mutations as the T. b gambiense series and ing levels, thus allowing relapse. an Angolan isolate (Matovu et al, 2001). Since the achievable CSF dose is close to the MIC of normal sensitive trypanosomes, a mere halving in Many instances of cross-resistance between mela- sensitivity of a line of parasite could mean that a mine-based arsenicals and diamidines have been larger number of individuals fail to accumulate ster- reported (reviewed in Barrett and Fairlamb, 1999). ilizing levels of arsenical in the CSF. As the degree of The fact that the P2 transporter appears to be sensitivity declines, the number of refractory cases responsible for the uptake of both of these classes of will rise in proportion. drug has suggested that transporter alterations could be the basis of cross-resistance. The fact that According to this model, a combination of parasite some lines of pentamidine resistant parasite have no drug-sensitivity and host factors, including the per- reduction in P2 transporter activity was explained meability of the blood-brain barrier to the drug, will by the discovery of additional pentamidine trans- determine whether a case is sensitive or refractory porters (De Koning, 2001) and by the fact that other, to arsenicals. This model is supported by recent data non-transport, related events could underlie pen- from northern Uganda which indicate that alter- tamidine resistance (Berger et al, 1995). The recent ations to the TbAT1 gene that encodes the P2 trans- discovery that loss of the P2 transporter through porter correlate with resistance to melarsoprol gene knockout rather than drug selection leads to a (Matovu et al, 2001). rather modest decrease in sensitivity to melamine-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 99 based arsenicals suggests that other biochemical Elimination is slow, with estimates of 70-80% of the changes must be occurring in resistant cells. These drug reported binding to plasma proteins (Sands other factors contributing to resistance remain to be et al, 1985). The plasma half life is 12 days (but identified. varies), and the drug is probably metabolized by humans since only around 11% is eliminated in the urine. The drug is thought not to cross the blood- Pentamidine brain barrier, although there are reports suggesting that small amounts may enter the CSF. Summary points on pentamidine resistance • Use of the drug in the field against early-stage Intramuscular injection can cause reactions at the sleeping sickness has been extensive, although its site of administration. Other reactions include withdrawal from use as a prophylactic in the mid- hypotension, abdominal pain, hypersalivation, ver- twentieth century might have slowed the appear- tigo, nausea and chest pain. Nephrotoxicity is com- ance of resistance. mon; hypoglycaemia is also seen in significant num- • Anecdotal reports about resistance in the field are bers of patients. Diabetes mellitus may ensue sever- on the increase. al months after therapy. Rapid intravenous injection • Laboratory isolates resistant to the drug have should be avoided as it induces a number of effects been selected. including hypotension, tachycardia, nausea and • There can be cross-resistance to other drugs, vomiting (Anon, 1998). including melamine-based arsenicals and other diamidines, which may to be related to loss of Mode of uptake and action drug uptake via the P2 transporter. Pentamidine is concentrated to high levels by the • Resistance need not necessarily involve cross- parasites. It seems that pentamidine can enter T. bru- resistance. For example, one laboratory derived cei via the same P2 amino-purine transporter which line selected for melarsen resistance which had accumulates melaminophenyl arsenicals (Carter lost the P2 transporter was not cross resistant to et al, 1995). Loss of this transporter can render para- pentamidine. Moreover, another line selected for sites cross-resistant to both diamidines and arseni- pentamidine resistance was not cross resistant to cals. However, some parasites without P2 remain arsenicals or other diamidines. sensitive to pentamidine (Carter and Fairlamb, 1993). In T. brucei, three transporters that can carry • Pentamidine can enter T. brucei via several trans- pentamidine into the cell have been identified porters (P2, HATP1, LAPT1) and it accumulates to (De Koning, 2001). In addition to P2, a high affinity high intracellular levels. Resistance in some cases transporter HAPT1 (K for pentamidine = 36nM) correlates to reduced uptake, but in others m and a low affinity transporter LAPT1 (K for pen- reduced uptake does not appear to be involved. m tamidine = 56 µM) are responsible for the uptake of • The mode of action is not known. pentamidine. This could explain why the P2 deficient line, RU15, which is resistant to melamine-ba- The drug and its use sed arsenicals and some diamidines, was not resist- Pentamidine is an aromatic diamidine that has been ant to pentamidine (Carter and Fairlamb, 1993). in use for treatment of trypanosomiasis for over fifty Moreover, another line selected for pentamidine years (Sands et al, 1985). It is supplied as a white resistance was not resistant to other diamidines powder in 200 mg ampoules. It was developed after (Berger et al, 1995); a better understanding of the the observation that a related compound, synthalin, different routes of uptake for different diamidines is which induces hypoglycaemia in mammals, had desirable. prolific anti-trypanosomal activity. Diamidines actually work directly against the parasites, inde- The mode of action of the drug has not been estab- pendently of their physiological action on the host. lished. As a polycation, the molecule interacts elec- Pentamidine is active against early stages of the trostatically with cellular polyanions, including the gambiense form of sleeping sickness. It is also used unique intercatenated network of circular DNA against antimony refractory leishmaniasis and molecules which make up the mitochondrial Pneumocycstis carinii pneumonia (Sands et al, 1985). genome of all kinetoplastid flagellates termed the kinetoplast. In the case of African trypanosomes, an Maximum plasma concentrations are reached with- interaction with the kinetoplast may appear to be of in an hour of intramuscular injection. Extensive limited interest as these organisms do not have a variation in plasma concentration is found between classical mitochondrial metabolism. However, it is individuals (0.2-4.4 mg l-1 following a 4 mg kg-1 wrong to consider the mitochondrion as inert since injection have been reported). Each daily dose leads some kinetoplast genes are known to be expressed to an increase in residual drug concentration. and the membrane is maintained in an energized

100 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 form indicating that several mitochondrial enzyme • Suramin resistance in T. evansi lines appears to be systems must be active. stable in the field. • Numerous laboratory isolates (T. brucei and T. T. brucei can retain viability when the kinetoplast evansi) have been selected for suramin resistance. has been removed (dyskinetoplastidy). On the other • Most laboratory resistant isolates are not cross- hand, dyskinetoplastid parasites have been shown resistant to other drugs. to be somewhat less sensitive than wild-type cells to • Neither a mode of action nor mechanisms of diminazene (Agbe and Yielding, 1995). Fluorescent resistance are known. analogues of the diamidines, e.g. DB75 and stil- bamidine, accumulate rapidly in the kinetoplast and The drug and its use have also been shown to accumulate in small vesic- Suramin, a colourless polysulphonated symmetrical ular structures in the cytosol. naphthalene derivative, was first used against sleeping sickness in 1922 (Voogd et al, 1993). It is Numerous other potential targets have been pro- useful for the treatment of early-stage infection due posed but none have been verified. Given that the to either T. b. gambiense or T. b. rhodesiense, when drug reaches millimolar concentrations within try- there is no central nervous system involvement. panosomes, it could be that its toxic effect arises Other naphthalene dyes, including trypan red and from inhibition of multiple cellular targets, although trypan blue, were initially developed for their the fact that one resistant line (Berger et al, 1995) marked trypanocidal activity. The drug has recently does accumulate drug to millimolar concentrations been used in clinical trials against hormone-refrac- without adverse effects might indicate that there is a tory prostate cancer and has also been used in the specific target that has yet to be identified. chemotherapy of some helminth infections. During the 1950s, there were reports that treatment failure Resistance in patients infected with T. b gambiense was relative- Pentamidine resistance did not emerge during ly high (around 30% [Neujean and Evens, 1958]). large-scale chemoprophylaxis campaigns in the Consequently the drug lost favour as a treatment for middle part of the twentieth century in west Africa, gambiense sleeping sickness although the factors underlying these treatment failures were never possibly because the drug was withdrawn from identified with any certainty. widescale use in the 1950s thus removing selection pressure. In this regard, it is perhaps of note that one Poor intestinal absorption, and a local irritation if laboratory derived line resistant to pentamidine had given intramuscularly, mean that the drug should be substantially diminished viability in mammals administered by slow intravenous injection (Anon, (Berger et al, 1995). 1998). Dosing at 1 g per week over six weeks main- tains levels at 150-200 mg l-1. Most of the drug binds It is not clear, nor easy to ascertain from available lit- to serum proteins. It does not cross the blood-brain erature, whether melarsoprol resistant field isolates barrier to levels capable of killing trypanosomes in are pentamidine cross-resistant. However, it seems the CSF at doses given during treatment of early- that both pentamidine and melarsoprol enter try- stage disease. Plasma concentrations decline expo- panosomes via the P2 transporter and anecdotal evi- nentially with a half-life of up to 60 days. About 80% dence has indicated that treatment failures with of the dose is eliminated in the urine. pentamidine are growing more common as they are with melarsoprol. Possibly the presence of trans- Nausea, vomiting, urticaria and loss of consciousness porters, in addition to P2, which can accumulate can be immediate side-effects (Anon, 1998). Fever (up pentamidine means that changes to P2 will not to 40oC within a few hours) is common as are photo- underlie pentamidine resistance in the field. phobia and lacrimation. Renal damage can occur sev- Mechanisms for resistance to pentamidine are cur- eral days after treatment. Other adverse reactions rently not known. include exfoliative dermatitis, stomal ulceration, agranulocytosis and, rarely, haemolytic anaemia. Suramin Mode of uptake and action Summary points on suramin resistance The drug is a highly charged molecule containing • Use of the drug in the field against sleeping sick- six negative charges at physiological pH and it ness is not extensive. binds with high avidity to many serum proteins • Few reports about resistance in the field have including low density lipoprotein (LDL), for which been published. trypanosomes have a receptor (Vansterkenburg • Veterinary use was more widespread than human et al, 1993). Suramin accumulates in trypanosomes use in the mid to late twentieth century. relatively slowly and may be taken up bound to

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 101 LDL by receptor-mediated endocytosis, although been reported. Information from these species (par- definitive evidence proving this is absent. The same ticularly T. evansi) might be useful in helping to experiments that showed that uptake might depend ascertain resistance mechanisms in man. on LDL also showed that suramin inhibits LDL uptake and that this inhibitory effect could be the The relative scarcity of reports of suramin resistance cause of suramin’s toxic effect on trypanosomes in sleeping sickness has made it difficult to compile (these organisms get most of their lipids from the field data on this subject. The large incidence of breakdown of exogenous LDL). The highly charged reported treatment failures of T. b gambiense in west nature of the drug enables it to bind to many pro- Africa in the 1950s could have been due to multiple teins through electrostatic interaction. factors of which parasite resistance is just one Consequently, when the drug is tested for inhibition (Neujean and Evens, 1958). Many laboratory of a variety of purified enzymes, it shows activity. derived lines have been selected for resistance to This has led to many hypotheses regarding its mode suramin over the years (since the 1930s). Several of action. studies have focused on T. evansi (Mutugi et al, 1995). One laboratory study pointed to the fact that Treatment of trypanosomes with the drug does lead resistant lines were more difficult to clone and grow to a reduction in the glycolytic rate. This inspired a than wild-type T. evansi. This prompted suggestions study into its inhibitory effect against the enzyme that resistance might not be a stable phenotype glycerol phophate oxidase, which is present in try- (Mutugi et al, 1995). However, T. evansi isolated panosomes but absent from the mammalian host from the Sudan some twenty years after suramin (Fairlamb and Bowman, 1977). Suramin inhibited had been withdrawn from use due to the advent of the enzyme and some textbooks mistakenly report resistance (El Rayah, 1999) was still highly resistant that this enzyme is the target. The discovery of clus- to this drug, indicating that resistance can be very ters of positively charged amino-acids within sever- stable. al of the T. brucei glycolytic enzymes led to the hypothesis that the drug’s action was dependent No evidence for a reduction in drug uptake associ- upon this interaction. However, none of these spec- ated with resistance has been reported and mecha- ulative ideas have been proven and, at this stage, it nisms of resistance are not known. Most laboratory should be emphasized that the drug’s mode of studies, stretching back 70 years or more, have action is not known. failed to identify cross-resistance between suramin and other trypanocides. One study did show that a The drug is highly active against bloodstream forms line selected for resistance to melarsen oxide (33- of the parasite in vitro, but around a hundredfold fold resistance) (Fairlamb et al, 1992) had a nearly less active against procyclic forms of the organisms sixfold decrease in susceptibility to suramin. (Scott et al, 1996). This indicates that either its However, most other studies have concluded that uptake, or its direct or indirect targets, are differen- there is no cross-resistance between suramin and the tially regulated in the different forms of the parasite. melamine based arsenicals or other drugs. Early suggestions that receptor mediated endocytosis did not occur at all in procyclic forms have proven to be incorrect as this form of the parasite Eflornithine does endocytose a number of macromolecules (Liu Summary points on eflornithine resistance et al, 1999). However, specific receptors may be dif- • Use of the drug in the field against sleeping sick- ferent. The fact that procyclic form organisms, ness has not been extensive. unlike bloodstream forms, are not totally dependent • No reports about resistance in the field have been upon glycolysis has also been used to fuel ideas on published. modes of action. • T. b. rhodesiense is innately refractory to eflornithine, possibly due to a shorter half life of the Fang et al (1994) recently re-introduced the old target enzyme ornithine decarboxylase compared notion that the immune system may play a role in to the susceptible T. b. gambiense. the action of suramin, since immunosuppressed • Laboratory isolates resistant to the drug have mice needed higher doses (around threefold) to been derived. clear T. evansi infections. • Model organisms (e.g. Leishmania, Neurospora) resistant to the drug have also been derived. Resistance • In model organisms, resistance has been shown to Field reports on sleeping sickness resistant to relate to different phenomena e.g.: suramin are rare. In animal diseases, however, para- - Increase in ornithine decarboxylase levels (gene sites of the brucei group resistant to this drug have amplification).

102 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 - Decreased drug uptake (loss of basic amino acid the membrane (Erwin and Pegg, 1982). It has been transporter in Neurospora; unknown mechanism reported that uptake of DFMO in T. brucei also behind reduced uptake in procyclic T. brucei). occurs via passive diffusion across the plasma mem- • T. brucei resistant to DFMO have not been report- brane (Bitonti et al, 1986; Bellofatto et al, 1987). ed to be cross-resistant to other drugs. These observations were based on the fact that • The mode of action of the drug relates to its inhi- uptake appeared to be unsaturable over a wide bition of ornithine decarboxylase. A functional range of DFMO concentrations and that internal immune system is required to kill cells in vivo. concentration of drug equilibrated with external Whether it is simply loss of polyamines or other concentration. Other features reported in these stud- indirect effects, e.g. increase in decarboxylated ies, e.g. temperature sensitivity of uptake, might be S-adenosyl methionine and inappropriate methy- interpreted as evidence for transport although the lation, that is behind cytotoxicity is not clear. authors did not consider this. A separate report did Uptake has been reported to be via passive diffu- note a saturable process typical of transport-associ- sion in bloodstream forms and via carrier mediat- ated uptake in procyclic organisms with a Km of ed uptake in procyclic forms, although further 244 µM (Phillips and Wang, 1987). In the yeast studies on this question are needed. Neurospora crassa, a basic amino-acid transporter has been implicated in uptake of DFMO (since this The drug and its use transporter is lost in strains selected for resistance to Eflornithine, or D,L-α-difluoromethyl ornithine the drug) (Davis et al, 1994). None of the studies in (DFMO), is an analogue of ornithine which acts as a trypanosomes have indicated that DFMO shares an specific suicide inhibitor of the enzyme ornithine uptake mechanism with ornithine, arginine or decarboxylase (ODC). It was developed as an anti- lysine. The mode of uptake into trypanosomes cancer reagent, however, it remains at the trial stage remains uncertain. against neoplastic disease. The drug also has activity against sleeping sickness caused by T. b gambiense, DFMO has similar affinity for both the mammalian even in the late CNS-involved stage. It was regis- and trypanosomal ornithine decarboxylases. Its tered in the USA in 1990 and the UK in 1991 (Pépin specificity against the parasite apparently arises and Milord, 1994). By 1995 it was registered in seven because T. b. gambiense ODC is degraded within the African countries. Fourteen daily intravenous injec- cell and replenished at a rate much slower than its tions of 400 mg per kg of body weight are recom- mammalian counterpart (Phillips et al, 1987). Thus, mended (Anon, 1998). It has also been licensed for a pulse of DFMO can deprive trypanosomes of ODC use as a topical application to prevent facial hair and polyamine synthesis for a prolonged period growth and is now marketed for this purpose compared with mammalian cells, leading to a cessa- (Hickman et al, 2001). Fifty four per cent of the dose tion of growth. becomes bioavailable after oral administration (Anon, 1998). The mean half-life in plasma follow- Inhibition of ornithine decarboxylase has other ing intravenous injection is 3 hours, with 80% of the results besides a reduction in putrescine and further drug excreted unchanged in urine after 24 hours. polyamine biosynthesis. For example, it leads to an Little of the drug binds to serum proteins. increase in cellular levels of S-adenosyl methionine, Immediately after a 14-day course, the CSF to plas- which might have toxic effects (Byres et al, 1991). ma ratio is 0.91 in adults and 0.58 in children. Inappropriate methylation of proteins, nucleic Children retain less of the drug than adults. acids, lipids and other cell components is thus being implicated. Few side effects are apparent although anaemia and other blood cell reductions (leukopenia, thrombocy- Trypanothione is a glutathione-spermidine conju- topeania) are known. Diarrhoea is a common prob- gate unique to trypanosomatids and it plays a criti- lem to those on oral eflornithine. Convulsions, fever cal role in maintenance of cellular redox potential and vomiting have also been reported in low num- (Fairlamb and Cerami, 1992). Trypanothione levels bers of cases (Anon, 1998). are diminished after DFMO treatment, which might render parasites more vulnerable to oxidative stress. Mode of uptake and action A functional immune system is required to kill the Eflornithine is a specific suicide inhibitor of the growth-arrested trypanosomes (De Gree et al, 1983). enzyme ornithine decarboxylase (ODC), which is a It has also been reported that T. brucei lacks key enzyme in the biosynthesis of polyamines polyamine transporters, rendering the parasite aux- (McCann and Pegg, 1992). Some early studies in otrophic for polyamines (Fairlamb and Le Quesne, mammalian cells indicated that DFMO uptake was 1997). Conversely, many mammalian cells can scav- a passive process involving simple diffusion across enge polyamines from plasma using transporters, allowing them to bypass the lack of endogenous

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 103 biosynthesis while T. brucei cannot tolerate this situ- boxylase activity associated with an amplification in ation. copy number of the gene (probably associated with amplification of an episome) (Sanchez et al, 1997). In order to be effective against sleeping sickness, the Elevated putrescine uptake in L. infantum exposed drug needs to be given in large doses. An addition- to DFMO has also been reported (Balana-Fouce et al, al drawback is the drug’s lack of activity against 1991). rhodesiense sleeping sickness (Iten et al, 1995), which appears to contain an ornithine decarboxylase that Increased ornithine decarboxylase activity (associat- is relatively quickly turned over (Iten et al, 1997). A ed with a rise in transcript levels but not, this time, seven-day course appears to be somewhat less effi- gene copy number) has also been reported in arsen- cacious than the 14-day course; however, the cost- ite resistance, associated with increased trypanoth- benefit ratio appears to favour the shorter course ione biosynthesis in Leishmania tarentolae (Haimeur (Pépin et al, 2000). et al, 1999). The significance of this observation lies in the fact that, should a similar route to arsenical Resistance resistance be possible in T. brucei, the possibility of No reports of resistance in the field were found in cross-resistance to DFMO would materialize. the literature, which is not surprising since the drug However, it should be stressed that so far a similar has not been widely used in very large-scale treat- mechanism has not been noted in trypanosomes, ment regimes. T. b. rhodesiense appears to be innate- and to date, lines selected for resistance to DFMO ly less susceptible to the drug than T. b. gambiense were not cross-resistant to other trypanocides. (Iten et al, 1995) since it has a higher overall ODC activity and the enzyme has a shorter half life than the gambiense counterpart (Iten et al, 1997). Another Nifurtimox explanation involving relative levels of S–adenosyl methionine, which accumulate to lower levels in The drug and its use refractory but not sensitive cells treated with Nifurtimox was originally licensed for use against DFMO, has been proposed. Alternative explana- South American trypanosomiasis. The drug con- tions related to drug uptake, or polyamine uptake tains a nitro group which is central to its activity. It allowing bypass of the inhibitory effect, have not has also been used in trials, with only limited suc- been subject to extensive analysis. cess (50-80% cure), against T. brucei gambiense in West Africa, although since it is apparently active In procyclic cells selected for DFMO resistance, putrescine uptake was noted to be three-four times against melarsoprol refractory parasites it may still higher than in wild-type lines (Phillips and Wang, be used and as treatment failures with arsenical 1987). Putrescine at >1 mM allowed parasites to sur- increase (Pépin et al, 1992). vive DFMO treatment in vitro while 0.1 mM did not (Phillips and Wang, 1987). Moreover, T. brucei para- Serum levels are reportedly low when given orally, sites from which the ornithine decarboxylase gene peaking one-three hours after administration. The had been removed were also viable and capable of drug can accumulate across the blood-brain barrier growth provided external putrescine was abundant (Anon, 1998). (Li et al, 1988) (far more abundant than in mammalian serum, where it is around 220 nM [Cooper Toxic effects to the central nervous system and et al, 1978]). A similar result was obtained with peripheral nervous system have been reported. Leishmania parasites from which ODC had been knocked-out, i.e. putrescine enabled bypass of Mode of uptake and action DFMO inhibition of polyamine synthesis, and also No reports about the mechanism of action against enabled the cells to dispose of accumulated T. brucei could be found in the literature, although S–adenosyl methionine (Jiang et al, 1999). reports relating to activity against T. cruzi exist. Uptake of nifurtimox into Trypanosoma cruzi has Lines selected in the laboratory for resistance to the been reported to occur via passive diffusion across drug have been studied. Reduced drug accumula- the plasma membrane (Tsuhako, et al.,1991). Studies tion was noted in procyclic parasites resistant to have not yet been extended to T. brucei but it is also eflornithine (Bellofatto et al, 1987). However, likely to enter these cells via passive diffusion. whether this was due to decreased uptake or increased efflux was not determined. Nifurtimox is a nitrofuran compound. One electron reduction of the nitro-group generates a potent free A Leishmania line selected for resistance to DFMO radical which may interact with cellular con- was shown to have an increase in ornithine decar- stituents or generate reduced oxygen metabolites

104 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 believed to cause death of the parasite (Docampo The drug can be given orally. Peak plasma levels fol- and Moreno, 1984). The reduction potential of the lowing a 100 mg kg-1 dosing (Enanga et al, 2000) in compound (-260 mV) is such that it is relatively eas- primates yielded plasma levels of between 0.2 µg ml ily reduced in many cell types. The specificity and 46 µg ml-1 24 hr after dosing. The drug or its towards the parasite (which is not great, nifurtimox metabolites can be found in the CSF at levels 5.5%- being quite toxic to mammals) is thought to be asso- 10.6% of those in plasma. The elimination half time ciated with its being more readily reduced by the was around 2.5 hours. Suramin substantially parasite than the host cells. Moreover, mammalian increases the half-life of the drug and also increases cells may have better protection against oxidative the amounts which can accumulate across the damage. Specific targets or enzymes capable of blood-brain barrier. reducing the drug cannot be ruled out. Some pathways which might lead to the preferential reduction No published reports of the effect of megazol on of these compounds in trypanosomes have been humans exist. When the drug was administered to studied. An intriguing hypothesis was that trypan- two patients with Chagas’ disease, anecdotal evi- othione reductase might be responsible for the dence indicated there was little toxicity. However, reduction (Henderson et al, 1988). Certainly, a num- the compound is positive in Ames’ tests (Ferreira ber of nitro-containing compounds can act as “sub- and Ferreira, 1986) and this fact alone appears to versive-substrates” for the enzyme. However, nifur- have served as a deterrent to further development, timox was one of the less successful substrates in spite of the excellent safety record of another (Henderson et al, 1988). and it is unlikely that try- Ames’ test positive anti-protozoal 5-nitroimidazole, panothione reductase-mediated nitro-reduction metronidazole. Toxicological studies in mammals underlies the activity of the clinically used nitro- should be performed under recognized good labo- heterocyclics. ratory practice (GLP) conditions as a matter of Resistance urgency. If toxicity proves to be no worse than for nifurtimox, then the case for pursuing the drug as a African trypanosome lines selected for resistance to potential reagent for clinical use would be strong. nifurtimox have not been reported in the literature. It is not clear why treatment failure is high. Further pharmacokinetic studies should be made to deter- Mode of uptake and action mine the degree to which the drug reaches the CSF. Megazol possesses part of the motif recognized by the P2 amino-purine transporter which is responsi- Trypanosoma cruzi isolates show various levels of ble for the uptake of several anti-trypanosomal sensitivity to the drug (Filardi and Brener, 1987). drugs (Barrett et al, 2000). Should this drug share the There appears to be a correlation between drug P2 transporter as a portal of entry, it would be of uptake and sensitivity (with lines accumulating limited use against arsenical resistant parasites. least drug being least sensitive to it). No systematic However, strains of parasite lacking the P2 trans- study on susceptibility of different sub-species or porter and resistant to other drugs which use this strains of T. brucei have yet been conducted. portal of entry into trypanosomes, were not cross- Interestingly, another nitroheterocycle called mega- resistant to megazol drugs (Barrett et al, 2000). zol (discussed below) was equally active against Uptake of radiolabelled megazol revealed that this lines of T. cruzi showing different sensitivities to drug, although capable of interacting with the P2 nifurtimox (Filardi and Brener, 1987). transporter, enters cells predominantly via passive diffusion (Barrett et al, 2000).

Megazol The mode of action of the drug is not clear. The fact that a nitro group is central to its function does not The drug and its use necessarily imply that the mode of action will be the Megazol is a 5-nitroimidazole which has good effi- same as for nifurtimox. Indeed, its reduction poten- cacy against both T. cruzi (Filardi and Brener, 1987) tial of -438 mV (Viodé et al, 1999) is far lower than and T. brucei (Enanga et al, 2000). Its synthesis was that of nifurtimox (-260 mV), and 5-nitroimidazoles first reported in 1968 (Berkelhammer and Asato, are not normally reduced by aerobic cells. However, 1968). The activity of the drug against African try- megazol is susceptible to nitro-reduction in the panosomes is striking. A single dose clears parasites presence of several enzymatic systems including from the blood of rodents and a primate model. some found in T. cruzi extracts. How it exerts a lethal Administration of the drug following a single dose action against parasites, however, is not certain of suramin cleared parasites from the CSF of an although it seems likely that trypanosomes possess infected primate (B. Enanga, personal communica- a specific enzyme capable of reducing this com- tion). pound.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 105 Resistance T. b. gambiense is much more difficult to grow in Both procyclic and bloodstream forms of the para- rodents, although Mastomys rats do allow prolifera- sites have been selected for resistance to megazol in tion of some isolates (only 20% of T. b. gambiense iso- the laboratory (Enanga & Barrett, unpublished lates from a recent study in northern Uganda could results) (procyclic forms with >100-fold resistance, be grown in Mastomys - Matovu, personal commu- bloodstream forms with about 20-fold resistance to nication). Standardization of rodent protocols is the drug). Modest levels of cross-resistance to the important, i.e. similar parasite numbers should be other nitroheterocycle, nifurtimox, were apparent inoculated, similar drug dosing post-inoculation (about 7-fold in both cases). Even more modest lev- should be given, and there should be similar follow els of cross-resistance to diamidines (2 to 4-fold) and up with regard to checking mice for parasitemia. melamine based arsenicals (3 to 5-fold) were also observed. Preliminary evidence suggests that there The protocols established at the Swiss Tropical is not a role for an efflux pump or for increased lev- Institute for each of these procedures may be rec- els of trypanothione in the procyclic lines. The ommended as the standards which should be fol- mechanism of resistance is currently unknown but lowed, although consensus agreement is required under investigation. for this and other procedures may be considered (for example those recommended for testing of vet- DETECTION OF DRUG RESISTANCE erinary trypanocides in a recent PAAT document) Treatment failure and drug resistance are by no (Geerts and Holmes, 1998). means synonymous. Resistance is best defined as “the heritable, temporary or permanent loss of the Drug Sensitivity Testing in Vitro initial sensitivity of the population of microorgan- It is also possible to cultivate some lines in vitro isms against the active substance” (Schnitzer and using established culture media. Laboratory adapt- Grunberg 1957). It is essential that drugs are admin- ed lines are relatively easy to establish in culture. istered according to the recommendations put for- However, many field isolates, particularly of ward based on regimens optimal for activity, T. b gambiense, do not adapt readily to in vitro culture although further studies on optimizing the dose are and few reports of axenically cultured T. b. gambiense required for most drugs. This is particularly impor- could be found. Lines which have successfully been tant in late-stage sleeping sickness where the quan- passed through Mastomys can proliferate in rich tity of drug that accumulates in the extravascular medium (containing human serum) over a mono- compartment may not extend far beyond the MIC layer of Mastomys embryonic fibroblast cells (Brun required to kill the parasites. Treatment failure can et al, 1989). Until better conditions for the in vitro come about for a number of reasons including cultivation of T. b. gambiense are established, it is administration of sub-curative doses of drug or host important that all drug tests against all isolates factors including metabolism and distribution with- should be performed under the same conditions. If in the body. T. b. gambiense is to be compared with T. b. rhodesiense, it is of limited use to use different cultivation It is important to determine whether patients who systems to determine drug sensitivity. This is partic- have not cleared all parasites after treatment actual- ularly the case when using mammalian cell mono- ly carry drug resistant trypanosomes. Therefore layers as part of the culture system since these may tests for resistance should be performed. affect the drug and its activity against parasites. Isolation of Parasites, Propagation and Drug Sensitivity Testing in Rodents Molecular Approaches to the Identification of Drug Resistance Blood (or CSF) can be taken from infected patients Alterations to the gene TbAT1 that encodes the P2 and injected directly into a suitable rodent model. In the case of T. b. rhodesiense, standard laboratory transporter have been identified, (Maser et al, 1999) white mice or rats are adequate for this process. and many parasite lines which are less sensitive than High parasitemias are readily achieved in these normal to melarsen-based arsenicals possess similar hosts and highly parasitemic blood isolated from mutations (Matovu, personal communication). these hosts can be isolated, mixed with a suitable Particular mutations appear to have been selected on cryopreservant, and frozen in liquid nitrogen. multiple independent occasions. This opens the pos- Preserved stocks can be re-injected into rodents and sibility of using a polymerase chain reaction (PCR) then treated with trypanocidal drugs over a range of based approach to detect the presence of particular concentrations to determine the MIC and effective mutant alleles which correlate to drug resistance. This approach will be limited if different types of dose 50 (ED50) of drug useful against these parasites in vivo. Protocols must be standardized for this mutation affect the status of expression of the TbAT1 purpose. gene. More studies to investigate the frequency of

106 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 particular mutations that correlate to resistance are pentamidine appears to have alternative routes of needed if such a test is to be useful. The possibility entry into the cells (De Koning, 2001). Nevertheless, that different types of mutation could affect the P2 pentamidine should also be administered prudent- transporter in such a way as to reduce sensitivity to ly, and curative doses ensured, to restrict the oppor- drug, and the possibility that other biochemical tunities for selection of resistance to this drug which changes not related to the P2 transporter could also might cause cross-resistance to melarsoprol. induce resistance to arsenicals, means that a simple PCR based test might produce an unacceptably high A Possible Role for Veterinary number of false negative results. Trypanocide Use in the Selection of Melarsoprol Resistance in Sleeping GUIDELINES ON THE DELAY OF THE Sickness DEVELOPMENT OF DRUG RESISTANCE Microbes selected for resistance to drugs during Recent evidence indicates that the rise in the num- unsupervised treatment of livestock have become a ber of sleeping sickness cases proving refractory to major route of introduction of resistance and resist- melarsoprol treatment (Legros et al, 1999) is at least ance genes into human pathogens. partially caused by the emergence and spread of parasites resistant to the drug in the field (Matovu It seems that cross-resistance between diminazene et al, in press). However, the global quantities of and melamine based arsenicals occurs more consis- melarsoprol administered are far lower than, for tently than between the latter and pentamidine example, those for chloroquine in malaria prophy- (Barrett and Fairlamb, 1999; Barrett et al, 1995). This laxis, or for many of the antibiotics to which resist- could be because diminazene appears to enter pre- ance is now widespread. It is also recommended dominantly via the P2 transporter and not via the that melarsoprol is administered in a clinical setting, alternative transporters that carry pentamidine thus improving the likelihood that a full curative (De Koning, 2001). The emergence of diminazene- dose is given. As well, the dynamics of transmission melarsoprol cross-resistance can have profound via tsetse flies make it far less likely that human try- implications for the development of drug resistant panosomes will be transmitted between human sleeping sickness in the field. hosts at the same frequency as are Plasmodium parasites by anopheline mosquitoes. Therefore, it is per- To date, no studies have been conducted to assess haps surprising that resistance to the drug has whether a drug resistant parasite selected in an ani- emerged, albeit apparently with a substantially mal can be transferred to humans – this is a specu- slower time of onset than with chloroquine (chloro- lative scenario. However, it is clear that diminazene quine was introduced in 1945 and melarsoprol in is administered to trypanosome infected cattle 1949). These factors also need to be set against the (Geerts and Holmes, 1998) and that several generic fact that the quantities of melarsoprol reaching the versions of this product have appeared on the mar- parasites within the CSF are only marginally higher ket which are of highly variable quality. Ad-hoc, than the MIC of the drug, so that parasites which are unsupervised administration of sub-curative doses only slightly less sensitive than wild-type to drug of diminazene to cattle is reportedly widespread, may be selected with relative ease. While the param- and resistance to diminazene (in T. congolense and eters that can lead to selection of resistance are com- possibly other trypanosome species) is rife in parts plex, there is no doubt that it is critical to ensure of Kenya and elsewhere. that the recommended dose of the drug is given to every patient. Up to 20% of trypanosomes isolated from cattle in western Kenya and parts of Uganda are infectious to The potential of cross-resistance between pentami- humans (MacLeod et al, 2001). Therefore, human dine and melarsoprol must also be considered. This infectious trypanosomes are present in areas where is because both of these trypanocides can enter conditions for selection of resistance to diminazene T. brucei via the P2 nucleoside transporter (Barrett are prevalent. Indeed, one isolate from south-eastern and Fairlamb, 1999). Selection of resistance to one Uganda was clearly resistant to diminazene and drug could therefore, in principle, lead to cross- isometamidium (Matovu et al, 1997). It has yet to be resistance to the other. Interestingly, the relatively demonstrated whether resistance is associated with widescale use of pentamidine as a prophylactic in loss of the P2 transporter, or whether cross-resistance west Africa up until the 1950s appears not to have to melarsoprol occurs. In spite of this lack of data, selected for resistance. Some laboratory derived there are reasons for concern about the selection of lines have been shown to be pentamidine-melarso- drug resistant parasites in animals which can then be prol cross-resistant but this has not been shown to transferred to human infections. It should be stressed be so in all cases. This could be due to the fact that at this point that the wider host range of T. b. rhode-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 107 siense makes it more likely that such a scenario might References arise for this form of sleeping sickness more readily Agbe A, Yielding KL. Kinetoplasts play an impor- than for the gambiense form of the disease. tant role in the drug responses of Trypanosoma brucei. Journal of Parasitology, 1995, 81:968-973. Studies should be conducted to assess the degree to which drug resistance selected in animals can be Anon. Control and surveillance of African trypanosomi- transferred to humans. In the meantime, efforts asis. WHO Technical report, 1998. aimed at limiting the spread of drug resistance among trypanosomes in cattle should be given Balana-Fouce R, Escribano MI, Alunda JM. additional impetus so as to minimize the risk of this Putrescine uptake regulation in response to alpha- occurring. difuoromethylornithine treatment in Leishmania infantum promastigotes. Molecular and Cellular Biochemistry, 1991, 107:127-133. Widespread implementation of the rationally derived ten-day administration protocol for melar- Barrett MP. Problems for the chemotherapy of soprol in gambiense patients (Burri et al, 2000) human African trypanosomiasis. Current Opinion in might also reduce the probability of transmission of Infectious Diseases, 2000, 13:647-651. resistant lines. Barrett MP et al. Uptake of the nitroimidazole drug GUIDELINES ON THE CONTROL OF megazol by African trypanosomes. Biochemical DRUG RESISTANCE ONCE PRESENT Pharmacology, 2000, 59:615-620. Melarsoprol resistance appears to be on the increase. However, the only other drug licensed to treat late- Barrett MP, Fairlamb AH. The biochemical basis of stage sleeping sickness, namely eflornithine, is not arsenical-diamidine crossresistance in African try- currently available, although it seems that Aventis panosomes. Parasitology Today, 1999, 15:136-140. will produce it for the coming five years so melarsoprol refractory patients should be treated with this Barrett MP et al. A diamidine-resistant Trypanosoma drug if they are infected with gambiense parasites. equiperdum clone contains a P2 purine transporter If eflornithine is not available, then melarsoprol with reduced substrate affinity. Molecular and refractory patients can be treated with recently rec- Biochemical Parasitology, 1995, 73:223-229. ommended doses of nifurtimox (Bisser et al, 2000). Combination chemotherapy can also be considered Bellofatto V et al. Biochemical changes associated and, in this regard, melarsoprol/eflornithine, melar- with D,L-alpha-difluoromethylornithine uptake soprol/nifurtimox and nifurtimox/eflornithine have and resistance in Trypanosoma brucei. Molecular and all been used to treat drug resistant T. b gambiense Biochemical Parasitology, 1987, 25:227-238. (Jennings, 1993). Berger BJ, Carter NS, Fairlamb AH. Characterisa- For early-stage disease, resistance to suramin has tion of pentamidine-resistant Trypanosoma brucei not yet been identified. It needs to be confirmed brucei. Molecular and Biochemical Parasitology, 1995, whether contemporary lines of T. b gambiense are 69:289-298. susceptible to suramin but, if there is no pentamidine-suramin cross-resistance, then one drug Berkelhammer G, Asato G. 2-Amino-5-2-imidazolyl- should be suitable to treat parasites resistant to the 1,3,4-thiadazole. A new antimicrobial agent. Science, other drug. Melarsoprol can also be used to treat 1968, 162:1146. early-stage disease which is resistant to suramin, as Bisser S et al. New therapeutic regimen for sleeping can eflornithine, if available. Caution should be sickness. Description, results and lessons from the exercised in treating melarsoprol refractory para- clinical trial established in Bwamanda (Equateur, sites with pentamidine, or pentamidine refractory RDC). Proceedings of the trypanosomiasis and leishma- parasites with melarsoprol, since cross-resistance niasis seminar and 3rd COST-B9 meeting in anti-proto- due to the lack of the P2 transporter is a possibility. zoal chemotherapy. Bruges, Belgium, 28-31 May, 2000. Acknowledgments Bitonti AJ et al. Uptake of alpha-difluoromethylor- I am grateful to Dr Enock Matovu for sharing nithine by Trypanosoma brucei brucei. Biochemical unpublished results, also to Drs Reto Brun, Mike Pharmacology, 1986, 35:351-354. Turner, Harry De Koning, Dominique Legros and Anne Moore for critical reading of this manuscript. Brun R, Baeriswyl S, Kunz C. In vitro drug sensitivity of Trypanosoma gambiense isolates. Acta Tropica, 1989, 46:369-376.

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TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 111 III HUMAN AFRICAN that more than 15% of all patients in the nervous TRYPANOSOMIASIS system phase of the disease, treated in Côte (Original manuscript in French) d’Ivoire, can be excluded from the fatal side-effects of arsobal therapy. Felix Doua Clinical Research Project into Trypanosomiasis, BP 1425 Treatment and Clinical Trials using Daloa, Côte d’Ivoire Melarsoprol Treatment regimens using melarsoprol in countries INTRODUCTION where sleeping sickness is endemic date back to In the last 20 years there has been a resurgence of colonial times and consist of the administration of interest in human African trypanosomiasis (HAT) three to four series of two to four injections of melar- following a recrudescence of the disease in most soprol spaced out at seven to ten day intervals. countries of sub-Saharan Africa. Although there are These regimens, developed on an empirical basis, thousands of new cases every year in countries such have helped control HAT epidemics but could be as the Democratic Republic of the Congo (DRC), the cause of the growing number of recorded treat- Angola and Uganda, no noteworthy progress has ment failures. Recent pharmacokinetic data on been recorded in the development of new drugs to arsobal, obtained by bioassay and computer simula- fight the condition. tions (Burri et al, 1993), have shown that an alternative, more rational, therapy regimen, consisting of The drugs currently used in the treatment of sleep- ten injections at low doses, can be used without ing sickness were first marketed in the 1950s. increasing the risk of fatal accidents. The new regi- Pentamidine and suramin are the drugs of choice men (Burri et al, 1995) is currently under clinical for the early-stage disease, while melarsoprol evaluation in seven African countries. (arsobal), despite its toxicity, is the drug of choice for the late-stage disease, when the central nervous sys- Treatment and Clinical Trials using DFMO tem is involved. However, a rise in treatment fail- DFMO (ornidyl, eflornithine) is an irreversible spe- ures with melarsoprol has been noted in the last few cific inhibitor of ornithine-decarboxylase, a key years (Ginoux et al, 1984; De Gros et al, 1999), there- enzyme in the synthesis of polyamines, which are by hampering efforts to combat sleeping sickness physiological substances implicated in cell multipli- despite the development of sensitive diagnostic cation (Sjoerdsma et al, 1984). tools and versatile low-cost anti-vectoral control methods such as insecticide impregnated screens. Clinical trials carried out using DFMO show that a seven-day regimen is effective in treatment of Difluoromethylornithine (DFMO, ornidyl, eflor- T. b. gambiense HAT. The side effects, recorded in nithine) has been undergoing clinical trials in the parenteral DFMO administration, are generally re- last two decades but is not yet on the market versible (Doua et al, 1987; Taelman et al, 1987) and because its cost is considered prohibitive for user the relapse rates after treatment are normally about countries. There is therefore an urgent need to use 1% (Doua et al, 1993). However, the cost of DFMO better the available trypanocides and to develop remains a limiting factor to its wider use. Clinical new ones that are cheap, well tolerated and effective trials are ongoing in Côte d’Ivoire, to determine the at every phase of the disease. efficacy after oral administration, and the pharmacokinetics. The intended result is to develop an oral formulation of the product at an affordable cost to TREATMENT AND CLINICAL TRIALS patients. CONDUCTED ON THE T. B. GAMBIENSE FORM OF SLEEPING SICKNESS Treatment and Clinical Trials using Nifurtimox Treatment and Clinical Trials using Pentami- This use of nifurtimox for the treatment of T. b. gam- dine biense HAT has not been studied in large-scale clini- Pentamidine, an aromatic diamidine, has a success cal trials, although results obtained with four late- rate of 95% when used for early stages of T. b. gam- stage patients in the DRC show that the product is biense HAT (Doua et al, 1993), and of 94% when effective but very toxic (Jansens et al, 1977). used for patients suffering from the early nervous system phase (Doua et al, 1996). These results indi- CONCLUSIONS AND cate that pentamidine passes the blood-brain barri- RECOMMENDATIONS er and is capable of halting the multiplication of In the last 20 years there has been rapid develop- trypanosomes in cerebrospinal fluid. This suggests ment of both serological and parasitological HAT

112 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 diagnostic techniques, but in 2001, treatment of the Doua et al. Human tryponosmiasis in the Ivory disease is still based only on pentamidine and Coast: therapy and problems. Acta tropica, 1993, suramin for the early stages of the infection, and on 54:163-168. melarsoprol for the central nervous system stages. Treatment schedules with these trypanocides were Doua et al. Efficacy of pentamidine in the treatment derived empirically, without pharmacokinetic stud- of early late stage trypanosoma brucei gambiense try- ies. Thus, the treatments applied to patients vary panosomiasis. American journal of Tropical Medicine from one country to the next and sometimes from and Hygiene, 1996, 55:586-588. one treatment centre to another in the same country, which only aggravates the high number of failures Ginoux et al. Les échecs du traitement de la try- observed, in particular with melarsoprol, the drug panosomiase à T. b. gambiense au Congo. [Failures of of choice for HAT. T.b. gambiense trypanosomiasis treatment in the Congo]. Médecine tropicale, 1984, 22:149-154. DFMO, which has given rise to great hopes for the treatment of T. b. gambiense HAT, still has not been Jansens et al. Clinical trials with Nifurtimox in commercialized because of its cost. To make efforts human trypanosomiasis. Annales de la Société Belge to control HAT more effective, and to make for de Médecine Tropicale, 1977, 57:475-479. rational treatment of patients, we recommend the following: Sjoerdsna A, Schecter PJ. Chemotherapeutic impli- • Encouragement of research aimed at developing cations of polyamine biosynthesis inhibition. Cli- new, effective, well-tolerated, cheap trypanocides. nical Pharmacology and Therapeutics, 1984, 35:287-300. • Harmonization of treatment schedules for HAT, particularly those for melarsoprol. Taelman H et al. Difluoromethylornithine, a new ef- • Conduct of multicentre clinical trials using pen- fective treatment of Gambian trypanosomiasis. Re- tamidine for patients in the early nervous system sults in five patients. The American Journal of stage of HAT, with a view to evaluating its effica- Medicine, 1987, 82:607-614. cy in different HAT foci. • Conduct of multicentre clinical trials using orally administered DFMO, after the current study on pharmacokinetics is completed. • Conduct of multicentre clinical trials using melarsoprol-DFMO combination in patients suffering relapse after treatment with melarsoprol. • Introduction of nifurtimox for the treatment of HAT cases that are resistant to melarsoprol.

References Burri C et al. Pharmacokinetic properties of the trypanocidal drug melarsoprol. Chemotherapy, 1993, 39:225-234.

Burri C et al. Alternative application of melarsoprol for treatment of T.b. gambiense sleeping sickness. Annales de la Société belge de médecine tropicale, 1995, 75:65-71.

De Gros et al. Echecs thérapeutiques du melarsoprol chez des patients traités au stade avancé de la tryponoso- mose humaine africaine à T. b. gambiense [Therapeutic failures of melarsoprol among patients treated at an advanced stage of T. b. gambiense HAT in Uganda]. 25th CISRLT, programme provisoire 1999, pp. 3.

Doua et al. Treatment of human late-stage gambiense trypanosomiasis with alpha-difluoromethylornithine (eflornithine). Efficacy and tolerance in 14 cases in Côte d’Ivoire. American journal of tropical medicine and hygiene, 1987, 37:525-533.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 113 IV TREATMENT AND ed to be more than US$100 per adult patient (WHO CLINICAL STUDIES 1998). In addition, sleeping sickness patients are almost always from poor rural backgrounds within Martin Odiit their countries (Okia et al 1994). Drugs for the treat- Livestock Health Research Institute, P.O. Box 96, Tororo, ment of sleeping sickness are not readily available in Uganda drug stores and are most often procured by the governments and non-governmental organizations SUMMARY through the assistance of the World Health Organi- Diagnosis and treatment of sleeping sickness zation. Stocks of drugs are carefully monitored at remains the major control strategy of the disease. national levels. However, it is conceivable that, in The current diagnostic kits are insensitive, the diag- the event of an epidemic, it may not be possible to nostic and treatment centres inadequately equipped have enough drugs immediately available, a con- and staffed, and the patients poor and often in inac- straint already experienced by some African coun- cessible rural locations. The drugs used for the treat- tries during sleeping sickness epidemics. Most drug ment of human African trypanosomiasis are toxic, companies appear to be reluctant to produce drugs necessitating a positive demonstration of parasites, for sleeping sickness because they will not obtain including the invasive, painful lumbar punctures returns that are adequate to finance further drug for disease stage determination. The treatment of development (Barrett, 2000; Stephenson and the disease is lengthy, parenteral and costly, and the Wiselka, 2000). drugs used toxic and not readily available. These traditional problems of sleeping sickness are com- SLEEPING SICKNESS CONTROL pounded by the emergence of drug resistance to Sleeping sickness control has traditionally been car- melarsoprol in T. b. gambiense patients, the increas- ried out through case detection and treatment, and ing uncertainty of sustained production of drugs for through vector control when epidemics occur sleeping sickness due to economic justification, and (WHO, 1998). Early diagnosis and treatment is the the increasing prices of some drugs due to their goal of control programmes, but is often hampered newly found use for HIV treatment. Clinical studies by the paucity of resources required to implement that address these problems which face the major these programmes. Most programmes of sleeping control strategy for sleeping sickness are urgently sickness control are donor dependent because the needed. For melarsoprol treatment of T. b. gambiense costs of sleeping sickness interventions are beyond late-stage patients, studies on reduction in length of the budgets of the affected countries. The need for treatment schedules, and therefore of costs, and cheaper control strategies is therefore a priority. improved compliance, are in the advanced stages. Decentralization of services, including health servic- Research into cost-effective and sustainable strate- es, is being advocated. In some countries, planning gies of improving diagnosis and treatment are nec- and implementation of the sleeping sickness control essary to reduce the level of under-reporting that is programmes has been moved to the district level. associated with certain death of sleeping sickness However, current sleeping sickness control strategies patients if untreated. This scientific working paper are expensive and often complicated, requiring qual- reviews some of the current problems of treatment ified personnel. Research into the roles of the nation- of sleeping sickness and suggests clinical studies al and sub-national levels in the decentralization of that may address these problems in the near future. sleeping sickness control should be reviewed.

INTRODUCTION DIAGNOSIS Human African trypanosomiasis (sleeping sickness) Detection of the parasite in gland or lymph node is found in the tsetse belt of tropical Africa, where it aspirate or blood is always followed by a lumbar is estimated that over 50 million people live (WHO, puncture to determine whether there is involvement 1998). Approximately 45 000 cases are reported of the central nervous system (CNS) (also known as annually, though it is believed that 300 000 persons the late stage), because drugs used when the CNS is are infected each year (WHO, 1998). There are two involved are different from those used when the types of sleeping sickness, caused by: Trypanosoma parasite is still restricted to the haematolymphatic brucei gambiense, found in west and central Africa, system (also known as the early stage). However, and by T. b. rhodesiense, reported in east and south- most of the currently used confirmatory diagnostic ern Africa. The gross national product per capita for techniques such as the thick blood smear and the the sleeping sickness affected countries (< US$500) haematocrit centrifugation test have poor sensitivity ranks them as amongst the least developed coun- (WHO 1998). The polymerase chain reaction (PCR) tries of the world (World Bank, 2000). However, the has been found to be very sensitive and specific costs of treatment for sleeping sickness are estimat- (Kyambadde et al, 2000; Penchenier et al, 2000), but

114 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 it is rather complicated and expensive for applica- series of injections each separated by an interval of tion as a routine diagnostic test, which restricts its one week, are used (WHO, 1998). One regime starts use to research laboratories and referral hospitals. with 2.5ml and the other with 0.5ml, but both total 35 mls of a 36g/litre solution of melarsoprol. The criteria used for determination of CNS involvement are as follows: cell counts above 5 cells/mm3 The most serious side effect of melarsoprol is reac- or the demonstration of trypanosomes or protein tive encephalopathy occurring between the third levels above normal (25mg per cent) in the cere- injection and the beginning of the second course. brospinal fluid (CSF). The first two criteria are gen- The onset may be sudden or the condition may erally used because they are simpler and do not develop slowly with fever, headache, tremor, slur- require reagents. ring of speech, convulsions, and finally coma. It occurs in 5% of patients, and the incidence of fatali- CHEMOTHERAPY OF SLEEPING ty due to these reactions is approximately 1% (Odiit SICKNESS et al, 1997). However, the fatality of untreated dis- Most of the affected countries use broad guidelines ease is believed to be 100%, making the decision to for the treatment of sleeping sickness as recommend- treat an ethical necessity. The treatment of these ed by the World Health Organization (WHO, 1998). reactions includes the use of corticosteroids, hyper- However, there is an urgent need for novel approach- tonic solutions to combat cerebral oedema, rapidly es in sleeping sickness treatment and control. acting anticonvulsants, and subcutaneous adrena- line. There are a few cases of diarrhoea, jaundice and Pentamidine is used for treating the early stage of dermatitis, but these are generally not life threaten- sleeping sickness caused by T. b. gambiense. It is ing. effective for treatment so long as there is no detectable involvement of the CNS. The dosage PATIENT FOLLOW-UP used is 4mg of pentamidine base per kg of body It is mandatory to systematically follow up all treat- weight. A total of seven injections are given daily, ed patients to ascertain that they have been cured. It intramuscularly. Drug resistance appears not to be is recommended that patients be seen every six an important problem in the use of pentamidine for months over a two-year period. In addition to a full the treatment of T. b. gambiense. The side effects clinical and parasitological examination, a lumbar include pain and induration or sterile abscess at the puncture should be carried out on the patients, to site of injection, vomiting, abdominal pain, hypo- allow the CSF to be examined for possible increase tension, syncope, hypoglycaemia, and peripheral in leukocyte counts or presence of trypanosomes neuritis. Treatment failure attributable to resistance that would indicate a relapse. However, due to the to pentamidine appears not to be an important pub- painful lumbar puncture procedure, most patients lic health problem. Studies of the pharmokinetics of resent returning for follow-up once they begin to pentamidine are required to determine if the length feel better, while active follow-up to ensure compli- of the prescribed treatment regime can be reduced. ance is not affordable. Therefore, less invasive and painful methods of ascertaining cure need to be Suramin is used for the treatment of the early stage developed. Changes in CATT titres or CIATT titres of T. b. rhodesiense because, though the duration of and haematological parameters such Hb, ESR etc. treatment is longer than that for pentamidine and it are suggested for investigation in assessing cure. In is given by intravenous treatment, no primary addition, the duration of post-therapeutic follow-up resistance to suramin has been reported for should be reviewed, especially for T. b. rhodesiense, T. b. rhodesiense. The dosage used is 20mg/kg body in the expectation that it may be shorter than that weight. Intravenous injections - a maximum of se- required for T. b. gambiense. ven - are given every seven days. Side effects observed include pyrexia, pains in the joints and soles of DRUG RESISTANCE the feet, skin rash and desquamation, hypersensitiv- There is no recent evidence for resistance of T. b. rho- ity reactions. Pharmacokinetic studies of suramin desiense to melarsoprol. In the early 1970s, melarso- should be undertaken to determine if the treatment prol treatment failure of 12 (3.4%) out of 358 treated regime can be shortened. cases was reported (Ogada, 1974). It is possible that, with low treatment failure rates and lack of system- Melarsoprol is the drug used to treat late-stage atic follow-up, patients with treatment failure may sleeping sickness due to both T. b. rhodesiense and be missed. There should be periodic active follow- T. b. gambiense in Uganda. The maximum dosage for up of cohorts of sleeping sickness cases to monitor each injection is 3.6 mg/kg body weight. Currently, drug efficacy. two regimes for the treatment, comprising several

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 115 Treatment failure of melarsoprol in T. b. gambiense east, the teams are used to increase the awareness disease is emerging. Legros et al (1999a and 1999b) of the population and detect cases who may not yet reported a melarsoprol treatment failure rate of have sought health care. The costs of maintaining 26.9% among 428 patients treated in north western mobile teams are not affordable by the countries Uganda. Melarsoprol treatment failure rates needs affected by sleeping sickness, and therefore the to be monitored and documented regularly to estab- teams are often used in epidemic situations and lish the magnitude of the problem. The cause of this with donor support. Operational research is treatment failure is under investigation to deter- required to optimize the distribution of fixed post mine whether it is due to variation of melarsoprol diagnosis and treatment. This strategy may be a pharmacokinetics between individuals or if it is more sustainable strategy, especially in between associated with reduced susceptibility of try- epidemics of sleeping sickness. panosomes to melarsoprol. Legros et al (1999b) emphasize the need for second-line drugs to treat NEWLY DESIGNED TREATMENT patients that have already received one or several REGIMES full course(s) of melarsoprol. In Uganda, melar- Pharmacokinetic data from studies carried out on soprol treatment failure is a more significant prob- T. b. gambiense patients indicate that the course of lem to the west of the river Nile than to the east, in melarsoprol treatment in an adult can be reduced the T. b. gambiense affected area in the north-west of from a period of 25-36 days (WHO, 1998) to a peri- the country. Possible risk factors explaining this dif- od of just ten days, greatly cutting the costs of hos- ference in geographical distribution here and else- pitalization (Burri et al, 1995). A randomized trial where on the continent should be examined. Sub- comparing the standard treatment schedule with clinical, inadequate treatments in the history of this new concise regimen showed the same levels of these melarsoprol treatment failure foci could be an parasitological cure (100%) and adverse effects (16- explanation. 17%) as well as better compliance with the new regime (Burri et al, 2000). Adaptation of the new HEALTH SYSTEMS AND SLEEPING concise regime is to be monitored. It would not be SICKNESS CASE MANAGEMENT advisable to adapt the model to treatment of late- Demonstration of the trypanosome is mandatory stage T. b. rhodesiense infection because the pharma- before treatment can be administered to a sleeping cokinetics may be dissimilar given that this disease sickness case, due to the toxicity of the drugs used. is much more severe than the gambiense form. The blood-brain barrier may be more affected, allowing Due to the costs of staffing and equipping sleeping for higher levels of melarsoprol in the central nerv- sickness treatment facilities, few health units are ous system and possible neurological side effects. A equipped for treating the disease. The endemic study of the pharmacokinetics of melarsoprol in area for sleeping sickness in most countries is vast, late-stage T. b. rhodesiense infections is therefore nec- with poor coverage by fixed post health units for essary. However, a seven-day course of eflornithine diagnosis and treatment of the disease. Therefore, for the treatment of late-stage T. b. gambiense infec- most sleeping sickness patients travel more than tion was found to be inferior to the standard 14-day 5km to receive treatment. The problem of poor regimen (Pépin et al, 2000). accessibility to diagnosis and subsequent treatment may be alleviated by the use of mobile teams. There have been attempts to adjust the criteria for However, this is more rewarding where a valid treatment and so reduce the number of patients screening test is commercially available. The card receiving melarsoprol treatment that is very toxic. agglutination test for trypanosomiasis (CATT) is To determine CNS involvement, it has been pro- such a test. It has good sensitivity and specificity posed that the number of cells be raised to 20 (Magnus et al, 1978) but it is only useful for the cells/mm3 (Doua et al, 1996). There is not yet gambiense form of sleeping sickness. A similar test enough evidence to change the cut-off point for the for the rhodesiense form of the disease is required number of cells/mm3 in the CSF (Doua et al, 1996); before mobile teams can provide a rewarding strat- therefore the WHO recommended criteria for stag- egy for routine diagnosis and treatment. A new ing are still maintained. test, the card indirect agglutination trypanosomiasis test (TrypTectCIATT®), is reported to have good Chemoprophylaxis is not currently used as a sleep- sensitivity for both forms of sleeping sickness ing sickness control strategy. A multicentre evalua- (Nantulya, 1997) and is to undergo further field tion of the positive predictive value of increasing evaluation (TDR, 1999). In Uganda, mobile teams dilutions of the CATT (Magnus et al, 1978) is being are used in the north-west with support from a carried out; results of this study will be used to see non-governmental organization (Médecins sans if mass chemoprophylaxis is indicated in instances Frontières, France); during outbreaks in the south- of high sero-prevalence.

116 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Eflornithine (difluoromethylornithine, or α-DFMO, lating in humans and livestock is similar. The cost- or Ornidyl®) is not effective against T. b. rhodesiense effectiveness and acceptability of the strategy to (Iten et al, 1995; Matovu et al, 1997) but is useful for control T. b. rhodesiense sleeping sickness by mass the treatment of late-stage T. b. gambiense infection chemotherapy of livestock should be investigated, (Doua et al, 1987). It is presently available for the taking into account other potential benefits to the treatment of relapse following melarsoprol treat- farmer. ment, but the production of this drug was recently suspended. It has been suggested that nifurtimox be In conclusion, the current problems in the treatment used in desperate situations where DFMO is not of sleeping sickness in Africa include: melarsoprol available for treating melarsoprol refractory cases. treatment failure of T. b. gambiense sleeping sick- Nifurtimox is very toxic but is reported to be effec- ness; unavailability of alternative drugs; unafford- tive for curing some T. b. gambiense patients, includ- able treatments; poor coverage by diagnostic and ing those with late-stage disease refractory to melar- treatment facilities. Studies to identify practical soprol treatment (WHO, 1998). solutions to these problems are urgently required to reduce the current high fatality due to sleeping Nifurtimox (Lampit®, Bayer) has been tested for the sickness, a disease that is regarded as always fatal if treatment of sleeping sickness with conflicting not treated. results (Pépin et al, 1992; Van Niewenhove, 1992; Doua and Yapo, 1993). It is a cheap drug and easy to SUGGESTED STUDIES administer but is not yet registered for use in the • Research into new, safe, effective and cheap drug treatment of this disease. With the current emer- combinations that are active for all stages of sleep- gence of melarsoprol resistance, the efficacy and ing sickness. Suggested drug combinations to safety of nifurtimox in the treatment of sleeping compare are melarsoprol and eflornithine, melar- sickness caused by T. b. gambiense should be re-eval- soprol and nifurtimox, nifurtimox and eflor- uated. nithine.

REDUCTION OF DRUG PRESSURE • Continued screening of new compounds that are In areas where drug resistance to melarsoprol exists, less toxic, easy to administer, cheaper and effec- the need to use the drug can be reduced by putting tive for all stage of sleeping sickness. emphasis on preventive control strategies such as vector control and mass chemotherapy of the • Research on cost-effective means to prevent out- domestic animal reservoir of the disease. breaks of sleeping sickness through methods such as targeted vector control, and to avoid the high TSETSE CONTROL costs of treatment, problems of drug manufacture, Although tsetse control is reported to reduce the and complications associated with treatment fail- transmission of sleeping sickness, it is rarely main- ure. Identification of markers for determining the tained because of the costs of the methods applied. probability of occurrence of sleeping sickness out- Currently, tsetse traps and targets are considered the breaks in space and time for use as a basis for dis- cheapest, and an environmentally friendly option, ease prevention. applicable through community participation, but they are still not widely used to control sleeping • Active follow-up of cohorts of sleeping sickness sickness. Pour-on insecticide for cattle is another cases to accurately monitor drug efficacy, and vector control option that may involve community document the results. Mapping of foci of melarso- participation. However, use of pour-on insecticides prol treatment failure to study the possible risk is dependent on the distribution and movement of factors of its evolution. livestock. • Research into other methods of control to reduce MASS CHEMOTHERAPY OF LIVE- the pressure on human trypanocidal drugs, given STOCK IN T. B. RHODESIENSE AREAS the development of drug resistance. Evaluation of Research on the role of the livestock reservoir in the the cost-effectiveness of mass chemotherapy of live- epidemiology of T. b. rhodesiense sleeping sickness stock in the control of T. b. rhodesiense epidemics. indicates that chemotherapy of livestock in endemic areas is an important policy for the control of sleep- • Operational research to optimize the geographical ing sickness. There is molecular evidence for the distribution of sleeping sickness diagnostic and similarity of the parasites of man and livestock treatment facilities, taking into account factors (Enyaru et al, 1992; Hide and Tait, 1991; Hide et al, such as current disease distribution, population 1994), suggesting that the parasite population circu- distribution, availability of health infrastructure,

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World Health Organization. Control and surveillance of African Trypanosomiasis. Report of a WHO expert committee on Sleeping Sickness, 1998, Technical report series 881.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 119 Annex 6 PATHOGENESIS, GENOMICS AND APPLIED GENOMICS African trypanosomiasis

120 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 I DISCUSSION DOCUMENT ON • Late-stage anti-inflammatory and immunosup- PATHOGENESIS / APPLIED pressive events that modulate the protective GENOMICS effects of host T and B cell responses: - Elucidation of mechanisms that promote type 2 John M. Mansfield cytokine responses, and that suppress host tis- Department of Bacteriology, University of Wisconsin- sue specific resistance. Madison, 1925 Willow Drive, FRI Building Madison, WI - Role of these modulatory events in controlling 53706, USA or exacerbating tissue pathology.

For the purposes of group discussion, I have arbi- 2. Changes in parasite cell biology during infection trarily selected several research areas relevant to that impact on host resistance, with an emphasis on: human African trypanosomiasis (HAT)-associated • Molecular basis of changes in trypanosome viru- pathogenesis; these areas are outlined below. Some lence for the host background information on trypanosome immunol- - Relatedness to other clonal differentiation events. ogy and cell biology is provided in the text that fol- - Association with tissue specific residence/tro- lows (excerpted from recent reviews by Mansfield pism. and Olivier (2001), Mansfield et al. (2001), Paulnock - Identification and targeting of parasite genes and Coller (2001), and related resources (Imboden and molecules that are differentially expressed et al, submitted for publication, 2001; Mansfield et al, in highly virulent trypanosomes. in press; Paulnock et al, 2000). It is anticipated that Melville and colleagues will provide more detailed • Molecular basis for the resistance and susceptibil- and appropriate information on the trypanosome ity of certain trypanosomes to the cytolytic effects genomics project and its practical applications. of human serum high density lipoproteins (HDL) and of tumour necrosis factor-alpha (TNFα).

DISCUSSION OUTLINE 3. Identification of stage-specific parasite molecules through genomics and proteomics approaches that 1) Stage-specific immunobiological changes that might be exploited, with an emphasis on: occur during infection, with an emphasis on the fol- • Potential trypanosome molecules/cell biological lowing subtopics: systems that could be targeted by highly specific • Early pro-inflammatory events that trigger innate chemotherapeutic agents: immune system responses: - BSF trypomastigote specific. - Glycosylphosphatidylinositol (GPI) substituents - Tsetse fly stage specific. of the variant surface glycoprotein (VSG) molecule shed into host tissues. • Highly conserved invariant antigens that could be - Trypanosome lymphocyte triggering factor (TLTF) used for either more accurate immunodiagnosis release from trypanosomes and induction of in- or for targeted immunotherapy. terferon-gamma (IFN-γ) production. - Macrophage activation events associated with exposure to parasite activation molecules and BACKGROUND INFORMATION host activation factors. The background information that follows is an opin- • Early-stage acquired immune mechanisms associ- ionated view of trypanosomiasis, excerpted from ated with parasite control in the vascular and this author’s writings and other resources, that is extravascular tissue sites: meant to serve only as background information for - Distinct roles of Th1 cells and B cells in provid- group discussion on immunology and selected ing tissue specific resistance to trypanosomes. aspects of parasite biology. As pointed out in the - Macrophage release of trypanocidal factors/ passages below, there may be some disagreement as cytokines. to the biological significance of certain observations. Additional background information on the geno- • Interrelationship of host innate and acquired mics projects will be provided by others. immune responses to host pathology throughout infection: Stage-Specific Immunobiological Changes - Elucidation of immunological events that pro- that Occur During Infection mote tissue specific pathology at all stages of Macrophages and innate immunity infection. Cells of the macrophage lineage provide the first line of host defense against infectious diseases, and also

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 121 modulate downstream events that impact on the tion, 2001; Paulnock et al, 1989); and, upregulation development of acquired immunity. Macrophages of mRNA or proteins for other markers that include are present in all tissues and possess the ability to TNFα, IL-1, IL-6, inducible nitric oxide synthase recognize and eliminate many microbes. It is well (iNOS), prostaglandins and IL-10 (Beschin et al, established that recognition of microbes by 1998; Darji et al, 1992; Imboden et al, submitted for macrophages results in cellular activation following publication, 2001; Magez et al, 1999; Mathias et al, the uptake or binding of microbial components to 1990; Schleifer and Mansfield, 1993; Sileghem et al, specific membrane receptors (Hoffman et al, Mosser, 1989). More importantly, the expression or release of 1992; Mosser and Karp, 1999). Receptor-mediated several of these activation markers is associated activation of macrophages represents one of the first with modulation of host immunity and resistance. events in the innate immune response to many For example nitric oxide (NO), prostaglandins and microbial infections, leading to the production of TNFα have been implicated in the suppressor cell pro-inflammatory cytokines that initiate an inflam- activity exhibited by macrophages at different time matory response and affect the downstream devel- points of infection (Beschin et al, 1998; Borowy et opment of activated T cells as well as other parame- al, 1990; Hertz and Manfield, 1999; Schleifer and ters of host immunity. Cytokines produced by acti- Mansfield, 1993; Sileghem et al, 1991; Sileghem et vated T cells, primarily IFN-γ, provide additional al, 1989; Sternberg and McGuigan, 1992; Sternberg activation signals for macrophages, unleashing effec- and Mabbot, 1996); although NO and TNFα have tor functions that can destroy a wide range of intra- been shown to kill trypanosomes in vitro and are and extracellular microorganisms (Bendelac and thought to be important for trypanosome control in Fearon, 1997; Fearon and Locksley, 1996; Medzhitov extravascular tissue sites (more below on this topic) et al, 1997). Thus, the processes of innate resistance (Lucas et al, 1994; Magez, et al 1997; Magez et al, and acquired immunity are intimately interdepend- 1999; Mnaimneh et al, 1997; Vincendeau and ent, with macrophages playing a dual role as the ini- Daulouede, 1991; Vincen-deau et al, 1992), neither tiators of acquired responses and as a major effector factor alone has been linked definitively to protec- component of cell-mediated immunity. tion in vivo (Hertz and Mansfield; 1999; Magez et al, 1999) and the expression of these factors may be Macrophage activation in linked to pathological changes during infection trypanosomiasis (Mabbott and Sternberg, 1995; Mabbott et al, 1994; Macrophage activation is one of the hallmarks of Sternberg and Mabbott, 1996). However, the pro- infection with the African trypanosomes (Askonas, inflammatory pattern of macrophage activation 1984; Askonas, 1985; Bancroft et al, 1983; Beschin appears to change over the course of infection to et al, 1998; Borowy et al, 1990; Clayton et al, 1979; become a counter-inflammatory pattern of activa- Darji et al, 1992; De Gee et al, 1985; Fierer and tion in which IL-10 predominates and Type 2 Askonas, 1982; Fierer et al, 1984; Grosskinsky and cytokine responses appear to emerge (Namangala Askonas, 1981; Grosskinsky et al, 1983; Mayor-Wi- et al, 2000; Namangala et al, 2000; Namangala et al, they et al, 1978; Murray and Morrison, 1979; 2001); these events have been associated with late Paulnock and Coller, 2001; Paulnock et al, 1989; stage disease (Imboden et al, 2001; Paulnock and Sacks et al, 1982; Schleifer and Mansfield, 1993, Coller, 2001; Sternberg, 2001). Sileghem et al, 1989; Wellhausen and Mansfield, 1979). There is extensive evidence that the numbers and activity of macrophages increase dramatically in the tissues of trypanosome infected animals, and are associated with tissue pathology. Within the first two weeks of experimental Trypanosoma brucei rhodesiense infection, for example, a large percentage of cells in the enlarged spleen exhibit membrane and functional characteristics associated with activated macrophages. These include: increases in the release of interleukin-12 (IL-12) and IL-18, known to be important in the development of the early polarized Th1 cell responses to trypanosome antigens Figure 1. Glycosylphosphatidylinositol (GPI) mem- (Mansfield, 1994; Mansfield et al, submitted for pub- brane anchor substituents of the trypanosome variant lication; Schleifer et al, 1993; Schopf et al, 1998), an surface glycoprotein (VSG) molecule. GPIs have been enhanced ability to serve as antigen processing cells termed “…one of the most potent microbial pro-inflam- coupled to increases in expression of membrane I- matory agents known”(Almeida et al, 2000). The GPI Aα, B7-1 and B7-2 (Imboden, submitted for publica- anchor of the Trypanosoma brucei rhodesiense LouTat 1 VSG

122 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 molecule is depicted in this figure, showing glycosylinos- mechanisms, the amounts of GPI substituents (GPI- itolphosphate (GIP) substituents associated with sVSG mfVSG, GIP-sVSG and DMG) saturating host tis- after cleavage from membrane-anchored GPI-mfVSG by a sues during infection are quite substantial. trypanosome membrane-associated phospholipase C Conservative calculations estimate that experimen- (GPI-PLC), as well as dimyristoylglycerol (DMG) sub- tally infected animals may be exposed to 15-20 µM stituents that remain associated with the trypanosome VSG with each wave of parasitemia, which is an membrane. Figure adapted from Menon (1999 and 1994), inordinate amount of parasite material with intrin- Magez (1998) and others (Varela-Nieto et al, 1996). sic macrophage activation potential to be released into host tissues. Role of the variant surface glycoprotein GPI anchor in macrophage activation Limited in vitro studies by several labs have begun The source(s) and mode of action of activating fac- to characterize the activating effects of GPI sub- tors delivered to macrophages during trypanosome stituents on macrophages. It appears that GIP-sVSG infection are only partially understood. One major and GPI-mfVSG (containing the DMG lipid sub- activation factor is of parasite origin; this is the gly- stituent) have similar macrophage activating capa- cosylphosphatidylinositol (GPI) membrane anchor bilities in terms of TNFα, IL-6 and NO production, of the trypanosome VSG molecule (see Figure 1). but that there may be subtle differences in the abili- The GPI anchor precursor is synthesized in the ty of GPI-mfVSG to more effectively induce IL-1 and endoplasmic reticulum and subsequently is cova- IL-12 production (Magez et al, 1998; Schofield et al, lently attached to newly synthesized VSGs after 1996; Schofield and Tachado, 1996; Tachado et al, proteolytic cleavage of a VSG C-terminal GPI 1996; Tachado and Schofield, 1994). An extension of attachment signal sequence (Bangs et al, 1988; these initial studies regarding the ability of GIP- Cross, 1990; Doering et al, 1989; Doering et al, 1990; sVSG to interact with macrophages demonstrates Englund, 1993; Ferguson et al, 1988; Masterson that the GIP-sVSG component binds directly to et al, 1989; Menon, 1999; Menon, 1994, Menon et al, macrophages and induces expression of a specific 1997; Menon et al, 1990; Menon et al, 1990; Patnaik subset of activation genes in an IFN-γ independent et al, 1993; Raper et al, 1993; Sharma et al, 1999; manner (Imboden et al, submitted for publication Vidugiriene and Menon, 1995; Werbovetz and 2001; Paulnock and Coller, 2001). Studies have also Englund, 1997). After further modifications in both shown that GPI substituents exhibit signaling activ- the glycoprotein and GPI anchor residue, the ities (Tachado et al, 1997). That specific signal(s) are mature VSG is transported to and anchored in the delivered to the cell is apparent from the resultant trypanosome plasma membrane as membrane-form activation phenotype of macrophages exposed to VSG (GPI-mfVSG). During the course of infection, a GPIs, and by recent in vitro studies showing that trypanosome membrane-associated phospholipase GIP substituents (specifically the core glycan C (GPI-PLC) becomes activated and cleaves the GPI sequence) activate a specific protein tyrosine kinase, anchor as shown in Figure 1; this results in the while the DMG substituent may independently acti- release of substantial soluble VSG (GIP-sVSG) that vate a protein kinase C isoform in macrophages retains only the glycosylinositolphosphate (GIP) (Tachado et al, 1997). However, it is not yet known substituent of the original GPI anchor and leaves the how GPIs interact with the macrophage membrane dimyristoylglycerol (DMG) lipid component nor what receptor(s) may be important in delivering remaining behind in the membrane (Armah and GPI-mediated activation signals to the cell nucleus. Mensa-Wilmot, 2000; Bulow et al, 1989; Butikofer et al, 1996; Hereld et al, 1988; Hereld et al, 1986; During trypanosome infection, however, it is clear Mensa-Wilmot and Englund, 1992; Mensa-Wilmot, that host cells are exposed to biologically active lev- 1995; Paturiaux-Hanocq et al, 2000). Parasite num- els of GIP-sVSG, DMG and GPI-mfVSG, although bers routinely fluctuate during infection, both in the the timing of exposure to these molecules and the blood and extravascular tissues, primarily as the nature of their delivery to the macrophage mem- result of host B and T cell responses to variant deter- brane could be very different (see Figure 2). While minants of the VSG molecule. Since a) trypanosome the effects of such GPI substituents on uninfected numbers may approach 108 organisms per ml blood macrophages have been partially characterized and may also be quite high in the extravascular tis- in vitro, the impact of tissue saturation with these sues during peak parasitemias, b) there are approx- activating agents in vivo during infection is just imately 107 VSG molecules per cell, c) GIP-sVSG is being fully appreciated (Imboden et al, submitted clipped and released from both viable and for publication 2001; Paulnock and Coller 2001). stressed/damaged trypanosomes, and d) try- Also, macrophages are not the only cell type that panosomes are episodically destroyed by antibody- can be targeted by GPIs; recent evidence demon- (Ab-) and Th1 cell/macrophage-dependent effector strates that natural killer (NK) 1.1 T cells may

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 123 express T cell receptors (TCR) specific for GPI deter- panosome growth (Bakhiet et al, 1996; Bakhiet et minants and that B cells may be stimulated to under- al, 1990; Hamadien et al, 1999; Olsson et al, 1991; go cell differentiation by GPIs (Bento et al, 1996; Scho- Olsson et al, 1993; Vaidya et al, 1997). Thus a factor field, et al, 1999). Thus, GPI substituents released by secreted from the parasite, TLTF, is visualized as pathogens such as the African trypanosomes may inducing an essential trypanosome growth factor, have broad effects on the host immune system that IFN-γ, from host cells. surpass central activating effects on macrophages. Experiments partially in conflict with these pro- Role of IFN-γ in macrophage activation posed hypotheses exist, however. First there has and early host protection and/or pathology been no independent confirmation that IFN-γ serves The other major macrophage activation factor pro- as a growth factor for African trypanosomes; duced during trypanosomiasis is IFN-γ, which is of unpublished studies from several labs have failed to host origin. Small amounts of IFN-γ may be pro- substantiate the claim that IFN-γ serves as a growth duced very early during infection as the result of factor and no critical biochemical studies of IFN-γ TLTF activation of CD8 T cells (Bakhiet et al, 1996; binding to or utilization by trypanosomes have yet Bakhiet et al, 1993; Schleifer and Mansfield, submit- been published. Furthermore, parasitemias are ted for publication 2001; Vaidya et al, 1997). First higher in IFN-γ knockout mice, which are highly described by Bakhiet, Olsson and colleagues susceptible rather than more resistant to infection (Bakhiet et al, 1993; Bakhiet et al, 1996; Bakhiet et al, with T. b. rhodesiense (see discussion below on the 1990; Olsson et al, 1991; Olsson et al, 1993; Olsson role of IFN-γ in early host resistance [Hertz et al, et al, 1992) and subsequently cloned by the Do- 1998]). One might reasonably expect that parasi- nelson laboratory (Vaidya et al, 1997), TLTF is a 453 temias would be lower in IFN-γ deprived animals if amino acid protein with potentially important bio- this cytokine served in any substantial manner as a logical effects. TLTF was discovered when resear- growth factor. Additionally, TLTF expression is chers noted that rodents injected with T. b. brucei, or identical in trypanosomes expressing high and low lymphoid cells cultured with trypanosomes in vitro, virulence attributes (Mansfield, 2001), suggesting exhibited an increase in the number of antigen non- that there is no modulation of the gene or protein in specific IFN-γ secreting cells; depletion of CD8+ T organisms known to exhibit rapid growth character- cells in animals or cultures abrogated the effect and, istics and virulence attributes for mammalian hosts. interestingly, also resulted in less trypanosome growth (Bakhiet et al, 1990). Use of a chamber sys- Initial studies asserted that TLTF was a secreted pro- tem separating lymphoid cells and trypanosomes tein (Vaidya et al, 1997), but subsequent characteri- showed that a soluble factor was responsible for zations showed that the amino acid sequence does induction of IFN-γ synthesis (Olsson et al, 1991). not contain a hydrophobic region typical of membrane-transported trypanosome proteins, though it Several Trypanosoma species appear to express TLTF did appear to be targeted to the flagellar pocket but may possess different IFN-γ stimulating abilities region through an apparent conformational signal as measured by the relative increase of IFN-γ pro- within a specific 144 amino acid domain (Hill et al, ducing cell numbers in the presence of extracts or 1999). To date there is no clear evidence that TLTF is culture filtrates of species including T. evansi, T. b. rho- a secreted protein, though the predicted protein desiense, and T. b. gambiense (Bakhiet et al, 1996). Subse- does possess unique internal targeting sequences. quent characterization of CD8+ T cell IFN-γ activa- More recent studies on the cell biology of TLTF have tion by TLTF showed that tyrosine protein kinases suggested an alternate (or coincident) role for the are necessary for activation but protein kinase C and protein. The gene sequence identified as TLTF is protein kinase A specifically are not (Bakhiet et al, expressed in both insect and bloodstream forms of 1993). Interestingly, TLTF may stimulate other cells T. b. brucei and the protein appears to be tightly asso- to release IFN-γ, such as rat dorsal root ganglia, and ciated with the flagellar cytoskeleton (present in this secretion apparently also is dependent on tyro- detergent-resistant and Ca2++-resistant cytoskeletal sine kinase(s) (Eltayeb et al, 2000). These types of fractions of trypanosome extracts) (Hill et al, 2000); studies and their experimental extension over the modification of TLTF gene expression in the pro- past decade have led investigators to posit the fol- cyclic form resulted in an unusual motility defect, lowing hypotheses with respect to the role of TLTF: suggesting that TLTF may be an integral part of the trypanosomes secrete TLTF which binds to CD8 trypanosome cytoskeletal architecture. Surprisingly, molecules expressed on CD8+ T cells, thereby in- TLTF-like genes are present in a number of diver- ducing antigen non-specific activation and produc- gent eukaryotes including Drosophila and zebra fish. tion of IFN-γ; TLTF-induced release of IFN-γ subse- Notably, the human growth arrest specific gene quently serves as a growth factor that promotes try- (GAS)11, closely related to TLTF (Vaidya et al, 1997),

124 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 is a possible tumor suppressor molecule with a sub- linked to early host resistance during T. b. rhodesiense molecular region that may localize to cellular micro- infection, and is thought to be associated with tubules. macrophage activation characteristics responsible for control of the parasites in extravascular tissue Thus, it is difficult to see how TLTF, a tightly bound sites (De Gee et al, 1985; Hertz et al, 1998; Imboden cytoskeleton-associated molecule, would be secret- et al, submitted for publication, 2001; Paulnock and ed or released in biologically active levels during Coller, 2001). infection or in cell cultures containing viable trypanosomes to affect the release of IFN-γ from host cells. Yet, it is clear that trypanosome infections and Figure 2. Macrophage activation in African try- trypanosome extracts are capable of inducing IFN-γ panosomiasis is mediated by exposure to host and release from naive host lymphoid cells in an antigen parasite factors. nonspecific manner; the levels are low and occur independently of antigen specific induction of IFN- γ from host Th1 cells (Mansfield, 2001; Schopf et al, 1998). Thus, IFN-γ secretion induced by parasite Trypanosomes material(s) has been a repeatable phenomenon and is clearly of some interest; there is the distinct possibility that release of biologically active TLTF (or a DMG similar molecule with closely related effects) occurs during periods of cataclysmic elimination of try- GIP-sVSG panosome variant antigen types (VATs) by host Ab and Th1/macrophage cell responses throughout LEVELS RELATIVE infection (see below), rather than by an active secre- IFN-γ tory pathway, to induce IFN-γ. Given that trypanosomes / trypanosome extracts are capable of 0 1 2 3 4 5 6 7 8 9 10 inducing nonspecific IFN-γ release from host cells, DAY OF INFECTION and that IFN-γ may be an early and critical factor in host protection (probably important in regulating parasite numbers in the extravascular tissues—see (Figure adapted from Paulnock and Coller, 2001) Figures 2 and 3), it may be that TLTF is important in a different context than originally suggested: by The theoretical timing of exposure to host- and parasite- inducing low levels of IFN-γ that control an early derived macrophage activating factors during an early potentially explosive spread of trypanosomes in peak of parasitemia in trypanosome infection is shown infected host tissues, regardless of the genetically- here. GIP-sVSG homodimers, released from the try- based resistance status of the host. Such an early panosome plasma membrane by the action of GPI-PLC, control over parasite expansion and, in susceptible are detectable within several days of infection throughout animals or individuals, a delay in host death, would the body (Diffley and Jayawardena, 1982; Diffley and permit the host to survive for a period of time so Straus, 1986; Diffley et al, 1980; Norton et al, 1986). that the possibility of trypanosomes being taken up Although DMG is liberated by the same GPI-PLC cleav- in a blood-meal and transmitted to new hosts could age event as GIP-sVSG, host tissues may be exposed ear- occur. Regardless of one’s view of TLTF, the hard lier to GIP-sVSG since effective membrane transfer of work of providing functional and genetic linkages DMG would largely be dependent on high concentrations of parasites in contact with macrophage membranes. GIP- between TLTF expression and biological effects on sVSG can activate macrophages independently of residual the host remain. DMG (Imboden et al, submitted for publication 2001; Magez et al, 1998; Paulnock and Coller, 2001; Schofield Clearly, however, the major source of IFN-γ during and Tachado, 1996; Tachado et al, 1997). During immune infection appears to be parasite antigen stimulated clearance of trypanosomes, macrophages take up para- Th1 cells, which appear in significant numbers sites and presumably are also exposed to intact GPI- somewhat later than early TLTF induced IFN-γ mfVSG as well as DMG remaining from GPI-PLC cleav- responses. Th1 cell responses to VSG (and to invari- age of the anchor. Low levels of IFN-γ may also serve as ant antigens of the parasite) have now been charac- an early activating agent during infection, presumably γ terized and result in strong IFN- responses in derived from TLTF-stimulated CD8 T cells. Cytokine lev- genetically more resistant animals but not in sus- els subsequently rise substantially over time due to CD4 ceptible animals (Hertz et al, 1998; Hertz and Th1 cell stimulation by VSG and other parasite antigens. Mansfield, 1999; Schleifer et al, 1993; Schopf et al, The patterns shown here may differ markedly later in γ 1998). The IFN- response has been definitively infection.

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 125 The IFN-γ/IFN-γ receptor interaction and down- that susceptible mice, which normally do not make stream subcellular signaling pathways have already a sufficient Ab response to VSG and do not clear been well characterized in other model systems, and VATs from the blood, were afforded by donor cells macrophage activation events triggered by IFN-γ from resistant mice a functional B cell response that have been extensively studied (Adams and enabled them to clear parasitemia during infection; Hamilton, 1984; Adams and Hamilton, 1987; Adams however, despite the ability to eliminate try- and Hamilton, 1986; Hamilton, 1989; Paulnock, panosomes from the blood, these animals were just 1992; Paulnock, 1994; Paulnock-King et al, 1985). In as susceptible as mice receiving susceptible donor African trypanosomiasis, it is clear that some bone marrow cells that failed to make protective macrophage activation events are dependent on VSG-specific B cell responses (De Gee and IFN-γ exposure and others on exposure to try- Mansfield, 1984). Subsequent genetic studies with panosome-derived molecules, including GIP-sVSG crosses between Ab+ resistant and Ab- susceptible (Imboden et al, submitted for publication 2001; mouse strains showed that the F1 hybrids all were Paulnock and Coller, 2001). The activation patterns able to make VAT-specific Ab responses and control observed in the presence of both factors are different parasitemias, but all such hybrids were as suscepti- from either one alone, are dependent on the genetic ble as the susceptible parental strain (De Gee et al, background of the infected host, and may be impor- 1988). Taken together, these types of results showed tant in the control of infection. Why trypanosomes that the VSG-specific B cell response, although induce such broad macrophage activation effects is linked to trypanosome clearance from the blood, unknown. It may be important for trypanosomes to was not by itself functionally or genetically linked to induce early temporal protection against the infec- overall host resistance. tion regardless of the genetically-based resistance status of the host (e.g. so the host isn’t killed by Th cell responses to trypanosome antigens infection before natural transmission of the disease This information led the way to studies that first can effectively occur). Alternatively, it might be elucidated Th cell responses to VSG and other try- linked to the early generation of suppressor panosome antigens during infection (Hertz et al, macrophage activity so as to depress host T cell 1998; Hertz and Mansfield, 1999; Mansfield, 1994; responses to parasite antigens. Or it may result in Schleifer et al, 1993; Schopf et al, 1998). T cell deregulation of IFN-γ-induced activation events in responses to trypanosome antigens were not discov- macrophages in order to avoid parasite elimination. ered previously because of several interesting char- Clearly, a goal of unraveling the cellular and molec- acteristics of trypanosome infections. First, a non- ular basis of macrophage activation by try- specific immunosuppression of T cell responses in panosome-derived antigens or other factors (e.g. trypanosomiasis had been recognized for many DNA released from dead trypanosomes [Shoda years (Mansfield and Wallace, 1974), and, although et al, 2001]) in the context of overall host resistance earlier studies revealed that T cell responses to try- to infection and tissue pathology is important. panosome antigens could be induced in immunized animals (Campbell et al, 1982; Finerty et al, 1978), Tissue specific immune control such responses were not readily detectable in infect- mechanisms in early infection ed animals (Mansfield and Kreier, 1972; Paulnock There are clear differences in the ability of various et al, 1989). For example, not only were spleen or host species, and strains within species, to display lymph node T cells from infected mice unable to relative resistance to African trypanosomiasis proliferate in response to mitogens or antigen, they (Levine and Mansfield, 1981; Mulligan, 1970). also failed to produce significant amounts of IL-2 or Studies over the past twenty years have revealed IL-4, and these events could be shown to impact on that the host Ab response plays only a partial role in T-dependent B cell responses to a variety of antigens such relative resistance against trypanosomes. (Mansfield and Bagasra, 1978). This generalized While VSG-specific Ab clearly is responsible for the immunodeficiency was shown to result in part from cataclysmic elimination of VATs from the blood- the presence of macrophage “suppressor cells” in stream of infected hosts, it is now known that this lymphoid tissues (Sacks et al, 1982; Wellhausen and event is not linked, functionally or genetically, to Mansfield, 1980; Wellhausen and Mansfield, 1979; overall host resistance (De Gee et al, 1988; De Gee Wellhausen and Mansfield, 1980); in fact, and Mansfield, 1984; Mansfield, 1995; Mansfield, macrophages from infected mice had the capacity to 1990; Mansfield and Olivier, 2001). The seminal actively suppress the proliferative responses of nor- studies were those in which H-2 compatible radia- mal T cells to mitogens and antigens in vitro and in tion chimera mice, reconstituted with reciprocal vivo. A breakthrough in recognizing that Th cell bone marrow cell transplants from relatively resist- responses to trypanosome antigens occurred during ant or susceptible donors, revealed the following: infection came with the finding that functional com-

126 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 partmentalization of such responses occurred sons for the relative tissue compartmentalization of (Schleifer et al, 1993). It was revealed that Th cells Th cell cytokine responses (e.g., IL-2 and IFN-γ pro- reactive with VSG were predominant in the peri- duction by peritoneal Th cells, but mostly IFN-γ pro- toneal T cell population; when stimulated with duction by Th cells in the peripheral lymphoid tis- VSG, these cells made a substantial IL-2 and IFN-γ sues) have not been elucidated, the reason for inhi- cytokine response but failed to proliferate. Subse- bition of T cell clonal expansion has now been quently, it was discovered that Th cells in the resolved. Suppressor macrophages were shown to peripheral lymphoid tissues also made an IFN-γ elaborate several factors that inhibited the prolifera- response (but little IL-2) when stimulated with VSG. tive (but not the cytokine) responses of VSG activat- Thus, it was apparent that VSG-reactive T cells were ed Th1 cells: NO, prostaglandins and TNFα (Darji et present in infected animal tissues but that they al, 1996; Hertz and Mansfield, 1999; Schleifer and exhibited a restricted cytokine response and mini- Mansfield, 1993; Sternberg and McGuigan, 1992). mal evidence for clonal expansion (Schleifer et al, Macrophages were activated to produce these sup- 1993). Since these VSG-reactive T cells displayed a pressive factors primarily as the result of exposure CD4+ αβ TCR+ membrane phenotype, expressed to GIP-sVSG and to IFN-γ released by parasite anti- Type 1 cytokines, were major histocompatibility gen stimulated Th cells (Hertz and Mansfield, 1999; class II antigen (MHC II) restricted and antigen pre- Mansfield et al, submitted for publication; Schleifer senting cell (APC) dependent (Hertz et al, 1998; and Mansfield, 1993). The full impact of NO and Schleifer et al, 1993; Schopf et al, 1998), it was clear prostaglandins on host immunity to trypanosomes that they represented a classical Th1 subset of T cells has not been completely resolved, but studies with that recognized VSG during infection. More recent iNOS KO mice have shown that, although NO is the work has begun to elucidate the submolecular tar- main “suppressor” factor that limits clonal expan- gets of VSG-reactive Th cells. In preliminary studies sion of T cells (and maybe also modulates cytokine it has been shown that Th cell specificities are direct- responses to a degree) the absence of NO did not ed against a defined hypervariable subregion of affect overall host resistance (Hertz and Mansfield, VSGs that is not exposed when VSG homodimers 1999). are assembled into the surface coat structure (Mansfield, 2001; Mansfield and Olivier, 2001), ful- Early and strong trypanosome-specific Th1 cell filling earlier predictions that VSG sequence vari- responses may provide an essential component of ability in nonexposed regions of the molecule might host resistance; this realization emerged from stud- be driven by T cell selection (Blum et al, 1993; Field ies with cytokine gene knockout mice. The central and Boothroyd, 1996; Reinitz et al, 1992). finding in one study was that mice with a resistant genetic background but which lacked a functional The extreme polarization of the Th1 cell cytokine IFN-γ gene were as susceptible as scid mice to try- responses seen in some experimental systems is due panosome infection, even though those mice pro- in part to the early production of IL-12 by duced Abs sufficient to control parasitemia (Hertz et macrophages exposed to trypanosome GPI sub- al, 1998). In contrast, the same genetic strain of stituents (Mansfield et al, submitted for publica- mouse with the IL-4 instead of the IFN-γ gene tion). That IL-12 is not the only polarizing factor is knocked out were as resistant as wt mice to infection seen from preliminary studies with IL-12 knockout (Hertz and Mansfield, 1999). These results under- (KO) mice and mice exposed to Abs against IL-12; in scored earlier studies demonstrating that the VAT- each case, early temporal depression of the Type 1 specific Ab response and control of parasitemia cytokine response did not result in a compensatory were not capable of providing resistance alone, and Type 2 cytokine response and, after a period of 10 that the production of a single cytokine, IFN-γ, in days or so, the Th1 cell response emerged in both response to infection was found to be a critical ele- groups (Mansfield et al, submitted for publication). ment in host resistance. The mechanism(s) associat- That IL-12 is not the only polarizing factor is seen ed with IFN-γ-mediated resistance are not yet clear, from preliminary studies with IL-12 knockout (KO) but seem to involve macrophage factors induced by mice and mice exposed to Abs against IL-12; in each IFN-γ activation. Several candidate factors have case, early temporal depression of the Type 1 been proposed, such as NO and TNFα, both of cytokine response did not result in a compensatory which have been shown to kill trypanosomes in vitro Type 2 cytokine response and, after a period of 10 (Lucas et al, 1994; Lucas et al, 1993; Magez et al, days or so, the Th1 cell response emerged in both 1997; Magez et al, 1999; Mnaimneh et al, 1997; groups (Mansfield et al, submitted for publication). Vincendeau et al, 1992). Recent studies suggest, Thus, there are complex features of infection that however, that neither factor alone is capable of promote the production of Type 1 cytokines and the mediating resistance in vivo; results with try- outgrowth of antigen-reactive Th1 cells. While rea- panosome infected iNOS KO mice and TNFα KO

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 127 mice showed that such mutations on a resistant phenotype of pro-inflammatory responses and parasitici- mouse genetic background do not significantly dal macrophage activation to a phenotype of counter- affect the course of infection (Hertz and Mansfield, inflammatory responses and Type 2 cytokine responses 1999; Magez et al, 1999; Millar et al, 1999), although that do not promote parasite elimination from tissues and it is possible that the combination of NO and TNFα perhaps also from the blood. Adapted from Mansfield and is required for functional resistance. Clearly, IFN-γ Olivier (2001). inducible events in macrophages must carefully be evaluated for their impact on trypanosomes during Thus, relative resistance to African trypanosomes early stages of infection. Since these events occur may be mediated by two major components of host independently of B cell mediated resistance mecha- immunity, neither one of which by itself is adequate nisms that are known to control trypanosomes in to provide resistance (Figure 3A). First, VSG specif- the vasculature, and since IFN-γ activated ic Ab responses control trypanosomes present in the macrophage control mechanisms are presumed to blood. Second, T cell production of IFN-γ and sub- be important in regulating trypanosome numbers in sequent macrophage activation events are necessary the extravascular tissue spaces (but this by itself is to control trypanosomes in the extravascular tissues. inadequate to provide protection [Mansfield, 2001]), Animals that make weak B cell and/or T cell it appears that multiple arms of the host immune responses to trypanosome variant antigens invari- system are required to control trypanosomes and to ably will demonstrate relative susceptibility; in con- provide relative resistance during infection. trast, animals making pronounced B and T cell responses (including appropriate macrophage acti- Figures 3A and 3B. Innate and acquired immune vation events) will display relative high resistance. elements in early- versus late-stage trypanosomiasis. However, this picture is evolving considerably due to evidence that the later stages of experimental infection display a different pattern, a pattern that is associated with loss of resistance to trypanosomes (see Figure 3B). Overview Events that impact on B, T or macrophage cell responses during infection can be expected to cause modulations in host resistance and tissue pathology. These events need to be clarified in both animal model systems and in clinical HAT.

Changes in Parasite Cell Biology during Infection that Impact on Host Resistance The African trypanosomes display considerable biological variation during their life cycle. This biological variation is directed by specific patterns of gene and protein expression. For example, bloodstream trypomastigote forms display a number of different surface antigenic phenotypes during infection of their mammalian hosts; this phenotypic variation has as its basis the differential expression of VSG genes and molecules (Borst et al, 1998; Borst and Rudenko, 1994; Cross, 1990; Donelson, 1987; Van der Ploeg et al, 1992; Vickerman and Luckins, 1969). Additionally, the differentiation of long slender (LS) trypomastigote forms to short stumpy (SS) These figures portray the cell and molecular elements trypomastigotes during infection results in pro- important in controlling trypanosome infection of host tis- found morphological and functional changes in sues. In early infection stages, IFN-g cytokine responses these cells. Such changes include mitochondrial bio- activate macrophages to produce factors cytotoxic for try- genesis and the acquisition of new metabolic path- panosomes, limiting the spread or survival of parasites ways; it is the differential expression of specific outside the vasculature. B cell responses appear to selec- genes and proteins that presage these biological tively control trypanosomes within the vasculature. In changes (Bienen et al, 1983; Bohringer and Hecker, late-stage infections, there appears to be a shift from the 1975; Hua et al, 1997; Mulligan, 1970; Mutomba and

128 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Wang, 1998; Tschudi, 1995; Vanhamme and Pays, genetically distinct populations of trypanosomes, or 1995; Vickerman, 1971; Vickerman et al, 1993). whether there is intraclonal biological variation Furthermore, the differentiation of SS forms to pro- within trypanosome populations that impacts on cyclic forms in the insect vector also results from the the course of disease in a genetically defined host. differential expression of specific genes and proteins (Butikofer et al, 1997; Roditi, 1996; Ruepp et al, 1997; This question was addressed in an earlier study in Vanhamme and Pays, 1995; Vickerman et al, 1988). which mice of a relatively resistant phenotype were Finally, the transformation of insect forms to meta- infected with a single trypanosome of T. b. rhode- cyclic trypomastigotes results in different morpho- siense clone LouTat 1 (Inverso and Mansfield, 1983). logical and functional changes in the parasites that Several different VATs were isolated from para- permit infection of a new mammalian host; these sitemic peaks at intermediate and late time points changes also occur as the result of differential gene during infection of a single mouse; these VATs were and protein expression (Turner et al, 1986; subcloned and characterized as to VSG phenotype. Vanhamme and Pays, 1995; Vickerman, 1985; Three different VATs, which represented antigeni- Vickerman et al, 1993). Thus, all trypanosomes cally distinct daughter cell populations clonally exhibit considerable clonal plasticity in terms of derived from a single LouTat 1 parental cell, were their antigenicity, morphology and biological func- used to infect the same mouse strain; the courses of tion during the life cycle; this plasticity occurs as the infection were monitored in comparison with mice direct result of differential expression of specific infected with LouTat 1. The interesting result was subsets of genes and proteins. It follows that the fail- that each of the daughter cell populations exhibited ure of trypanosomes to undergo biological variation a different virulence profile compared to the at critical points in the life cycle may result in elimi- parental clone (Inverso and Mansfield, 1983). For nation by the mammalian host, failure to differenti- example, LouTat 1 caused death in approximately ate within the intermediate host and vector, or 62 days post-infection, while LouTat 1.3, LouTat 1.4 inability to establish new animal infections. and LouTat 1.5 caused death in approximately 44, 30, and 28 days, respectively. These results demon- The molecular mechanisms that regulate changes in strated that VATs arising during infection expressed VSG phenotype and stage of cellular differentiation virulence phenotypes different from the infecting are shared in common among African trypano- VAT. In essence, daughter cells arising within a try- somes; however these are not the only mechanisms panosome population expressed the capacity to that regulate biological differences in these para- transcend host genetic resistance characteristics and sites. New evidence is emerging that differential render a relatively resistant animal into a more sus- susceptibility of brucei group trypanosomes to host ceptible one. factors such as trypanosome lytic factor (TLF) (De Greef and Hamers, 1994; Hager et al, 1994; These seminal observations have been repeated with Hajduk et al, 1992; Rickman and Robson, 1970; consistent results, and related observations on clonal Rifkin, 1978; Smith et al, 1995); may occur as the heterogeneity among trypanosomes have been made result of clonal variation among trypanosomes in other studies (Diffley and Mama, 1989; Inverso (Hager and Hajduk, 1997; Hajduk et al, 1995); the et al, 1988; Joshua, 1990; Mamman et al, 1995; Ortiz basis for such changes has not yet been defined, but et al, 1994; Reinitz and Mansfield, 1988; Sacks et al, is believed to encompass specific clonal modifica- 1980; Seed, 1978; Seed and Sechelski, 1996; Turner, tions in gene or protein expression. Host specificity 1990; Turner et al, 1995). Additionally, the general may also be defined by the clonal expression of dif- observation has been made that many other daugh- ferent transferrin receptor genes (Bitter et al, 1998). ter cells/VATs arising naturally from LouTat 1 also While these examples reflect on trypanosome infec- expressed differences in virulence (see Table 1, tivity for a host species, related observations and below), and that the most virulent VATs seemed to mathematical modeling predict that considerable arise at later time points in infection, just prior to biological variation occurs among trypanosomes host death. Thus, the apparent result of infection during infection that is not related directly to infec- with relatively low virulence trypanosomes is a pro- tivity or cyclical differentiation in the life cycle gressive increase in population virulence with time, (Turner et al, 1995; Vassella et al, 1997). For example, rather like the turning up of a “virulence rheostat”. it is well known that different isolates, species and Based on these observations, it was speculated that subspecies of trypanosomes exhibit remarkable the longer a trypanosome population existed in a variation in pathogenicity and virulence for geneti- mammalian host, the more virulent that population cally defined host species (Mulligan, 1970). A key might become. Such a virulence rheostat may have question has been whether such differences in viru- evolved as a programmed mechanism to overcome lence are immutable characteristics associated with different levels of host resistance that might be

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 129 encountered in nature, where there is a pool of coat structure (Inverso et al, 1988), transcribed iden- genetically disparate mammalian hosts available for tical VSG genes (Reinitz et al, 1992; Uphoff et al, sub- infection. Implicit in this speculation, however, is the mitted 2001) and expressed their VSG genes by a idea that a virulence rheostat must somehow be duplicative transposition event from the same chro- reset, perhaps upon cyclical passage through the mosomal telomeric expression site (Uphoff et al, sub- intermediate host and vector, the tsetse fly. mitted 2001).

Since virulent trypanosomes seem to arise after a Table 1. Virulence phenotypes of prolonged period of replication in an infected host, LouTat 1-derived VATs. it may be possible to generate highly virulent trypanosomes by rapid subpassage of low virulence Variant Antigenic Types Virulence trypanosomes through mice for a substantial time (Mean Survival Time) period; in essence, the effect would be one mimick- (A) VATs isolated from natural infections*

ing a single, prolonged course of infection. This was LouTat 1 (parental clone) 62 achieved by infecting irradiated mice with LouTat 1 LouTat 1.9 46 and subpassaging the trypanosomes into different LouTat 1.3 44 LouTat 1 4 30 mice every three days for approximately six months. LouTat 1.5 28 At the end of this time, trypanosome stabilates were LouTat 1.7 25 LouTat 1.2 13 made from sublines and subclones, and were LouTat 1.6 11 assessed for their virulence characteristics. One rep- LouTat 1.10 6 resentative subclone, designated LouTat 1A, was examined in some detail. These trypanosomes dis- (B) VATs derived by subpassage** played a single uncontrolled peak of parasitemia LouTat 1A 4 LouTat 1D 4 and were able to kill a resistant mouse strain (as well LouTat 1X 4 as all other resistant or susceptible strains of mice) in LouTat 1n 6-60 approximately four days post-infection; in contrast the parental clone LouTat 1 gave rise to multiple Therefore, it is unlikely that either VSG molecules or peaks of parasitemia and a prolonged survival time the active VSG gene expression site are important of over 60 days in the same mouse strain (Inverso for virulence regulation in trypanosomes (see et al, 1988). Thus a model system of comparative Figures 4-6). Confirmation that VSG genes and VSG trypanosome virulence was developed from this gene expression sites are not involved in virulence approach, in which the relatively low virulence came from additional experimental approaches. clone LouTat 1 and the relatively high virulence sub- New sublines and subclones were derived by rapid clone LouTat 1A represent different ends of a viru- subpassage of LouTat 1 through irradiated mice, as lence spectrum, with the virulence of other natural- above, and examined for VSG phenotype and gene ly arising VATs existing somewhere between these expression. Many of the subclones expressed the two extremes (Table 1). same VSG gene as LouTat 1 and all were highly virulent like LouTat 1A (Table 1). In another approach, A natural question that arose from these types of LouTat 1A was used to infect rabbits and goats; studies was whether the VSG molecules expressed although these animals exhibited pathology sooner by virulent VATs acted as virulence factors, with spe- than LouTat 1 infected controls, they did not die as cific VSG isotypes exerting defined biological effects early as mice and trypanosomes were able to under- on the host. This idea was not unfounded since sev- go antigenic variation. The VATs generated from eral biological traits associated with VSG molecules LouTat 1A in these animals were isolated and sub- have been described in the literature (Mathias et al, sequently used to infect mice; the results showed 1990; Musoke and Barbet; 1977; Schofield et al, 1999; that the LouTat 1A-derived VATs were as virulent Tachado et al, 1997; Tizard et al, 1978). Alternatively, for mice as LouTat 1A (Inverso et al, 1988). Thus one could also speculate that expression site-associ- high levels of virulence, once expressed in mam- ated genes (ESAGs) co-transcribed with certain VSG malian hosts, appear to be a constitutive trait that is genes at specific chromosomal expression sites may unaffected by further VSG gene switching. be responsible for virulence expression or regulation. This idea was based on observations of others concerning potential growth or differentiation regulatory roles associated with ESAGs (Cross, 1990; Vickerman et al, 1993). However, an analysis of the LouTat 1/LouTat 1A model system revealed that both organisms displayed the same antigenic surface

130 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Figure 4. clones. A few chromosomes were altered in size, as Amino acid sequence of the T. b. rhodesiense LouTat 1/ LouTat 1A determined by pulsed field gel electrophoresis; VSG molecule. The protein sequence was deduced from the identical full-length nucleotide sequences (Accession no. X56643) however, there was no net loss of cellular DNA nor (Reinitz et al, 1992) as well as from partial amino acid sequencing were any differences apparent in restriction frag- data (Inverso et al, 1988). The N–terminal signal sequence and the ment length polymorphism (RFLP) patterns utiliz- C-terminal hydrophobic extension are underlined; threonine-27 ing a number of random and known non-VSG is the start site of the mature protein. cDNA probes. Thus, the chromosomal size variations observed may largely be subtelomeric or simply do not involve chromosome regions to which the probes hybridized. Two-dimensional gel electrophoresis of 35S-labeled proteins showed not only that different proteins were expressed in LouTat 1A compared to LouTat 1, but also that there were different proteins expressed in LouTat 1 compared to LouTat 1A. Competitive Northern analyses in which labeled total cDNA from LouTat 1A, in the presence of excess unlabeled LouTat 1 competitive total cDNA, was hybridized to mRNA from LouTat 1A showed that that there were numerous mRNA species unique to LouTat 1A. Taken together, these observations suggested that a Figure 5. Southern blot analysis of LouTat 1 subset of genes and proteins was being expressed and 1A VSG genes. Genomic DNA from LouTat 1, 1.5 and 1A was digested with in LouTat 1A that was not being expressed at the XmnI and hybridized in a Southern blot with a labeled LouTat 1 same level in LouTat 1. VSG cDNA probe (BC, basic copy of VSG gene; ELC, expression linked copy). Overall, preliminary observations on the biological behaviour of LouTat 1 and LouTat 1A, and on the subcellular differences detectable between LouTat 1 and LouTat 1A, as well as biological variation observed with other subspecies of T. b. brucei in terms of TLF susceptibility or TNFα sensitivity, have led to the hypothesis that African trypanosomes have the capacity to regulate clonal expression of virulence. Specifically, it has been proposed (a) that trypanosomes have evolved the programmed capacity for clonal upregulation of virulence as a means to successfully subvert host resistance mechanisms, regardless of host genetic background; (b) that this capacity to modify virulence phenotype occurs independently of changes in VSG gene expression; and (c) that the level of virulence expressed is determined by differential gene and/or protein expression during trypanosome growth in an infected host.

Overview Figure 6. Restriction map of the LouTat 1 It seems therefore an important goal to characterize and LouTat 1A VSG gene expression sites. the molecular mosaic associated with the virulence The expressed copies are in a telomeric site while the basic copy phenotype and to determine whether or not specific of the VSG gene is found at an internal chromosomal site that is subsets of genes or proteins linked to virulence can the same for both trypanosomes. The solid bar denotes the VSG gene. be identified; the prediction is that such approaches may open new doors in the understanding of trypanosome-mediated pathogenesis, and candidate Subsequently LouTat 1 and 1A were examined for virulence factors could be targeted for specific non-VSG related cellular differences (Mansfield, immuno- or chemotherapies. 2001). Comparative analysis of several traits revealed significant differences between the two

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TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 139 II APPLIED GENOMICS: PROSPECTS and for research on the development of new drugs. FOR CONTROL OF AFRICAN TRYPANO- While this is encouraging news, treatment alone for SOMIASIS VIA THE TSETSE VECTOR a zoonosis for which there is no evidence of acquired immunity is likely to be extremely costly in the Serap Aksoy1, Wendy Gibson2, Ron H. Gooding3, long run even if effective drugs were to be available. Elliot Krafsur4, Anna Malacrida5, Michael J. The fact that the parasite relies on a single insect for Lehane6, Scott L. O’Neill1, Terry Pearson7, Alan S. its transmission opens up many avenues for control Robinson8, Philip Solano9, Antigone Zacharo- via the control of its vector. It is important to note poulou10 and Liangbiao Zheng.1 that trypanosomiasis is a disease based on interactions among at least three organisms, the human, 1Department of Epidemiology and Public Health, Section the parasite and the tsetse fly. Interference with any of Vector Biology, Yale University School of Medicine, 60 of these interactions can prevent disease. In fact, College St., 606 LEPH, New Haven, CT 06510, USA. tsetse control strategies have been widely used for 2School of Biological Sciences, University of Bristol, management of animal diseases. Most of these Bristol BS8 1UG, U.K. efforts however have been on a small scale, involv- 3Department of Biological Sciences, University of ing trapping and the use of insecticide sprays, and Alberta, Edmonton, Alberta, Canada. have been costly and difficult to sustain. A recent 4Department of Entomology, Iowa State University, 407 study looking into the cost/benefit analysis of vari- Science 2, Ames, Iowa 50011, USA. ous strategies clearly identifies large-scale, area- 5Dipartimento di Biologia Animale, Zoological wide methods as being far more efficient and affor- Laboratory, Universita di Pavia, Pavia, Italy. dable in the long run for tsetse control (Budd, 1999). 6School of Biological Sciences, University of Wales, In Lome, Togo, July 2000, the Organization of Bangor LL57 2UW, U.K. African Unity (OAU) endorsed a strategy of tsetse 7Department of Biochemistry and Microbiology, fly and trypanosomosis eradication on the African University of Victoria, Victoria, BC V8W3P6, Canada. continent by using an integrated approach includ- 8Entomology Unit, FAO/IAEA Agricultural and ing area-wide control strategies. The recent advan- Biotechnology Laboratory, A-2444 Seibersdorf, Austria. ces in molecular technologies and their application 9Institut Pierre Richet, 01 BP 1500 Bouke 01, Côte to insects have revolutionized the field of vector d’Ivoire. biology although progress in the tsetse field has 10University of Patras, Department of Biology, been slow. In this paper, we review the current sta- Laboratory of Genetics, Patras, Greece. tus of knowledge on the molecular aspects of tsetse with particular attention given to interaction with INTRODUCTION trypanosomes. We highlight areas where research Despite many decades of research on vector-borne efforts are needed and are likely to contribute either parasites and their development in mammalian to the development of new vector control strategies hosts, effective strategies have yet to materialize for or to the improvement of the efficacy and afford- control of any of the diseases with which they are ability of existing approaches. associated. At the same time, a heavy reliance on insecticides and therapeutic drugs has resulted in TSETSE GENOMIC STUDIES the spread of insecticide resistance in many vectors The trypanosome has been most studied in efforts to and the emergence of drug resistance in parasites, develop a mammalian-based vaccine. While these threatening the availability of effective tools to com- research efforts have turned trypanosomes into a bat these diseases. This scenario is also apparent for model eukaryote to study novel mechanisms of sleeping sickness in Africa where there are current- gene expression and cell biology, there are no effec- ly at least half a million people estimated to have tive products forthcoming for disease control in the contracted the fatal disease. Antigenic variation in foreseeable future. There are now genome sequenc- the mammalian host has hampered efforts for vac- ing programmes for the parasite, and the genome of cine development. The current strategies for man- the human host is being deciphered. In contrast, to agement of trypanosomiasis depend on active sur- date, only a handful of tsetse genes and proteins veillance and treatment of infected hosts, and on have been characterized. This is also in sharp con- limited vector control measures. These efforts are, trast to the vast level of knowledge available for however, restricted by the lack of effective drugs, mosquitoes such as Anopheles gambiae, for which an their high cost, adverse side effects, and the emer- international genome sequencing effort is now in gence of drug resistance in patients (McNeil, 2000). place, and Aedes aegypti, where a similar genome Thanks to recent international effort, there are now project is currently being planned. The many molec- several programmes in place that will allow contin- ular markers developed in A. gambiae have formed ued provision of the existing drugs for treatment the basis of a rapidly increasing number of popula-

140 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 tion genetics and ecological studies. The availability na, maternally inherited factors appear to be in- of contemporary genomics information will provide volved in hybrid male sterility (Gooding, 1990). Suc- us with essential tools for investigating parasite- cess in hybridizing members of the subgenus Glos- host interactions as well as vector population struc- sina is often highly asymmetric, with one cross being tures. This information is central for vector-based significantly more productive than the reciprocal control strategies to be effectively developed. cross. Gooding (1987) attributed this asymmetry to maternally inherited factors, which are now TSETSE POPULATION GENETICS believed to be Wolbachia (O’Neil, et al, 1993). These Population Structure factors can lead to unusual situations. For example, The recent availability of several microsatellite loci using G. m. morsitans and G. m. centralis it has been and their application to population analysis has possible to produce backcross males with maternal- revealed extensive genetic structuring in tsetse pop- ly inherited factors from G. m. morsitans and chro- ulations of the morsitans and palpalis groups at vari- mosomes from G. m. centralis, and yet such males ous geographical scales (Krafsur, et al, 2000; Krafsur can fertilize G. m. morsitans but not G. m. centralis and Griffiths, 1997; Krafsur, et al, 2000; Luna, et al, (Gooding, 1987). It was suggested that such flies 2001; Solano, et al, 2000; Wohlford, 1999). In the case could be used as genetic control agents against of G. palpalis gambiensis, these populations were G. m. centralis, but the phenotypic expression of shown to transmit different species of trypanosomes these maternally inherited factors is unstable with different efficiencies (Solano, et al, 2000; Solano, (Gooding, 2000; Gooding, 1990). However, if the sta- et al, 1997). Additional microsatellite loci and the bility problem can be overcome, this approach may development of efficient oligonucleotide primers are be useful for control of certain members of the sub- required to achieve the level of genetic resolution genus Glossina. necessary to measure accurately the differentiation of taxa and populations and gene flow within and Most hybridization studies on tsetse have used clo- among them. In addition, randomly amplified poly- sely related species and subspecies. There have been morphic DNAs (RAPDs) have been used successful- few “intra-taxon crosses”, using flies from colonies ly to examine paternal lines of gene flow, an impor- that were established from widely separated geo- tant tool in studying historical lines of dispersion graphic areas. Without exception, the latter studies (Malacrida, et al, 1999). These studies are important found that F1 males were fertile and this has been for vector control approaches such as the sterile interpreted as indicating that, within a nominal insect technique (SIT), which is being considered for taxon, there are no genetic barriers between geo- use in upcoming tsetse eradication campaigns. For graphically separated populations. However, hy- the successful application of SIT, information is re- brid breakdown has recently been found in tsetse quired on the tsetse species represented in the area, (Gooding, unpublished). When G. p. palpalis from the degree of reproductive isolation among field colonies that originated from widely separated geo- populations, the existence of natural barriers to dis- graphic areas were crossed, the first generation off- persion, and the locations of dense, stable popula- spring had normal fertility, but a high proportion of tions from which immigration is likely. males in the second and subsequent generations, and in backcrosses to the parental lines, were sterile. Hybrid Sterility The molecular basis for the hybrid breakdown is Hybrid sterility has been considered as a potential unknown, but its elucidation will be most helpful if control method for tsetse ever since Vanderplank hybrid breakdown is to be exploited for tsetse con- eradicated an isolated population of G. swynnertoni trol. Hybrid breakdown may be a useful genetic by release of G. m. centralis into its habitat (Vander- control approach for tsetse, especially if its manifes- plank, 1948; Vanderplank, 1944). In the two subgen- tations include reduced fecundity of females and/or era of Glossina that contain the most important try- reduced longevity of adult flies. In any case, the panosome vectors, Glossina sensu stricto and Nemo- existence of hybrid breakdown in tsetse suggests rhina, there are several groups of closely related taxa that there may be cryptic species of tsetse, a possi- that hybridize readily both in the field and in the bility that could have implications for evaluating laboratory (Gooding, 1990). Hybridization of tsetse what now appears to be simply geographic varia- almost always results in production of hybrid fema- tion in vector competence. Further work with other les with lower than normal fecundity and males that widely distributed species is clearly needed. are completely sterile (Gooding, 1990). The sterility in males is attributable mainly to incompatibility Polytene Chromosomes between sex chromosomes from two taxa, but auto- Polytene chromosomes have proven especially va- somal genes are also involved in some cases. In the luable in studies of chromosome structure and func- subgenus Glossina, but not in the subgenus Nemorhi- tion. They provide a means for the accurate map-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 141 ping of chromosome rearrangements and for the (Acosta-Serrano, et al, 2001). It has also been shown precise localization of genes by using both chromo- that the binding of a lectin (concavanalin A) to the some rearrangement analysis and the technique of procyclin coat molecules of the procyclic forms in situ hybridization. This has been technically chal- induces multinucleation, a disequilibrium between lenging in tsetse, but photographic polytene maps nuclear and kinetoplast replication and a unique have now been constructed for three species, form of cell death (Pearson, et al, 2000). Those sur- G. austeni, G. m. submorsitans, and G. pallidipes. Com- viving procyclic cells eventually proliferate in the parison of the homologous chromosomes between gut (establishment phase) and flies can be scored the three species indicates that, in addition to simi- with infections seven-ten days after acquiring an larities in their banding patterns, there are also var- infectious meal. The subsequent maturation phase ious major differences, especially between G. austeni occurs in the salivary glands for T. brucei (Vicker- and the other two species (Dr A. G. Papalexiou, man, et al, 1988). Here, they first differentiate into University of Patras, unpublished). Studies can now attached proliferating epimastigote forms which be undertaken to identify and characterize chromo- then yield the infective, free-living metacyclic forms somal rearrangements in field populations to which are transmitted to the next host during blood- understand genetic variation. The development of feeding by the fly (Vickerman, et al, 1988). It is at in situ hybridization using FISH analysis with this stage that parasites are thought to undergo cloned tsetse genes and selected molecular probes genetic exchange (Gibson and Whittington, 1993). such as microsatellites will facilitate the eventual The factors triggering this differentiation step are mapping of genes of interest (e.g. genes affecting unknown. There is believed to be a critical period vector competence, refractoriness, etc.). for maturation between days 8 and 11 after infection (Ruepp, et al, 1997; Sinkins, et al, 1995). One sugges- VECTORAL CAPACITY OF NATURAL TSETSE tion is that lectins have a role, since feeding lectin POPULATIONS inhibitory sugars can block maturation (Sinkins, et Trypanosome infections with T. brucei ssp. complex al, 1995). However, it is clearly crucial to investigate parasites are typically detected in less than 1% of the the role of other components of the immune system. field population of tsetse flies (Lehane, et al, 2000; Msangi, et al, 1998; Woolhouse, et al, 1994). Even The first physical barrier to ingested parasites in the under ideal conditions in the laboratory, transmis- gut is the peritrophic matrix (PM), which is a promi- sion rates are between 1-10% depending on the fly nent feature of the digestive tract of insects. While in species/strain and parasite strain (Moloo, et al, many adult insects, PM components are secreted by 1994; Moloo and Kutuza, 1988; Moloo, et al, 1992). midgut cells in response to a blood-meal, tsetse The basis for this refractoriness is not known, but adults have a PM constitutively synthesized from depends on tsetse species/strains and their symbi- cells in the proventriculus (cardia) in the foregut otic bacteria as well as the genotype of the strain of prior to feeding (Lehane, et al, 1996; Lehane and the parasite acquired. In laboratory infections, the Msangi, 1991). How trypanosomes cross the PM to age and sex of the fly and the source of the blood- move from the endo- to the ectoperitrophic space of meal also contribute to the outcome of the infection. the gut has been controversial, with penetration of Field data are limited, however infections with mul- the thick chitinous PM and migration around its tiple parasites are common, indicating that they open posterior end in the hindgut both having been were acquired in different blood-meals, hence fly proposed (Ellis and Evans, 1977; Welburn and age may not be as significant a factor as once Maudlin, 1999). It is generally thought that during thought. normal development in the fly there are no intracellular stages, although reports of intracellular The life cycle of T. brucei in the tsetse fly begins T. b. rhodesiense (Ellis and Evans, 1976; Evans and when it feeds from an infected mammalian host. Ellis, 1975) and T. congolense (Ladikpo and Seureau, The non-proliferating short stumpy parasites that 1988) in anterior midgut cells have been published. are pre-adapted for life in tsetse fly rapidly differen- It is also thought that during normal infection, try- tiate into procyclic forms in the gut lumen, lose their panosomes do not cross an epithelial barrier in the variant surface glycoprotein, and express a new coat fly, although once again there are several reports of composed of procyclin proteins. The procyclin coat trypanosomes in the hemolymph of flies (Mshel- contributes to the establishment of infections in the bwala, 1972; Otieno, 1973). Also unknown is how fly (Ruepp, et al, 1997). It has recently been shown procyclic parasites move from the gut to the mouth that procyclic cells express different procyclin coats parts and/or the salivary glands of the fly. The clas- during establishment in the gut and that the N-ter- sical route involves crossing back into the gut lumen minal domain of all procyclins are quantitatively across the proventriculus in the foregut and thence removed by proteolysis in the fly, but not in culture to the mouthparts and salivary glands (Ruepp, et al,

142 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 1997; Turelli and Hoffmann, 1991). This is indeed an panosomes can be significantly reduced (Zhen- incredible journey, which few trypanosomes appar- grong, et al, 2001). Further characterization of these ently embark on or complete (Ruepp, et al, 1997). immune mechanisms or products and their interac- Presumably the increased length and motility of the tion with trypanosomes are needed. In addition, a trypanosomes observed en route are adaptations study of other tsetse molecules, such as receptors enabling them to succeed (Ruepp, et al, 1997). involved in interactions with parasites, or molecules that are simply required for survival of tsetse or that TSETSE VECTOR COMPETENCE influence their fecundity, will be important for At the center of vector competence in insects are developing strategies to interfere with transmission immune reactions, which include a diverse set of of trypanosomes. These studies are of applied inter- mechanisms ranging from phagocytosis, activation est as they support the development of strategies to of proteolytic cascades, such as coagulation and block parasite transmission in vivo via transgenic melanization and production of various antimicro- approaches. bial peptides (Barillas-Mury, et al, 2000; Dimo- poulos, et al, 2001). While much of this immune TRANSGENESIS IN TSETSE response is initiated in the fat body, effector mole- Much effort has gone into developing genetic trans- cules expressed in the midgut are increasingly being formation systems for medically and agriculturally recognized as playing a role in immune reactions important insect vectors. There is no doubt that the (Dimopoulos, et al, 1997; Dimopoulos, et al, 1998; availability of this technology will revolutionize in- Lehane, et al, 1997). Discrimination between patho- sect genetics by allowing basic studies relating to gen groups is well known in Drosophila and mosqui- the functional characterization of various genes and toes, where it is explained by the use of different their products. It might also allow for the develop- receptor/signalling pathways. Because dipterans ment of alternative control strategies such as the use including mosquitoes and tsetse are largely refracto- of transgenic refractory insects. It is thought that ry to parasite transmission, much effort has gone such genetically engineered refractory insects can be into understanding the immune mechanisms of driven into natural populations to replace their sus- mosquitoes. If the genetic basis of this refractoriness ceptible counterparts and hence reduce disease tran- could be understood, then flies might be engineered smission. While the efficacy and feasibility of this to completely block parasite transmission using var- strategy are being widely debated among scientists ious recombinant approaches currently being devel- at large, in the case of tsetse, the development of try- oped. Immunity genes are also being studied as they panosome-refractory strains will have an immediate are strong candidates to confer such transmission application for at least one effective control strategy, blocking phenotypes for inducing refractoriness. As the sterile insect technique (SIT), as described below. a result of the application of molecular approaches, a good deal of information is now available about At the core of transgenesis is the process of genetic the response of mosquitoes to Plasmodium species. transformation, which in many insects relies on the microinjection of transposable elements that insert There is little known about the genetic basis for themselves into insect DNA, resulting in germ-line refractoriness in tsetse or the molecular basis for the transformation. Marker genes carried by the trans- wide ranging differences observed in parasite transposable element help identify transgenic individu- mission rates of different tsetse species. Lectins, als. It has now been possible to introduce foreign which are known immune molecules, might play a genes into several important insect vectors includ- role in the attrition of trypanosomes during estab- ing one important malaria vector in Asia, Anopheles lishment (Welburn and Maudlin, 1999). There is also stephensi (Cateruccia, et al, 2000), and others are like- some information demonstrating antimicrobial ly to follow using similar technologies. Tsetse, how- activity (Kaaya and Darji, 1988; Kaaya, et al, 1987; ever, have an unusual reproductive biology. There is Kaaya, et al, 1986), the presence of the phenoloxi- no free egg stage, females retaining each egg within dase cascade (Nigam, et al, 1997) in hemolymph. the uterus. Following hatching and in utero develop- Preliminary results indicate that the immune re- ment of the larva, one young larva matures and is sponse to different pathogens in tsetse is specific. finally expelled as a fully developed third instar These results also suggest that trypanosomes may larva. Each female can deposit four-six offspring use unprecedented novel mechanisms to achieve during its five-eight week average life span in the their transmission through the fly by blocking the field. This viviparous reproductive biology would expression of some of its immune responsive genes undoubtedly complicate any attempts to transform early in the infection process (Zhengrong, et al, tsetse through egg microinjection. However, tsetse 2001). When the immune system is upregulated flies naturally harbor a number of symbiotic micro- prior to an infectious meal, the transmission of try- organisms that have been exploited to express for-

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 143 eign gene products (Aksoy, 2000). Through such an symbionts to prevent parasite survival in the mid- approach, the insect cells are not transformed as in gut. The identification of monoclonal antibodies germ-line transformation, but instead, foreign genes (mAbs) with parasite transmission blocking charac- are expressed in the symbiotic bacteria. Since the teristics and their expression as single-chain anti- symbionts naturally live in close proximity to the body gene fragments in the symbionts provides an developing trypanosomes, anti-pathogenic gene alternative approach. Towards this end, several products introduced and expressed in these cells transmission-blocking antibodies targeting the could adversely affect trypanosome transmission. major surface protein of the insect stage procyclic trypanosomes have already been reported (Nantu- TSETSE SYMBIONTS AND EXPRESSION lya and Moloo, 1988). Recently, midgut-specific mo- OF FOREIGN GENES IN VIVO noclonals with transmission blocking activity have Many insects with limited diets such as blood, plant been characterized from A. gambiae (Lal, et al, 2001). sap or wood, rely on symbiotic microorganisms to It seems likely that similar molecules can be identi- fulfill their nutritional requirements. It has been fied in tsetse and ultimately expressed in the gut shown that tsetse harbour three distinct microor- symbionts. The relative ease of transformation and ganisms (Aksoy, 2000). Two of these are present in gene expression in bacteria, and the multitude of the gut tissue: genus Wigglesworthia (Aksoy, 1995; potential antiparasitic targets which can be explo- Aksoy, et al, 1995) and genus Sodalis, (Aksoy, et al, red, make this a desirable system for transgenic 1995; Cheng and Aksoy, 1999; Dale and Maudlin, approaches. Should resistance develop in parasites 1999) and both are closely related to Escherichia coli. against any of the expressed foreign gene products, The third symbiont harboured in certain tsetse it could be relatively easy to switch to express a dif- species is Wolbachia, an obligate intracellular bacteri- ferent gene product. Alternatively, several target um which is closely related to the genus Ehrlichia genes can potentially be expressed simultaneously (O’Neill, et al, 1993). During its intrauterine life, the in the symbionts to prevent the development of tsetse larva receives nutrients along with the two resistance against any one individual target. gut symbionts from its mother, via milk-gland secretions (Aksoy, et al, 1997; Ma and Denlinger, 1974), FIELD APPLICATIONS OF TRANSGENESIS while Wolbachia is vertically transmitted by transo- In order to interfere with disease transmission, the varial transmission. eventual goal of any transgenic approach is to replace the naturally susceptible population with It has been possible to culture the Sodalis symbiont their engineered refractory counterparts in the field. in vitro (Beard, et al, 1993; Welburn, et al, 1987) and At present, there are no proven mechanisms to a genomic transformation system has been devel- achieve this spread. One powerful potential driving oped (Beard, et al, 1993). Recently, a homologous system involves the use of Wolbachia symbionts, recombination approach has also been established which confer a reproductive advantage to their so that foreign genes can now be directly inserted hosts, including the engineered females. into the symbionts chromosome (Dale, et al, 2001). It has also been possible to reconstitute tsetse with the The functions of Wolbachia in their various hosts are recombinant Sodalis, which has been found to be variable. One common reproductive abnormality successfully acquired by the intrauterine progeny they induce has been termed cytoplasmic incompat- when microinjected into the haemolymph of the ibility (CI). This, when expressed, results in embry- female parent (Cheng and Aksoy, 1999). It is now onic death due to disruptions in early fertilization necessary to identify effective gene products which events (Bandi, et al, 2001). In an incompatible cross, have anti-trypanosomal effects when expressed in the sperm enters the egg but does not successfully Sodalis in tsetse midgut. Using a similar symbiont- contribute its genetic material to the potential based insect transformation approach, it has been zygote. In most species, this results in very few eggs possible to block the transmission of Trypanosoma hatching. Wolbachia infected females have a repro- cruzi in Rhodnius prolixus in vivo by expressing the ductive advantage over their uninfected counter- antimicrobial peptide cecropinA (cecA) in its sym- parts because they produce progeny after mating biont, Rhodococcus rhodnii, in the hindgut of the bugs with both infected and uninfected males. This repro- (Durvasala, et al, 1997). It has also been possible to ductive advantage allows Wolbachia to spread into express a single-chain antibody gene fragment in populations. In Drosophila simulans in the central the bacterial symbionts of R. prolixus (Durvasala, California valley, a natural Wolbachia infection et al, 1999). If the tsetse immune-responsive mole- invading naive uninfected populations has spread cules, which the trypanosomes apparently down- at a rate of over 100 km per year simply through the regulate to achieve their transmission, can be identi- expression of CI (Turelli and Hoffmann, 1991). To fied, they could be constitutively expressed in the understand the functional role of Wolbachia in

144 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 insects, most insects can be cured of their Wolbachia Cuisance, 1984; Vreysen, et al, 2000). Improvements infections by administering antibiotics in the diet. in two aspects of current tsetse SIT technology have This approach, however, is not feasible in tsetse the potential to enhance the efficacy of future pro- since antibiotic treatment results in the clearing of grammes (Aksoy, et al, 2001). all bacterial symbionts, including the obligate symbiont Wigglesworthia, in the absence of which the The first is the development of parasite refractory flies become sterile. In order to study CI expression strains. Since the large numbers of male flies re- in tsetse, uninfected flies need to be collected from leased can potentially contribute to a temporary the field and colonized so that appropriate mating increase in disease transmission, the incorporation experiments can be performed in the laboratory. As of refractory traits into the SIT release strains will Wolbachia infected insects replace naive populations greatly enhance the efficacy of this approach, espe- by virtue of the CI phenomenon, they can drive cially in human disease endemic foci. In the current other maternally inherited elements of tsetse, such field SIT programmes, male tsetse are provided, as the maternally inherited gut symbionts Sodalis, before release, with a blood-meal containing a try- into that same population (Beard, et al, 1993). It has panocide; no infections have so far been found in been proposed that multiple Wolbachia infections, in released sterile males caught in traps. The second is which an insect contains two or more different Wol- the use of Wolbachia mediated CI, or hybrid sterility, bachia strains that are incompatible with each other, as a method of inducing sterility - as an alternative could be used to generate repeated population re- to irradiation. With CI, the released strain of tsetse placements or to spread Wolbachia into target species would carry a Wolbachia infection that would induce that already contain an existing infection (Sinkins, CI when males mate with wild females. The com- et al, 1995; Sinkins, et al, 1997). The analysis of petitiveness of these males would be expected to be Wolbachia strain types infecting different species of much higher compared with irradiated males, and, tsetse has shown that they are different, and as such as a result, fewer insects would need to be released represent independent acquisitions (Cheng, et al, in order to achieve the same level of sterility in the 2000). wild population. This strategy is dependent on the use of a very efficient sexing system. If Wolbachia- In addition to CI, certain Wolbachia strains, such as infected females were released in sufficient quanti- wMelPop characterized from Drosophila melanogaster, ties, then Wolbachia would have the opportunity to induce an age-shortening effect in their host (Min invade the target population, which would render and Benzer, 1997). This age-shortening effect was subsequent releases ineffective. If it were impossible reversed when infected flies were cured of their to guarantee extremely low quantities of released Wolbachia infections with antibiotics (Min and females, then it would be possible to incorporate Benzer, 1997). It remains to be seen whether such low levels of irradiation with Wolbachia induced Wolbachia infections can be documented in tsetse. It sterility to prevent released females from successful- has also been possible to introduce Wolbachia into ly reproducing. This approach has been successfully insect species which do not harbour natural infec- tested in Culex mosquitoes (Shahid and Curtis, tions, and these approaches can be pursued in 1987). tsetse. Since the T. brucei group parasites require 14- 30 days to complete their developmental cycle in the CONCLUSIONS fly, tsetse flies need to be at least this age to transmit • Understanding the molecular/cellular basis of disease. Reductions in the life span of individual trypanosome transmission in tsetse is of funda- flies might have a large effect on disease transmis- mental significance, and will allow development sion in the field (Sinkins and O’Neill, 2000). of new applications for vector control. Since the symbiont-based transformation system can be STERILE INSECT TECHNIQUE AND used to express gene products in tsetse midgut, TRANSGENIC INSECTS such will aid in the identification of candidate SIT is a genetic population suppression approach genes that can be expressed to confer refractori- and involves sustained, systematic releases of irra- ness in tsetse. diated sterile male insects among the wild population. The sterile males fertilize wild females, which • A research plan should be developed to coordi- are then unable to produce progeny. By continually nate information on expressed sequence tag (EST) releasing sterile males in high numbers over a peri- sequences, genomic sequences and the physical od of three-four generations, after having previous- map locations of selected genes. For EST analysis, ly reduced the population density by other tech- the cDNAs to be sequenced can be obtained from niques (trapping, insecticide spraying, etc.), the tar- tsetse exposed to infected blood-meals containing get population can be eradicated (Politzar and eukaryotic pathogens, including trypanosomes,

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TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 149 III APPLIED GENOMICS AND contain the majority of genes, but the DNA content BIOINFORMATICS of the mini- and intermediate chromosomes should be investigated in the future. Sara E. Melville1 and Daniel Masiga2. 1Cambridge University, Department of Pathology, Most megabase chromosomes differ in size from Microbiology and Parasitology Division, University of their homologues by up to 15%, but homologous Cambridge,CB2, IQP, U.K. chromosomes in different stocks vary more - con- 2International Centre of Insect Physiology and Ecology siderably more than reported in other organisms. (ICIPE), Molecular Biology and Biotechnology Unit, PO Nevertheless, mapping studies show that syntenic Box 30772, Nyayo Stadium (00506), Nairobi, Kenya. groups are maintained in all stocks studied (Mel- ville, et al, 1998). Despite remarkable genomic plasticity (Melville, et al, 1999), studies reveal a signifi- OVERVIEW OF THE T. BRUCEI GENOME cant level of conservation of genome structure. AND THE HISTORY OF TRYPANOSOME Initial apprehension that the genome was so poly- GENOMICS morphic as to make sequencing of one strain of little use for cross-comparison to other strains has been The African trypanosome genome network was assuaged – although further studies could investi- formed by the UNDP/World Bank/WHO Special gate the role this plasticity is playing in the survival Programme for Research and Training in Tropical strategy of the trypanosome. Diseases (TDR) with a brief to coordinate the analysis and sequencing of the nuclear genome. It con- There is no perfect isolate that should form the basis sists of research laboratories worldwide, contribut- of all molecular experiments on T. brucei. It is indeed ing to different aspects of genome analysis. At the preferable that some genomic analyses are carried time of its formation, some aspects of genome out on multiple stocks, for example karyotyping structure and organization were known, especially and random gene sequencing. However, we can from studies on antigenic variation and transcrip- only aim to sequence one complete genome in the tion, but only one group had actively initiated immediate future. T. brucei stock TREU927/4 global genomic analysis. In the last five years, (GPAL/KE/70/EATRO1534) was cloned from a much has been learnt about the organization of the population of trypanosomes isolated from a tsetse nuclear genome of T. brucei, which consists of at fly in Kenya and was chosen as the reference least eleven diploid megabase chromosomes (1-6 genome for a variety of reasons, some optimal and Mb), a variable number of intermediate-sized chro- some pragmatic. The original stock exists and the mosomes of indeterminate ploidy (200-900 Kb), history of its isolation is documented. It was not iso- and 50-100 minichromosomes (25-100 Kb) lated from an infected human, but laboratory tests (Melville, et al, 1998). indicate that it has intermediate resistance to human serum (CMR Turner, personal communication). It is With the exception of some variant surface glyco- pleomorphic and may be replicated as the blood- protein (VSG) genes, most expressed genes are stream or procyclic form in laboratory animals, located in the megabase chromosomes. These are tsetse flies or culture. It has been used as a parent in inherited in a Mendelian fashion (Turner, et al, 1990) laboratory-controlled genetic crosses with other and have been assigned Roman numerals in order stocks, and cloned hybrids have been isolated of increasing size in TREU927/4 (Turner, et al, 1997). (Turner, et al, 1990; Tait, et al, in press), allowing the Many, perhaps most, genes are expressed in poly- creation of a genetic map. A high quality, arrayed cistronic transcripts (Vanhamme and Pays, 1995). genomic library of the megabase chromosomal Some chromosome ends carry VSG expression sites: DNA of TREU927/4 already existed (Melville, et al, only one is active at one time, resulting in a uniform 1996), and was used to determine the structure of a surface protein coat. Gene- or promoter-switching megabase chromosome (Melville, et al, 1999). results in antigenic variation (Barry and McCulloch, 2001), which is an effective way for the parasite to Participation in the network is dynamic because, as evade destruction by the host immune system and priorities change, researchers with different skills results in the characteristic fluctuating parasitemia are required. Many contribute without direct fund- and accompanying fever. The intermediate chromo- ing, and the network is dependent on the support somes also carry expression sites (Rudenko, et al, and active participation of the research community. 1998), and most mini-chromosomes contain non- It has been vital to promote universal commitment transcribed VSG and simple repeat sequences to the idea that this huge task can progress most effi- (Weiden, et al, 1991). The network has prioritized ciently if the community works together with open sequencing of the megabase chromosomes, as they sharing of data and resources.

150 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 The availability of the DNA sequence will benefit gle-pass sequence reads to aid gene discovery and many research programmes. Some benefits are al- facilitate the completion of contiguous chromosome ready obvious, some will only become clear as more sequences (http://ftp.sanger.ac.uk/pub/databas- researchers begin to use the available data. This is es/T.brucei-sequences). our task: to be forward-looking and imaginative in our plans for the use of this tremendous resource. The EST and GSS sequences have been clustered by collaborators at the Sanger Institute to provide CURRENT STATUS OF THE contigs. In total, 96 474 sequences (~45.87Mb) TRYPANOSOMA BRUCEI GENOME PROJECT were used for the clustering, achieved using the Expressed Sequence Tags (ESTs) sequence assembly programme Phrap - 12 251 Rapid gene discovery was achieved in the early contigs were generated and 8242 sequences could phase of the genome project by sequencing of ran- not be placed in a contig (singletons). One of the domly selected cDNA clones (El-Sayed, et al, 1995; contigs has 1926 constituent members (contig Djikeng, et al, 1998). At the time of writing, there length 9.342Kb), but this is probably due to are 5133 T. brucei ESTs in the public databases from repeated DNA and is an example of the care that four cloned stocks of T. brucei (dbEST: must be taken in interpreting these automatically http://www.ncbi.nlm.nih.gov/dbEST/index.html). generated data. The cluster data are available as a Most EST sequences were generated from cDNA searchable subcomponent of the T. brucei BLAST clones of bloodstream-form mRNA. There has been server (http://www.sanger.ac.uk/Projects/T_bru- no concerted effort to produce large EST datasets cei Toolkit/blast_server.shtml) and from the ftp site from each of the life-cycle stages as it was decided to as a gzip file (hftp://ftp.sanger.ac.uk/pub/databas- divert effort and resources to sequencing of genomes/T.brucei_sequences/GSS/clusters). ic DNA. The determination of life-cycle stage-specific expression will be undertaken by other meth- Physical and Genetic Mapping ods (see section 3.5). In addition to discovery of One chromosome was mapped thoroughly and to many novel T. brucei genes, the ESTs have provided completion (Melville, et al, 1999) prior to submitting a rich source of markers (Melville, et al, 1998) and sequencing grants. However, rapid progress in aid sequence annotation. methods for completion of genome sequences has reduced the requirement to produce prior contigu- Genome Survey Sequences (GSSs) for Gene ous physical maps of each chromosome (El-Sayed, Discovery et al, 2000). Nevertheless, mapping information is In a pilot project, approximately 500 random required for seeding BAC sequencing and to aid genomic clones were sequenced (El-Sayed and final reconstruction of the chromosome. Many Donelson, 1997) to show that this led to equally effi- hybridization data have been amassed by the cient gene discovery, due to the lack of introns and Cambridge group and also by the community the close spacing of genes in the T. brucei genome. through using centralized resources. So far, these Therefore, it was decided that a portion of the funds data have been made freely available but only by obtained for high-throughput sequencing at The email request. A database is being established to Institute for Genomic Research (TIGR) should be make the data web-accessible (see 4.4). allocated to sequencing of random, short pieces of DNA (GSSs). Sequencing of both ends of almost TDR and The Wellcome Trust have also supported 25000 clones (almost 50 000 short sequences) pro- the creation of a genetic map using classical genetic vided a total of 29 Mb of the TREU927 genome analysis of F1 hybrids (Tait and Turner, Glasgow). (http://www.tigr.org/tdb/mdb/tbdb/status.html). F1 hybrids have been isolated following simultane- A proportion of these sequences derive from the ous passage of genotypically distinct stocks through ends of large genomic clones (in bacteriophage P1 tsetse flies. This group has developed amplified and bacterial artificial chromosome (BAC) vectors), restriction fragment polymorphism (AFLP), mini- and these contribute to the mapping and sequenc- and micro-satellite markers for use in genetic analy- ing of whole chromosomes (see section 2.4) by pro- sis and, together with the genomics group viding paired markers of about 500 base pairs every (Melville), has been able to combine some of the 2.5Kb across the chromosomes (excepting the data with chromosome maps. The aims are to deter- minichromosomes). Many researchers have report- mine crossover frequency, estimate the physical size ed finding T. brucei homologues of known genes in of the recombination unit (Centimorgan), and inves- the GSSs, and this generated great enthusiasm for tigate variation in crossing-over in different genom- the rapid provision of more such sequences. In ic regions. Initial data of this type indicate that it is response to requests from the community, the indeed feasible to obtain sufficient hybrid progeny Sanger Institute has provided a further 47 000 sin- and genetic markers to aim towards using a genetic

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 151 map for positional cloning of genes underlying com- the data accessible and informative, and to stimulate plex phenotypes (Tait, et al, in press). new ideas in the search for novel approaches to combat African trypanosomiasis. Genomic Sequencing Sequencing of chromosome I of T. brucei strain 927/4 GUTat 10.1 commenced at the Sanger Institute in Access to Data 1998. Sequence was obtained from shotgun clones of The fields of genomics, databases and bioinformatics chromosomal DNA eluted from a pulsed field gel are dynamic and researchers may find it difficult to (PFG) (> 8 X coverage) and from mapped P1 clones learn how best to access the most recent data, or (1 X coverage), a combination of methodologies pio- where to find the most innovative new programmes neered by the malaria sequencing consortium. While for analysis. The field is developing rapidly and here approximately 75% of the chromosome sequence we aim to provide an outline of where data may be was contiguous by year two, 25% of the chromo- found at different stages of the sequencing projects. some has proved difficult due to repetitive DNA: Complementary DNA (cDNA) sequencing has been genes in VSG expression sites, gene families, retro- carried out in individual research laboratories and transposons, and some tandem repeats (Melville, the sequences are made available via the database et al, 1999). Following the random sequencing phase for expressed sequence tags (dbEST) at the National (see 2.3), TIGR commenced the sequencing of chro- Center for Biotechnology Information (NCBI). All mosome II in 1998. This sequence is derived from genomic sequence data are made available immedi- BAC clones, mapped to chromosome II using ately via the websites at the respective sequencing cDNAs from the EST project (Melville, et al, 1998; centres (TIGR and Sanger Institute). These are sin- El-Sayed, et al, 1995) and sequenced to ca. 7 X cover- gle-pass sequences and, although no error correction age. The end-sequences determined in the first or annotation are offered at this stage, this is an phase of the project (section 2.2) allow the selection important resource for researchers who are looking of BACs with minimum overlap for maximum effi- for genes in T. brucei that have homologues in other ciency. The progress on chromosome II has been sim- organisms. The clustering data provided by the ilar to that on chromosome I, with similar problems Sanger Institute are also generated automatically in regions of clustered multicopy sequences. and any errors will be incorporated. Therefore, use Chromosomes IV and VI are currently approximate- of such data always requires verification by the ly 75% completed at TIGR. Preliminary (and there- researcher. Search engines are provided, allowing fore in parts inaccurate and incomplete) annotation researchers to look for sequences with high similari- is provided by the sequencing centres prior to com- ty to the gene they seek (http://www.ebi.ac.uk/ pletion. It is best to follow progress by regularly blast2/parasites.html; monitoring both sequencing centre websites http://www.tigr.org/tdb/mdb/tbdb/seq_search.ht (http://www.sanger.ac.uk/Projects/T-brucei/; ml). Data on significant similarities to genes in the http://www.tigr.org/tdb/mdb/tbdb/index.html). databases are provided but researchers should note that new information becomes available in the cen- The funds to complete chromosomes IX, X and XI tral databases (European Molecular Biology Labora- were awarded to Barrell (Sanger Institute), Melville tory, EMBL/GENBANK/Database of Japan, and the network in 2000. Chromosome X shotgun DDBJ) all the time, and should check when the sequence is now available (http://ftp.sanger.ac.uk/ most recent BLAST searches were performed. At pub/databases/T.brucei-sequences) and chromo- intervals, sequences are submitted in batches to the somes IX and XI are in library preparation. The public databases at the European Bioinformatics funds to complete chromosomes III, V, VII and VIII Institute (EBI) and NCBI. Random genomic shotgun were awarded to El-Sayed at TIGR (collaborators sequences and end-sequences of genomic clones are Donelson, Ullu, Melville) in 2001. Each sequencing submitted to the database for genome survey centre will therefore provide approximately 50% of sequences (dbGSS) (http://www.ncbi.nlm.nih. the megabase chromosome DNA sequence. The proj- gov/dbGSS/index.html). Sequences of shotgun ects run until 2004 and all the sequence is likely to be clones derived from whole BACs are submitted to in the databases by 2003 at the latest; however it can- the database for high-throughput genomic se not be foreseen how long it will take to complete quences (dbHTGS) (http://www.ncbi.nlm.nih. contiguation. gov/dbHTGS/index.html) in three stages (at 3 X and 7 X coverage, and at completion). All these data To make full use of the substantial investment in will eventually be mirrored in the GENBANK, genome sequencing it is necessary to complete the EMBL and DDBJ databases, ensuring that all avail- task, to ensure there is complete information on all able T. brucei sequences may be found in a single enzyme pathways. It is also absolutely imperative to database. However, there are some advantages in invest substantial effort into bioinformatics to make looking at the data in the specialized genome data-

152 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 bases, as annotation is more extensive and sequence Predicted timeline to 2002-2004: similarity assignments are provided. • 1 x sequencing of IX, X and XI-specific BACs. • Chromosomes III, V, VII and VIII to be sequenced On completion of a chromosome sequence, the most BAC by BAC and appearing in the databases careful annotation is carried out. Great emphasis is piecemeal over the next two years. laid on ensuring the sequence is accurate and con- • Contiguation and annotation of chromosomes III tiguous (some areas of ambiguity may be tolerated in – XI over at least three years. the final sequence, if too many resources are required to provide final clarification), and on trying to identi- The timeline to complete sequencing is the most dif- fy all open reading frames (ORFs). Researchers ficult to predict, and depends to some extent on the should be aware that, however careful the annota- determination to complete subtelomeric regions, tion, some protein coding genes may not be predict- VSG arrays, etc. ed from sequence analysis alone and a few of those predicted may not be transcribed. The analysis of CURRENT STATUS OF FUNCTIONAL the chromosome is published in a peer-reviewed AND APPLIED GENOMICS PROJECTS journal and the sequence, with full annotation, can The African trypanosome genome network has ini- then be viewed with the Entrez browser at NCBI tiated discussions on the “post-genomic” agenda. (http://www.ncbi.nlm.nih.gov/Entrez/Genome/ This requires careful coordination and the involve- org.html). The sequencing centre responsible for the ment of appropriately skilled researchers. At the sequencing of the chromosome also places the same 1999 and 2000 network meetings, lists of priorities data on its website. drawn up (http://parsun1.path.cam.ac.uk/network.htm) included microarrays for transcription In an ideal world it would be possible to carry out analysis, techniques for gene knockouts, generation searches of all data at a single site but the curation of of mutants, RNAi and phenotype characterization, such a database would necessitate delay in access to and use of the genetic map for positional cloning of the data. Immediate access to raw, unfinished se- genes (some to proceed in collaboration with other quence is highly prized by a research community kinetoplastid genome networks). Recommendations waiting impatiently for gene sequences, and the of interest to this discussion include identification of sequencing centres have responded to this need. trypanosome-specific genes essential for infection This is only really possible via deposition on home and development, characterization of molecular websites prior to batch submission to NCBI. It is structures and parasite-specific metabolic path- therefore important for researchers to consider ways, elucidation of unique trypanosome-specific which databases contain the most valuable data (e.g. mechanisms of gene regulation and RNA process- the public genome databases because of their exten- ing, and analysis of the protein profile of the para- sive annotation, or the sequencing centre databases site to identify functionally important genes (pro- because they may contain sequences not yet proteomics). Perhaps one or more of these approaches cessed for transfer to NCBI) and, in some cases, to could lead to the identification of novel drug tar- perform searches at several sites. Long-term curation gets, and/or vaccine candidates, but only if those of interrelated genomic and functional data is a dif- researchers skilled in, and committed to, their deve- ferent consideration (section 3.1). lopment join the global collaboration and contribute to the post-genomic projects of the future.

Summary and Predicted Timeline. Sequence Analysis and Relational Databases The following sequence data are available in 2001: The Wellcome Trust Functional Genomics Initiative • 5133 EST sequences, providing approximately has provided funds to create a trypanosome data- 2000+ unique genes. base (genome databases for Schizosaccharomyces • 96 474 GSS sequences (~45.87Mb), including 10 pombe, Leishmania major and T. brucei ) to Drs B 990 BAC and P1 end-sequences. Barrell, Sanger Institute; M Rajandream (S. pombe), • Clustering data of all GSS and EST sequences, A Ivens (L. major); M Carrington and S Melville with data on significant similarities. (T. brucei). These databases will contain all genomic • The complete sequence of chromosomes I and II information, e.g. primary sequence annotation from (to be fully annotated in 2001). the sequencing centres, secondary (ongoing) se- • 75% of chromosomes IV and VI as complete BAC quence annotation, references, expression data, pro- sequences. tein characterization, knockout and RNAi pheno- • Chromosome X shotgun sequence (04/01); chro- type analyses. They will be relational databases mosomes IX and XI shotgun sequence to follow. using SQL (structured query language) and hosted by the Sanger Institute. The T. brucei database will

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 153 be developed in close collaboration with TIGR, and ture (M Turner, personal communication). The kary- is overseen by a management committee. Genome otypes are identical. The P1 library is prepared from databases of this type are long-term projects, pro- 927/4 and the BAC library from 10.1. The primary viding a single site to review ongoing genomic and sequencing substrate is 10.1 procyclic genomic functional genomic analyses long after sequencing DNA. of the reference strain is complete, and as such serve a different function to the rapid access sequence Stock 927 has the smallest nuclear genome of all deposition on the sequencing centre websites or the T. b. brucei or rhodesiense stocks examined so far with parasite genome BLAST server (see 2.5). News on approximately 10 Mb less DNA (Melville, et al, progress will be posted on relevant websites. 1998), reducing the amount of funding and sequencing required quite considerably. It is capable of com- The importance of ensuring that this database is pleting the life cycle, indicating that all vital genes readily available to African scientists hardly needs are present and it is likely that much of the extra explaining - they should not need to rely on collab- DNA consists of expansion of multicopy DNA – orations with laboratories in Europe or the Americas gene familes and repeats. Nevertheless we must to gain direct access to such comprehensive data. In always remember that trypanosomes are phenotyp- the long-term, the enabling of molecular biological ically variable, and that the basis of this variation research on trypanosomes will require that African must lie in the genome. It will require thought and laboratories have secure Internet connections of innovation to find methods to compare the geno- wide band-width. Until such time as this is possible, types of phenotypically variable strains. we should consider providing CD versions of the database at regular intervals. This may be proposed Both 927/4 and the derivative 10.1 may be trans- to the database committee via Dr Melville, and will formed with foreign DNA at the procyclic and require some coordination. bloodstream-form life cycle stages. Transformation of the bloodstream-form is less efficient than in Bioinformatics Projects strain 427, but this is common to most strains that The field of bioinformatics is changing rapidly and have not been replicated in vitro for long periods. A the databases described here will not remain static. line of GUTat10.1 expressing the tetracycline (TET) NCBI, EBI, the sequencing centres (and many oth- repressor has also been produced and tested. This is ers, including academic institutions) are actively useful for any experiment involving inducible developing better analysis tools, and it is necessary expression or inducible ablation of transcription/ to watch their websites for innovations and devel- translation, and this line is currently available from opments. Professor Christine Clayton, Heidelberg (van Deur- sen, et al, 2001). In addition, there is certainly scope for specific bioinformatic analysis projects, either based on the As stated in the introduction to this section, stock examples from more advanced genome projects 927/4 is found to be ‘intermediate’ in its resistance (yeast, C. elegans) or on novel approaches based on to lysis by human serum (Turner, personal commu- parasite-specific interests. For example, investiga- nication). The SRA (serum resistance-associated) tion of the components of metabolic enzyme path- gene has been found in the genome databases (Pays, ways (requiring skilled biochemists, but also novel personal communication), although it is not yet bioinformatics approaches to reduce the time known if it is expressed in the correct form. The- required for the largely manual approach currently refore, bearing in mind that T. b. brucei and T. b. rho- used); comparison of metabolic pathways to the desiense may only be genotypic variants of a single homologous pathways in humans and livestock species and that the basis of serum resistance may (again requiring biochemists and informaticians); not lie exclusively in SRA expression, it should be searching for commonalities in the biochemistry of assumed by all researchers using 927/4 and its the kinetoplastid parasites, that differ from the derivatives as bloodstream forms in experiments mammalian host; analysis of targeted or global/sin- that there may be human-infective cells in the po- gle-pass comparative sequencing of other trypano- pulation. some strains and species. Transformation Technology Biological Characteristics of the Reference Strain Transformation technology for genetic analysis of A minimally culture-adapted line of the original T. brucei is in routine use (Clayton, 1999). There are stock TREU927/4 has been generated with greater multiple vectors, several types of transformation stability of variant antigen types (TREU927/4 markers (drug resistance, fluorescence) and a sys- GUTat 10.1). This grows to a higher density in cul- tem of inducible expression. Foreign DNA inserts

154 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 into the genome by homologous recombination and post-translational controls on gene expression, and this can be precisely targeted if the exact sequence of will provide a firmer basis for the design of many the target site is known or determined, as recombi- experiments. For example, if transcription levels are nation occurs preferentially at sites of exact homolo- found to be relevant in at least some cases, microar- gy. Expression from episomal vectors is less success- ray technology could be very useful to compare ful. At Tri-Tryp 2000, there was some discussion of transcription in strains of different clinical pheno- the need for technology development. There was a type, or to examine mutant strains, to observe not proposal that enhanced transformation efficiency of only the ablation of the targeted transcript but also bloodstream forms is one requirement for high- any “knock-on” effect on other genes. For now, the throughput analysis (e.g. of gene knockouts or discriminative power of microarray technology in RNAi mutants) of the mammal-infective form, as some respects remains unproven. It also remains an transformation of the procyclic forms (which is open question whether an array based on a single more efficient) followed by transformation of the strain (927) is sufficient to detect all relevant differ- cells to bloodstream forms requires passage through ences; it is possible that strains carry environment- the tsetse fly. Of course, the ability to culture other specific genes and that strain 927 lacks the genes of life cycle stages, e.g. metacyclic forms, would also interest. Nevertheless, if arrays are made available represent a considerable improvement in the tools for use by the community, such preliminary (”look- available for genetic analysis. and-see”) investigations could be carried out at little cost. No doubt as production costs become less pro- Microarrays hibitive, the number of strains from which arrays Some doubts have been expressed regarding the are available will increase. usefulness of expression analysis in the Kinetopla- stidae. The observed polycistronic transcription The materials currently available for the creation of suggests that control of gene expression is entirely standard DNA-spot microarrays are the ESTs and post-transcriptional, a possibility supported by GSS clones. The ESTs almost all derive from blood- reports of protein products of genes within the same stream forms. Two GSS libraries have been used: polycistrons that were found to be up/down-regu- one with average inserts of 2 Kb and the other with lated at different life cycle stages. Nevertheless, inserts of of 4 Kb. The GSS libraries may prove as there clearly is some life cycle stage-specific tran- useful as EST libraries for microarray analysis due scription and this is useful data for investigation of to the high gene density in the genome, and they are processes such as infectivity, etc. It is easy to obtain certainly more representative of all the genes in the large amounts of cultured procyclic RNAs from genome than are EST libraries. However, the clones many strains, but perhaps prudent to perform some may contain fragments of more than one gene, thus experiments to compare cultured procyclics with complicating full analysis of the dataset. The useful- those extracted from tsetse flies. It is possible to ness of GSS arrays is being investigated (Clayton, obtain sufficient RNA from bloodstream forms Heidelberg). Meanwhile, El-Sayed (TIGR) has pro- replicating in laboratory rodents, although slow- duced the first ORF-specific array by amplifying growing (e.g. less virulent) strains can prove more fragments of ORFs identified from the sequence of problematic (but the rate of improvement in chromosome II (El-Sayed, et al, 2000). A UK group microarray techniques is still in the exponential (Melville, Matthews, Turner, Sanger Institute) is phase). Unfortunately, it is very difficult to obtain developing a pilot project to test the relationship sufficient DNA from epimastigotes or the infective between post-transcriptional and post-translational metacyclic forms. The metacyclic life cycle stage is processing. The long-term aim, if the usefulness of arguably the most relevant for studies of infectivity, microarrays is proven, is to establish whole genome and for vaccine development. With the involvement ORF-specific microarrays within four years as a col- of researchers with tsetse fly colonies, it may be pos- laboration between TIGR and the UK group. These sible to perform some careful experiments with would have to be made available as a resource - metacyclic DNA after full optimization of the array either the arrays themselves, the DNA, the oligonu- technology. cleotides, or simply the oligonucleotide sequence. It is hard to predict at the present time what the Still, for the examination of wild-type/reference demand will be or how the technology will develop. strains, it remains necessary to carry out specific experiments that detect changes in levels of individ- An additional aim of the network is to develop stan- ual mRNAs after processing of polycistronic tran- dard operating procedures for microarray analysis scripts, and to compare these to the concomitant lev- and presentation of data in collaboration with other els of the protein products. This will provide data on organizations, including the EBI, where they are the relative importance of post-transcriptional and establishing a public repository for microarray-based

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 155 gene expression data (http://www.ebi.ac.uk/array- database could include: express/). These organizations are, in turn, mem- • unique marker name/identifier. bers of the Microarray Gene Expression Database • type of marker. Group (MGED), whose aims are to facilitate the • oligonucleotide primer sequences. adoption of standards for DNA-array experiment • polymerase chain reaction (PCR) parameters. annotation and data representation, as well as to • size(s) of PCR product in genome project refer- introduce standard experimental controls and data ence strain 927. normalization methods (http://www.mged.org/). • brief description of population studied and results. Proteomics • list of allele variants in named isolates with Proteomics is a rapidly developing field that details of isolate history. encompasses small-scale 2-D gel analysis to high- • references. throughput techniques such as mass spectrometry • coordination with physical mapping/sequencing that are not available to all. Analysis of the T. brucei data to provide information on genomic location, proteome is ongoing in several laboratories (e.g. etc. Wellcome News issue 26, Q1, 2001, p. 19). However, for a coordinated network approach to global pro- Over time, sufficient information should accumu- teomic analysis, it is necessary to develop standard late to facilitate the choice of suitable markers for operating procedures and common marker proteins given areas and defined experiments, without before any proteomic data are presented on the web. extensive literature searching or unnecessary collab- This was discussed at some length at Tri-Tryp 2000, orations. and the first step towards this goal in the T. brucei network is to combine proteomic and array analysis RNA Interference as a Method to Investigate as discussed in section 3.5. Gene Function A common approach to the investigation of gene Novel DNA-Based Markers for Epidemiology, function is to remove the gene from the genome Diagnosis and/or Positional Cloning (gene knockout) or to prevent its transcription and DNA-based markers have become increasingly translation. Gene knockout requires two rounds of important in epidemiology and diagnosis in the last transformation and gene knockout, as each gene is decade. The availability of genome sequence offers present as diploid alleles. RNAi functions by trans- the capacity to identify a whole range of new mark- formation of a plasmid encoding a double-stranded ers (microsatellites, minisatellites, single nucleotide RNA that is homologous to part of the transcript polymorphisms or SNPs, indels) at much greater under study. By an unknown mechanism, the pres- efficiency than before. Previously, satellite markers ence of the exogenous dsRNA prevents the transla- were identified by cloning and hybridization of tion of the endogenous mRNA (Ngo, et al, 1998). individual oligonucleotides, or by serendipity, This requires only one round of transformation and whereas now they are identified using informatics. is useful for the analysis of function of single-locus The identification of SNPs and indels requires se- and multi-locus genes. To date, RNAi is most suc- quence from different strains, but may provide the cessful in procyclic forms, although many groups advantages of isoenzyme analysis – stability over are now working to improve efficiency in blood- millennia - at less cost in terms of money and resear- stream forms. cher time. There is no doubt that numerous research groups are considering using the DNA sequence in A group of UK researchers (M Field, Imperial this way, and we may see more papers appearing College, et al) has recently been awarded a grant using recently identified polymorphic markers. from the Wellcome Trust Functional Genomics Committee to begin analysis of gene function in It seems, then, timely to suggest that these data T. brucei by RNAi . This will involve analysis of phe- could usefully be collated at a central site rather notype following systematic disruption of individ- than leaving individual researchers to draw up their ual genes identified on the sequenced chromosomes separate lists from the literature. This may also pro- I and II. It is the requirement of these types of grants ve especially useful to scientists in institutions with that both the data and the biological resources are less access to journals. This is not to subvert the use made available to the community. The data will be of publication, which is vital to all researchers, but web-accessible and prepared in collaboration with rather to suggest that, on publication, all markers the functional genomics database (see section 3.1). should be submitted to a central database and that Again, it may be necessary to consider how this this database should be available to all – on the web information may be accessed from areas with inse- and, if necessary, on CD (see section 3.1). Such a cure access to the Internet. The DNA resources (con-

156 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 structs of vector plus gene sequence for transforma- leads for biochemists to investigate further at a tion) will be available from Melville/Cambridge molecular level and/or for design of high- (see section 4.5). It is hoped that the transformed cell throughput combinatorial screening program- lines (i.e. 927 mutants) will be available from mes. However, the latter will require funding for Turner/Glasgow (see section 4.6). the considerable work involved in such analyses, even in the preliminary stages. Applied Genomics: Discovery of • At this stage, the retention of such work in the Novel Drug Targets academic sector due to lack of interest from phar- The authors are not experts in drug target validation maceutical companies could be exploited to en- and, to avoid charges of naivety, first wish to ack- sure that such studies are coordinated, data sha- nowledge the considerable work by leading groups red, basic functional data all web-accessible, and of biochemists on parasite-specific pathways that effort is not wasted. (Although, future problems may represent valid targets for design of drugs of with intellectual property rights have to be con- greater specificity. No doubt such projects will be sidered.) brought to the discussion table by other contribu- • The establishment of the genome network has tors. However, under the maxim that no single provided one paradigm of how the research com- method guarantees the discovery of a better drug, munity can pull together to collaborate rather and that all methods (rational design and global than compete. There are already well-established approaches) have potential for both success and fal- groups with an interest in increasing funding for se leads, we wish to pose the questions: is it neces- the discovery of new drugs for diseases of the sary to expand our efforts now to increase the num- poor, e.g. TDR working groups, the Access to ber of leads on the table? and how can we best use Medicine Campaign. Is it now possible to bring the limited funds available? This discussion needs (sections of) these disparate groups together to to take place across a wide range of researchers with discuss a new “network”? different skills. As observed by members of the • Currently in the UK there are possibilities to pharmaceutical industry when faced with the mass apply for funding for “functional genomics”, a of new genomic information, we may yet need fur- fundamentally different activity to basic, hypoth- ther breaking down of the barriers between scientif- esis-driven research. Are funding agencies in ic disciplines (Browne and Thurlby, 1996). other countries initiating such programmes? Is this a good moment to use these openings to con- ”Until recently genes have been cloned and sider an “applied genomics” application, aimed expressed usually as the result of targeted pro- directly at drug discovery? If Wellcome Trust- grammes of research, their biochemistry and phar- based, this would involve UK researchers, but the macology evaluated, high-throughput mechanism- Trust recognizes the need to involve international based screens devised and leads identified for opti- groups. mization by chemists ... In this traditional model of • It is possible that an early-stage drug would be pharmaceutical research, gene identification had more attractive to manufacturers if it were useful often been considered rate limiting. The paradigm against several parasites, for example all kineto- has shifted.... We need to be able to predict which plastids. Some effort should be put into bioinfor- genes may be useful and why... there are potentially matics comparison of kinetoplastid genomes as many, many more proteins to work with (and) the they become available. This could provide infor- abundance of opportunities (must) impact on con- mation on common parasite pathways that differ ventional research strategies”... (extracted from substantially from those found in humans and Browne and Thurlby, 1996). mammals.

The following observations need critical assess- Applied Genomics: High-throughput Testing ment: of Vaccine Candidates • The mass of genomic sequence provides the pri- Discussion of the identification and testing of vaccine mary data for discovery of new proteins and new targets following analysis of genomic information is metabolic pathways. Initially, we will be faced even more subject to charges of naivety than feared in with thousands of novel genes for which we have section 3.9. To the limited knowledge of the authors, no function. At this stage, bioinformatic analysis attempts have been made by numerous skilled work- (of metabolic pathways, for example) and system- ers to identify trypanosome proteins that may serve atic (preferably high-throughput) analysis of gene as vaccine candidates, only to be thwarted by the function (as attempted in the RNAi analysis de- dense coat of variable surface protein that prevents scribed in 3.8) will be important. We would expect access by antibodies to invariant proteins, and the that these kinds of analyses will provide new efficiency of antigenic variation. It is not immediately

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 157 obvious how the availability of genome sequence how we can ensure that the entire research commu- and the discovery of novel genes and pathways may nity has access to information. In the near future this provide new approaches, given these results. The may necessitate distribution of CDs, in which case only high-throughput parasite vaccine testing project how should this be coordinated? In the more distant known to the authors is that of Professor J Blackwell, future, WHO and those institutions who see a role who is testing pooled DNA vaccines against leishma- for such web-based information must seek a path to niasis. All the resources for such a project applied to improved institutional infrastructure and electronic infection with T. brucei are available, but any assess- resources. ment of applicability should involve a wider range of researchers with appropriate skills. Access to Unannotated and Annotated Sequence Data All raw unannotated sequence data are made avail- SHARING OF INFORMATION AND ACCESS able immediately on the websites of the sequencing TO DATA AND RESOURCES: SUMMARIES centres, and then in the public databases. However, The T. brucei Genome ‘Network’ access to annotated data is more problematic. The It can seem difficult to obtain information from, or Sanger Institute is unable to annotate fully until to know how to “join”, the genome network. This is close to completion of a chromosome, due to their not in any way due to exclusivity as such, but be- shotgun approach. Their provision of preliminary cause it works as any other network – people who annotation of chromosome I caused considerable know each other and who have common interests confusion as researchers did not fully appreciate come together in subgroups to seek funding. Inevi- that the chromosome was discontiguous. TIGR is tably this is also driven by the availability of suitable able to provide some annotation of completed BACs funding for functional/applied genomics in differ- as they are finished, but this approach does not pro- ent countries – for example, the Wellcome Trust vide as many raw sequences as early on in the proj- (UK) is currently the most forward planning in pro- ect. This problem is unresolved and subject to much viding funds for global analyses, reducing its insis- discussion at network meetings. One interim solu- tence on hypothesis-driven research etc., so, pre- tion is provided by the clustering and BLAST analy- dictably, recent groupings have been UK-driven. For sis of discontinuous sequence data (GSS, shotgun, this reason it is very important that WHO continues ESTs)(http://www.sanger.ac.uk/Projects/T_bru- to direct some funds towards helping those African cei/Toolkit/blast_server.shtml; hftp://ftp.sanger. scientists with an interest in this work to travel to ac.uk/pub/databases/T.brucei_sequences/GSS/cl network meetings, to make contacts, to set up col- usters/). Many researchers annotate the small re- laborations and to talk about priorities. This latter gions of immediate interest to them, but for some sci- point is very important: there are many very good entists this requires some bioinformatics training. We biologists working on the fascinating basic biology hope that some of these problems will be addressed of T. brucei, but their priorities may differ somewhat by the creation of the relational genome database. from those of the endemic countries and only the presence of outspoken scientists whose interests lie Access to Comprehensive Genome in controlling the disease will ensure that we do not Information in Relational Databases lose sight of the necessity to direct funds towards As stated many times above, access to all databases disease-applied genomics. currently under development will ultimately require secure access to the Internet, although provision of Obtaining Information CDs may be an interim solution. In addition to the Until now, information dissemination has been genome database to be hosted at the Sanger Centre underfunded and very dependent on the commit- (section 3.1), a separate Wellcome Trust-funded proj- ment of certain individuals to spreading the word, ect (Melville, see 4.5) will also provide genome map- maintaining websites, and being approachable and ping data in a relational database that will be com- amenable to email requests for information and ex- munity-interactive and will facilitate some analyses planations. But it is increasingly recognized that col- before it is superceded by the complete genome lected information and properly run databases are sequence. Once again, this will be web-accessible becoming vital in our brave new world of collabora- and, while it is possible to provide copies on CDs, tive “big science”, and as more resources are made personnel resources are limited to providing full available towards these aims, it should become eas- explanations and guidance on a website. ier to obtain up-to-date information on the progress of genome network-based projects. However, it is Obtaining DNA Resources for Use also inevitable that these information resources will in Research Projects be web-based. It is therefore vital to consider now All biological resources used by the genome net-

158 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 work are available to researchers on request, includ- treatment of trypanosome infection. All scientists ing the TREU927/4 stock and 10.1 derivative, high- should have access to such data as will simplify density filters of genomic libraries, genomic DNA hypothesis generation and experimentation, whe- clones, cDNAs and karyotype blots (Melville, et al, ther their interests lie in basic biology or in applied 1998; http://parsun1.path.cam.ac.uk). The genome science. In addition to the promotion of good scien- website provides addresses and email links. The tific experimentation, it seems likely at the current genome network insists that hybridization and time that African scientists must help persuade inter- sequence data derived using its resources are re- national funding bodies and the scientific communi- turned to the database curator for inclusion in the ty to apply the data for the development of novel genome database (http://parsun1.path.cam.ac.uk). epidemiological tools, drugs and vaccines. These resources were provided with funding from WHO/TDR for five years. Since the beginning of 2001, The area of genomics and applied genomics has the Wellcome Trust Functional Genomics Initiative has expanded considerably over the past decade or so; provided a four-year programme grant to expand this the driving force in this being the development of facility to assist the growth of functional genomics bioinformatics tools that make access to the avail- projects within the network (Melville, Cambridge). able data possible. Quite important also is the wide range of publicly accessible tools developed to make Obtaining Reference and Mutant Strains use of these data. Yet many African scientists who for Use in Research Projects could use such information cannot, or do not, for As part of the above project, a collection of reference many reasons. While this Scientific Working Group strains, transformed lines and mutants will be main- (SWG) will address the problem of African try- tained and provided to researchers on request (Tur- panosomiasis, there is no doubt that the opportuni- ner, Glasgow). Current funds are not sufficient to ties and impediments are generic, and hence appli- consider curating large field collections. The sharing cable to virtually all disease problems in endemic of well-curated field collections may be of value in countries in Africa, and perhaps in most developing certain comparative genomic projects and could be countries. Increasing the ability of African scientists offered up for discussion. Many institutions have to participate in the application of data derived from good collections of primary or very limited passage genome projects will stimulate research activities isolates that could be harnessed as a valuable not only in this area, but also in a multidisciplinary resource. WHO is supporting efforts to establish manner, since such data have to be evaluated in good quality databases in a number of places, inclu- processes that require broad-based competencies. ding Kenya Trypanosomiasis Research Institute (KETRI). We propose here ways in which participation in A- frica, outside of international institutions, can be INVOLVEMENT OF SCIENTISTS FROM developed and strengthened. TRYPANOSOMIASIS-ENDEMIC COUNTRIES It is with some disappointment that we report the Bioinformatics (Present Competence low level of participation of African institutions in and Training Needs) the genome network to date. There may be a range Although there is some competence in bioinformat- of reasons for this: inappropriateness of the tasks to ics within the science community in Africa, it is institutional aims or facilities, competition from largely fragmented and inadequate. To quantify it other laboratories with access to materials and the would be virtually impossible. However, it is known facilities to perform high-throughput tasks with that this capacity is mainly concentrated in interna- greater efficiency, disinterest in genomics given the tional institutes, a situation that is inappropriate, more pressing problems of control of livestock infec- and efforts should be made to extend this to univer- tion and human epidemics or research on pathogen- sities and national research centres. One way of esis etc., or lack of sufficient contacts and encourage- beginning to do this is to develop a series of training ment. Maybe we need not be too disappointed that courses in bioinformatics, either continent-wide or African laboratories did not play a central role in the regionally, to develop Africa’s capacity in bioinfor- process of sequence generation itself (other than the matics. The value of this is two-fold: ESTs, the majority of which were generated in • It will equip participants with knowledge on what Kenya; and we should also note the employment of information is available, where it is, how it can be African scientists in European and US laboratories). accessed and utilized to develop tools and meth- But we should consider it a scandal if this tremen- ods to address public health problems. dous resource, initiated by TDR, is not made avail- • It would begin to expand the human resource able to the entire global research community and is base in bioinformatics among African scientists, not applied to the full to problems of control and hence contributing to the expansion of the

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 159 research activities in African trypanosomiasis and in the continent for within-continent exchange of other major disease situations. This would create expertise. In some cases, such limitations exist even an environment for continuous in-house training. within countries. One reason for this is that the value of bioinformatics is not well recognized by It is anticipated that such courses would bring to- managers of national institutions. Sharing of infor- gether expertise from within Africa and elsewhere mation across areas of disease interest is also not (in or outside the African trypanosomiasis commu- good, especially in African trypanosomiasis, where nity). Doing this would help build the necessary many institutes have a single disease mandate. partnerships (within and across diseases) to sustain Thus: the knowledge base in bioinformatics. It may be pos- • there could be closer interaction between national sible to bring in experts in computing who have lit- and the international institutions where there is tle biological sciences training but who are willing to greater capacity to apply data derived from geno- use their expertise in biology. me projects, to use the available resources more efficiently. Capacity building in bioinformatics can also be • there should be greater effort to promote broad enhanced through providing funding support (by multidisciplinary projects constituted in part by TDR and other donors) for Internet service provider the use of applied genomics. Such linkages will (ISP) and access charges within projects. Addressed enhance the use of available information from as a budget item in relevant proposals, this could various genome projects to address different bio- include visits to laboratories with expertise in bioin- logical questions. formatics (north or south). We refer you also to the proposal from the Pathogenesis and Applied Geno- Retention of Trained Personnel mics Committee for regional bioinformatics courses. It may be prudent to say that no single agency can If the funding is secure and consistent, these may go guarantee that good quality personnel are retained in some way to meeting these aims. research, and particularly in national systems within disease endemic countries. However, important con- Infrastructural Capacity tributions towards stemming the outflow of person- Worldwide, the capacity of personal computers nel can be made, even by a single stakeholder. (processor speed and storage space) continues to improve phenomenally. Africa is no exception. Elec- Perhaps the flight of personnel largely accounts for tronic access to information is often limited due to the paucity of good quality funding applications poor communication infrastructure; good, fast and from Africa to address the problem of African try- dedicated connections are few and far between, and panosomiasis. In the terms of reference for this often only reliable in international institutions. With SWG, TDR recognizes that “many well trained per- proper training, however, good quality information sonnel in African trypanosomiasis have left the field can still be obtained by email via a telephone line, for others, such as HIV/AIDS”. Personnel have even where institutions have a very limited number indeed left in many directions, including to other of computer terminals with on-line access (even if countries, primarily in the north, to other research only one terminal with limited time slots). Im- areas within Africa, or even out of science. provement of institutional capacity should continue to be an important objective; admittedly, maximal While it has been suggested that TDR could follow access to databases is only achievable in an environ- a strategy of choosing a few studies and the centres ment that recognizes the value of bioinformatics. to carry them out, the success of this will depend on the retention of good quality personnel in the field. TDR can help by giving small grants, perhaps about Within Africa, the SWG should consider: US$2000 annually, to national institutes to fund elec- • allowing African investigators working in their tronic access. Such funds would be used to pay ISP own countries to draw salaries or salary support and connection charges to individual scientists from projects funded by TDR, since poor remu- within national institutes and universities. Such neration is a major impediment to creating a criti- funding would not necessarily be linked to specific cal mass for research. project funding, although applicants could be • promoting collaborative initiatives between coun- encouraged to budget for this within projects. tries in the south, where this is scientifically sound, hence expanding the collaborations that Institutional Linkages exist currently. African scientists who have undertaken some train- • encouraging collaborative projects (north-south; ing in bioinformatics have experience that can be south-south; inter-institutional within countries) shared. However, there is not enough linkage with- that have a bioinformatics (and bioinformatics

160 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 training) component in order to strengthen the RECOMMENDATIONS capacity to apply genomics data to address public • Improve institutional capacity in Africa to access health problems. data from genome projects by acquiring computing hardware suitable for bioinformatics (through FUNDING AND HUMAN RESOURCES: projects or independently). In the long term, this REQUIREMENTS AND OPPORTUNITIES will be vital for full involvement in molecular bio- The genome sequence will be a valuable resource for logical research on trypanosomes. the research community, but there is much to be • Identify possibilities for adding value in the colla- done to turn its potential for new directions and new tion of data, for example in a central database for discoveries into reality. No researcher need feel there DNA-based markers for use in epidemiological is no opportunity to become involved. However, in- studies. volvement will depend heavily on having access to • Identify areas of bioinformatic analysis that lack the available information and contact with other support, e.g. identification of metabolic path- groups. One way to become involved in the genome ways, comparison of pathways across the Kineto- network is to develop an interest group, investigate plastidae. Identify the researchers required for the resources available, then look for funding to use such projects. the genomic data for a purpose – such a group may • Identify now the requirement for further geno- then be “hooked in”. The RNAi group is one exam- mic analysis and sequencing of other strains ple, a drug discovery group may be another. (e.g. gambiense and other clinical variants) and species (T. congolense, T. vivax). This may be The next phase of the sequencing revolution will be complete genomic sequencing or shotgun se- “comparative genomics”. Technologies that aim to quencing to a given level (e.g. 3x, 7x, 10x) and simplify the resequencing of different genotypes could be based, once again, in high-throughput (strains) are in development. Although it hardly or local centres. seems possible that we might be able to sequence • Consider the techniques available for comparison more than one genome, only five years ago we of strains, including the possibility of compara- didn’t believe we could sequence even one. Now is tive sequencing (e.g. of gambiense). the time to identify those species and strains that • Consider inclusion of genomics experts in ongo- would provide vital information to researchers if ing drug development working groups. comparative sequence information were available. • Ensure that the voices of African scientists and applied scientists are heard within the genome Funding agencies are increasingly realizing the va- network. Bring a drug development working par- lue of large collaborative groupings and high-throu- ty into the network. ghput techniques. It is timely to consider the poten- • Encourage the dissemination of information that tial of novel applications of genomic methods to may lead to the formation of new interest groups current problems, as the openings are there. The (where possible within existing groupings, e.g. Wellcome Trust is in the forefront of such develop- TDR, the Programme Against African Trypano- ments. The Trust also has an active international somiasis (PAAT), Global Forum on Agricultural programme in (inter alia) tropical disease research Research (GFAR), to discourage proliferation) to (http://www.wellcome.ac.uk/en/1/biosfgintintfu- access the genomic resources for new projects. nunkcrg.html) and has recently indicated its desire • Consider current possibilities for funding of geno- to achieve a higher profile for this programme. mics-based projects leading to drug target identi- Therefore, at this point in time, north-south col- fication and validation; in this context, reevaluate laborations may be seriously considered: both full the potential for inclusion of scientists from try- inter-institute collaborations and relatively minor panosomiasis-endemic countries. collaborations that facilitate technology transfer • Facilitate training in bioinformatics to increase and the kind of training and contacts that enable competency and promote the utilization of geno- scientists to develop an independent career. mics data in Africa. We take note of the call by TDR for proposals in bioinformatics and applied Some projects mooted here are large and costly, genomics to develop research and training cen- requiring coordination and commitment. While tres/networks in Africa, Asia and Latin America. TDR funds may not be sufficient to underpin coor- • Encourage the establishment of contacts between dinated applied genomics projects, it is important scientists/institutions (in Africa and elsewhere), that working groups such as this take a lead role in through exchange visits, training courses and joint defining the problems that need to be addressed, to proposals, to maximize additive competencies. ensure that the genomics revolution does not bypass • Improve the remuneration of African scientists those directly affected by trypanosomiasis. working on TDR-supported projects in order to

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 161 retain trained personnel, hence improving institu- Melville SE et al. The molecular karyotype of the tional and national research capacity. megabase chromosomes of Trypanosoma brucei and • Promote collaboration between countries (South- the assignment of chromosome markers. Molecular South; North-South) to take advantage of shared and Biochemical Parasitology, 1998, 94:155-173. experiences and of possibilities for shared funding. Melville SE et al. Selection of chromosome-specific Acknowledgements DNA clones from African trypanosome genomic Some of the text included here is extracted from: libraries. In: Analysis of non-mammalian genomes, Melville SE. Characterisation and sequencing of the Birren B, Lai E, eds. New York, Academic Press, African trypanosome genome. In: Guidelines and 1996, pp 257-293. issues for the discovery and development of drugs against tropical parasitic diseases, Vial H, Fairlamb A, Ridley Ngo H et al. Double-stranded RNA induces mRNA R, eds. World Health Organization (2001). We thank degradation in Trypanosoma brucei. Proceedings of the all our colleagues for discussion of the points pre- National Academy of Sciences, USA, 1998, 95:14687-92. sented here and especially the members of the genome network for their open sharing of informa- Rudenko G et al. Selection for activation of a new tion and progress. variant surface glycoprotein gene expression site in Trypanosoma brucei can result in deletion of the old References one. Molecular and Biochemical Parasitology, 1998, Barry JD, McCulloch R. Antigenic variation in try- 95:97-109. panosomes: enhanced phenotypic variation in a eukaryotic parasite. Advances in Parasitology, 2001, Tait A et al. Genetic analysis of phenotype in try- 49:1-70. panosoma brucei: a classical approach to potentially complex traits. Proceedings of the Royal Society (in press). Browne MJ, Thurlby PL, eds. Genomes, molecular biology and drug discovery. Academic Press Limited, Turner CMR, Melville SE, Tait A. A proposal for 1996. karyotype nomenclature in T. brucei. Parasitology Today, 1997, 13:5-6. Clayton CE. Genetic manipulation of kinetoplastida. Parasitology Today, 1999, 15:372-378. Turner CMR et al. Evidence that the mechanism of gene exchange in Trypanosoma brucei involves meio- Djikeng A et al. Generation of expressed sequence sis and syngamy. Parasitology, 1990, 101:377-386. tags as physical landmarks in the genome of Trypanosoma brucei. Gene, 1998, 221:93-106. van Deursen FJ et al. Characterisation of the growth and differentiation in vivo and in vitro of El-Sayed N et al. The African trypanosome genome. bloodstream form Trypanosoma brucei strain TREU International Journal for Parasitology, 2000, 30:329-345. 927. Molecular and Biochemical Parasitology, 2001, 112:163-171. El-Sayed NMA, Donelson JE. A survey of the Trypanosoma brucei rhodesiense genome using shot- Vanhamme L, Pays E. Control of gene expression in gun sequencing. Molecular and Biochemical Parasi- trypanosomes. Microbiological Reviews, 1995, 59:223-240. tology, 1997, 84:167-178. Weiden M et al. Chromosome structure: DNA nucleotide sequence elements of a subset of the El-Sayed N et al. cDNA expressed sequence tags of minichromosomes of the protozoan Trypanosoma bru- Trypanosoma brucei rhodesiense provide new insights cei. Molecular and Cellular Biology, 1991, 11:3823-3834. into the biology of the parasite. Molecular and Biochemical Parasitology, 1995, 73:75-90. for example, see Wellcome News issue 26, Q1, 2001, p. 19.

Melville SE, Gerrard CS, Blackwell JM. Multiple causes of size polymorphism in African trypanosome chromosomes. Chromosome Research, 1999, 7:191-203.

Melville SE, Majiwa P, Donelson J. Resources available from the African trypanosome genome project. Parasitology Today, 1998, 14:3-4.

162 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Annex 7 INSTITUTIONAL CAPABILITY STRENGTHENING African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 163 INSTITUTIONAL DEVELOPMENT training, research and control work, must remain a AND CAPACITY BUILDING IN priority. The first step would be to carry out an in- COUNTRIES ENDEMIC FOR ventory of existing research and training institutions, SLEEPING SICKNESS and identify areas of immediate and long-term concern. This should assist in the identification of nation- J. Mathu Ndung’u al and regional institutions in Africa which could be Kenya Trypanosomiasis Research Institute (KETRI), strengthened by the international community in col- P.O. Box 362 Kikuyu, Kenya laboration with national institutions and governments. Linkage arrangements, between national and INTRODUCTION overseas institutions with donor support, need to be This report is intended to stimulate discussion on the further explored, strengthened and extended. possible solutions available for improving research and training in, and strengthening institutions for, TRAINING effective delivery of services in the 37 countries In the past, training specifically addressed the prob- affected by trypanosomiasis and the tsetse vector. lem of tsetse and trypanosomosis in the laboratory and field with a view to ensuring long-term com- The financial crisis that occurred in Africa in the 1980s mitment and contribution towards solving the prob- and 1990s had an almost paralysing effect, leading to: lem. However, this training did not take fully into • lack of funds to maintain physical infrastructure, account some vital concerns, e.g. that African try- vehicles and equipment, or to replace outdated panosomiasis is just one of the many health prob- and broken-down equipment. lems Africans are exposed to, the solution of which • decline or lack of funding, at both national and demands a broad understanding and approach. institutional levels, to carry out field surveillance and control of tsetse, and field and laboratory To retain the interest and commitment of personnel research in African animal trypanosomiaisis and who have been trained, there is a need for career sleeping sickness. development. In recent times therefore, efforts have • poor remuneration of staff, hence internal and been directed at broadening of training. Yet much external brain drain of trained manpower and, remains to be done, especially in light of the devel- therefore, paucity of leadership. opments towards privatization of services, including in tsetse control. It is in this light, and in light of Given this background, and considering that deter- the competing demands by different sectors of the mined efforts made over the years to solve the prob- economy for limited funds, that institutional devel- lems have not had the desired impact, a more ration- opment and training in disease endemic countries al approach to research, training, and institutional should be viewed. development is indicated. Issues arising include: number and size of available research and training Four types of training are generally recognized: institutions; appropriateness or otherwise of any re- • Training at sub-professional level, leading to the search and training undertaken; incentives for staff to award of a diploma or certificate, e.g. animal remain on the job, e.g. career development, adequate health assistant, animal health technician. remuneration and an enabling environment. • Training at professional level, leading to the award of professional degrees and diplomas. INSTITUTIONAL DEVELOPMENT • Training leading to postgraduate degrees and One of the recommendations made at the 1974 FAO diplomas. Expert Consultation on Animal Production and • Short-term training leading to specialization. Health Research in Copenhagen, Denmark, was to provide facilities for training research workers ”in To these must be added the training of: their own environment” and to strengthen existing • auxiliary personnel, e.g. clinical officers and field national and regional institutes. Presently, most re- assistants, who need short courses and/or in-ser- search institutes established pre-independence or vice training for field work. immediately post-independence in Africa are in a • field agents, whose activities are coming more deplorable state due to breakdown of physical infra- and more into the limelight because of their clo- structure, equipment and vehicles, lack of funds for seness to the communities in community-based laboratory and field work, and severe depletion of primary health care. research and field staff. • contacts and farmers (general training).

Rehabilitation and strengthening of existing national For the first four types of training listed, a prerequi- and regional institutions to carry out appropriate site for admission is formal education at secondary

164 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 school level, while on-the-job and in-service training explore whether there is a place for private initiative are essential to develop and sharpen skills. In the in training. case of training of auxiliaries, the requirement is that they should have attended a secondary school, but STAFF RETENTION may have dropped out along the way. In the case of The problem of staff retention is a global one, not field agents, the ideal requirement is basic primary peculiar to the field of tsetse and trypanosomosis. school education, emphasizing numeracy and What makes the case of those in the field of tsetse enabling them to follow simple instructions and and trypanosomosis different, however, is that training and to be selected by the community whom career advancement can be painfully slow and frus- they will serve. trating in the public sector. Among the causes of inability to retain staff are: Training Needs • slow career advancement and lack of career The priority given to training of the different cadres prospects. will vary from country to country but will largely • poor salary and working conditions. depend on: • delay or non-payment of field allowances. • the prevalence of tsetse and trypanosomiasis. • lack of transport. • the impact of tsetse and trypanosomiasis on the • lack of self-worth. national economy. • few or no opportunities to take part in short-term • the human, material and financial resources avail- and/or in-service training programmes, which able for dealing with the problem. were a major incentive in the past but which are now scarce due to lack of funds. While there is a great need for experienced professional laboratory and field staff, there is, at the pres- The low remuneration and lack of career prospects ent time, much greater demand for specialized mid- makes recruitment of good staff for training very dle-level personnel to carry out the arduous task of difficult. Young, talented people tend to decline field and laboratory operations, and for auxiliary and appointment unless they lack other job opportuni- field agents who are crucial to the success of com- ties. Trained staff, on the other hand, either leave munity-based health care and field programmes. their employment in favour of more lucrative economic activities and/or political appointments Training Institutions totally unrelated to their training, or, because of the While there is no problem finding institutions to need to supplement their meagre pay through other train personnel at professional and postgraduate unrelated economic activities, tend not to devote levels, there are serious problems with training for attention to the job. middle-level specialized personnel. In the last few years, many national and regional training institu- SUMMARY tions which give emphasis to tsetse and trypanoso- Training is needed at all levels, but more so at the mosis have either been reduced in size or closed middle personnel and auxiliary levels. It is impor- down completely because of funding difficulties. tant to address the career prospects and other bene- Yet this is the training area of great need. The num- fits of trainees, e.g. short courses and in-service ber of trainees that can be deployed to serve in the training that could encourage staff to remain in the field depends largely on the number and size of the job for which they are trained. training institutions available, and whereas concern has been expressed, it appears that no solution to The issue of funding research and training institu- sustaining the necessary institutions is in sight. tions on a sustainable basis must be addressed, for which it may be necessary to review the present Although there are certain advantages of tying trend of guaranteeing funding of research projects training in institutions to ongoing projects, e.g. with for a period of 12 months only. The possibility of regard to the funding and training of personnel, the involving private initiative in training, and how this disadvantage is that the lifespan and funding of can be implemented without sacrificing quality, projects is uncertain. What is needed is a training should be examined. South-south research and institution with its own lifespan and funding, and training should be encouraged. There is an urgent with participation from ongoing projects. need to find out the present status of national research and training institutions with a view to What then are other viable options? Would a donor advising national governments on the role of such consider supporting two or more training institu- institutions in tsetse and trypanosomosis research tions in Africa on a sustainable basis? Another sug- and control. gestion, which at first may appear remote, is to

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 165 In order to provide an enabling environment for field and laboratory research and training, institutional development needs to be given new impetus. A possible starting point would be to carry out an inventory of the existing research and training institutions to provide insight as to what type of strengthening may be needed. Linking national institutions and overseas institutions with donor support is a possible area for expansion.

166 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Annex 8 LIST OF PARTICIPANTS African trypanosomiasis

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 167 Dr Serap Aksoy, Department of Epidemiology and Professor Krister Kristensson, Division of Neurodege- Public Health, Section of Vector Biology, Yale nerative Disease Research, Department of Neuro- University School of Medicine, 60 College St. 606 science, Retzius vag 8, B2:5, Karolinska Institutet, SE- LEPH New Haven CT06510 USA. Tel: 1 203 737 2180, 171 77 Stockholm. Tel: 46-8-728 7825; Fax: 46-8-32 53 25, Fax: 1 203 785 4782, E-mail: [email protected]. E-mail: [email protected].

Professor Cyrus Bacchi, Pace University, Haskins Dr Grace B Kyomuhendo, Department of Women Laboratories, 41 Park Row, New York, NY 10038 and Gender Studies, Makerere University, P. O. Box –1598, USA. Tel: 1 212 346 1246, Fax: 1 212 346 1586, 7062, Kampala, Uganda. Tel: 256 774 71 600 or 256 41 E-mail: [email protected]. 531484, Fax: 256 41 543539, E-mail: gendermu@swif- tuganda.com, or [email protected]. Dr MP Barrett, University of Glasgow Institute of Biomedical and Life Sciences, Division of Infection Dr Francis J Louis Institut de médecine tropicale, BP 46 and Immunity, Glasgow G12 8QQ, Scotland. Tel & Le Pharo, 13007 Marseille, France. Tel : 33 4 91 15 01 46, Fax; 44 141 330 6904, E-mail: [email protected]. Fax : 33 4 91 15 01 43, E-mail: [email protected].

Dr Kwabena Bosompem, University of Ghana, No- guchi Memorial Institute for Medical Research, P.O. Professor John Mansfield, Department of Bacte- Box LG581, Legon. Tel: 233 21 500 374, Fax: 233 21 riology, University of Wisconsin-Madison, 1925 502182, E-mail: [email protected], kbosom- Willow Drive/FRI Bldg Madison, WI 53706 USA. [email protected]. E-mail: [email protected].

Dr Reto Brun, Swiss Tropical Institute, Postfach, Dr Daniel Masiga, Kenya Trypanosomiasis Re- Socinstrasse 57, Basel, Switzerland. Tel: 41 61 284 82 search Institute, P.O. Box 362, Kikuyu, Kenya. 31, Fax: 41 61 271 86 54, E-mail: [email protected]. E-mail: [email protected].

Dr Christian Burri, Swiss Tropical Institute, Po- Professor Ian Maudlin, Centre for Tropical Veteri- stfach, Socinstrasse 57, Basel, Switzerland. Tel: 41 61 nary Medicine, the University of Edinburgh, Easter 284 82 47, E-mail: [email protected] Bush Veterinary Centre, Roslin, Midlothian, UK EH25 9RG. Tel: 44 131 650 7347, Fax: 44 131 650 7348, Dr Philippe Büscher, Instituut voor Tropische Genee- E-mail: [email protected]. skunde, 155 Nationalestraat, 2000 Antwerpen, Bel- gium. Tel: 32 3 2476371, Fax: 32 3 2476373, E-mail: Dr Dawson B Mbulamberi, Ministry of Health, P.O. [email protected]. Box 7272, Kampala, Uganda. Tel: 256 41 43087, E-mail: [email protected]. Dr Simon L Croft, London School of Hygiene and Tropical Medicine, Department of Infectious and Tropical Diseases, London WC1E 7HT. Tel: 44 171 Dr Honoré Meda, Projet SIDA 2, Benin (ACDI-Ca- 927 23 45, Fax: 44 171 636 87 39, E-mail: nada), 08 B. P. 900 Tri Postal, Cotonou, République [email protected]. du Benin. Tel: 229 31 36 02, Mobile: 229 957 169, Fax: 229 31 36 05, E-mail: [email protected]. Dr Peter de Raadt, Bruglaan 11, 3743 J.B. Baarn, The Netherlands. Tel: 31 35 541 21 21, E-mail: Dr Sara E Melville, Cambridge University, De- [email protected] partment of Pathology, Microbiology and Parasito- logy Division, Cambridge, CB2 1QP, UK. Tel: 44 Dr Felix Doua, Projet des Recherches Cliniques sur 1223 333335, Fax: 44 1223 333346, E-mail: la Trypanosomiase (P.R.C.T.), B.P. 1425, Daloa, Côte [email protected] d’Ivoire. Tel: 225 32 78 36 10, Fax: 225 32 78 3021: _ E-mail: doua [email protected] Dr C Miaka Mia Bilengé, Ministère de la Santé, Secrétariat Général à la Santé, B.P. 3040 Kinshasa, Dr John K Enyaru, Livestock Health Research In- République Démocratique du Congo. E-mail: stitute, P.O. Box 96, Tororo, Uganda. Tel: 256 45 [email protected]. 45050, Fax: 256 422 1070, [email protected].

Dr J Josenando, Instituto de Combate e Controlo Dr V Nantulya, Harvard School of Public Health, das Tripanosomiases, 168, Kwenha, Ingombota, CP 665 Huntington Avenue, Boston, 02115 MA, USA. 2657, Luanda, Angola. Tel: 244 2 399610, Fax: 244 2 Tel: 1 617 432 0659 , E-mail: [email protected] 399611, E-mail: [email protected]. vard.edu.

168 Report of the Scientific Working Group on African trypanosomiasis, 2001 • TDR/SWG/01 Dr Joseph Ndung’u, Kenya Trypanosomiasis Re- Dr M Schottler, Head, Health Policy, Bayer A.G., search Institute (KETRI), P.O. Box 396, Kikuyu, Business Group Pharma, Europe and Overseas, Kenya. Tel: 254 154 32960-4, Fax: 254 154 32397, D-1368 Leverkusen, Germany. E.-mail: [email protected] or [email protected] or [email protected]. Dr J Slingenbergh, Senior Officer, Animal Pro- duction and Health, FAO, Rome, Italy. E-mail: Dr Martin Odiit, Livestock Health Research Insti- [email protected]. tute, P.O. Box 96 Tororo, Uganda. Tel: 256 45 45050, Fax: 256 45 45052, [email protected]. WHO secretariat Dr Mary Bendig, PRD/TDR Dr Alexandra PM Shaw, A. P. Consultants, Upper Mr Erik Blas, Programme Manager, PPM Cottage, Abbotts Ann, Andover SP11 7BA, UK. Tel: Dr Charity Gichuki, Organizer 44 1264 710 238, Fax: 44 1264 710 759, E-mail: Dr Melba Gomes TDR/IDE [email protected]. Dr Win Gutteridge, Team Coordinator PRD Dr Beatrice Halpaap, TDR/PRD Dr Richard R Tidwell, Department of Pathology & Dr David Heymann, EXD/CDS Laboratory Medicine, University of North Carolina, Dr Jean Jannin, CSR/EDC Chapel Hill, NC, USA. Te1: 1 919-966-4294, E-mail: Dr J Karbwang, TDR/PRD [email protected]. Dr Jane Kengeya-Koyondo Team Coordinator TDR/IDE Dr Philipe Truc, Institut de Recherche pour le Déve- Dr Deborah Kioy, TDR/PRD loppement UMR 035 “Trypanosomoses Africaines”, Mr Felix Kuzoe TDR/PRD OCEAC, BP288, Yaoundé, Cameroun. Tel: 237 23 22 Dr Maryinez Lyons CAH/FCH 32 or 237 84 60 57 (mobile), Fax: 237 23 00 61, E-mail: Dr Carlos Morel, Director WHO/CDS/TDR [email protected] Dr MA Mouries, TDR/PRD Collaborators and partners Dr Paul Nunn, TDR TB & LEP Disease Coordinator Dr Alain Aumonier, Director, International Affairs, Dr Ayoade Oduola Team Coordinator TDR/STR Aventis Pharma International, Tri E1/385 - 20 Dr Mark Perkins TDR/PRD Avenue Raymond Aron, F-92165 Antony Cedex, Dr Richard Pink, TDR/PRD France. Tel: 33 1 5571 6087, Fax: 33 1 5571 4185 Dr J Sommerfeld, TDR/STR E-mail: [email protected]. Dr J Vanreas, TDR/PRD Dr Simon Van Nieuwenhove, Regional Adviser, Dr Jean-Pierre Helenport, Consultant, Medécins WHO Regional Office for Africa, B.P 1899, Kinshasa sans Frontières/World Health Organization 1, Republique Démocratique du Congo. Tel: 243 88 Eflornithine Project, 2 Impasse des Bambous, Villa 03204, Fax: 1 321 953 9097, E-mail: omskin@joban- Maéva, 13600 La Ciotat, France. Tel: 33 4 42 981042, tech.cd or [email protected] E-mail: [email protected]. Mr S Wayling, TDR/RCS Dr F Zicker, Team Coordinator, TDR/RCS Dr Anne Moore, Division of Parasitic Diseases, F22, Centers for Disease Control and Prevention, 4770 Buford Highway, Atlanta GA 30341, USA. Tel: 1 770 488 7776, Fax: 1 770 488 7761, E-mail: [email protected].

Dr TC Nchinda, Senior Public health Specialist, Global Forum for Health Research, c/o WHO, Geneva, Switzerland. Tel: 41 22 791 3808, E-mail: [email protected].

Dr Bernard Pecoul, Project Director, MSF Access to Essential drugs Project, Médecins sans Frontières, 12 rue du Lac, C.P. 6090, 1211 Genève 6, Switzerland. E- mail [email protected].

Dr Michaleen Richer, Medical Coordinator, Sudan Program, International Medical Corps, P. O. Box 67513, Nairobi, Kenya. Tel: 254 2 574386/88/89, Fax: 254 2 573973, E-mail: [email protected].

TDR/SWG/01 • Report of the Scientific Working Group on African trypanosomiasis, 2001 169

Mailing address: TDR World Health Organization 20, Avenue Appia 1211 Geneva 27 Switzerland

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