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TDR/SWG/04 Scientific Working Group

Report on Leishmaniasis Report Report on

Mailing address: TDR World Health Organization 20, Avenue Appia 1211 Geneva 27 Switzerland 2–4 February 2004 Street address: TDR Geneva, Switzerland Centre Casai 53, Avenue Louis-Casai 1216 Geneva Switzerland www.who.int/tdr Tel: (+41) 22-791-3725 Fax: (+41) 22-791-4854 E-mail: [email protected] TDR/SWG/04 Web: www.who.int/tdr Original: English Report of the Scientific Working Group meeting on Leishmaniasis Geneva, 2–4 February, 2004 TDR/SWG/04

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Design: Lisa Schwarb Layout by Inís: www.inis.ie Contents

1. Rationale for convening the Scientific Working Group on Leishmaniasis ...... 1 2. Recommendations ...... 3 3. Introduction ...... 5 4. Generating evidence to improve the effectiveness of control programmes . . . . 7 IMPROVED DIAGNOSTIC TOOLS AND STRATEGIES FOR VISCERAL LEISHMANIASIS ...... 7 IMPROVED THERAPIES AND PATIENT MANAGEMENT ...... 9 IMPROVED PREVENTION STRATEGIES ...... 9 PARTNERSHIPS AND CAPACITY BUILDING ...... 12 2. Challenges and opportunities ...... 13 CHALLENGES POSED BY GAPS IN BASIC KNOWLEDGE AND LIMITATIONS OF CURRENT TOOLS . . 13 RESEARCH OPPORTUNITIES TO CLOSE THE GAPS IN BASIC KNOWLEDGE AND TO DEVELOP NEW TOOLS...... 14 3. High priorities ...... 17 INTERVENTION METHODS ...... 17 STRATEGIES & POLICIES ...... 17 PARTNERSHIPS & CAPACITY BUILDING ...... 17 BASIC KNOWLEDGE ...... 17 NEW AND IMPROVED TOOLS ...... 17

Annex 1. AGENDA: Scientific Working Group on Leishmaniasis ...... 19 Leishmaniasis Annex 2. LIST OF PARTICIPANTS: Scientific Working Group on Leishmaniasis. . . . 23 Annex 3. STRATEGIC RESEARCH ...... 29 3.1 FROM GENOME TO DISCOVERY IN LEISHMANIA: RECOMMENDATIONS FOR THE DEVELOPMENT OF DRUGS, VACCINES AND DIAGNOSTICS ...... 30 3.2 DRUG RESISTANCE IN LEISHMANIASIS (SUMMARY) ...... 33 3.3 HOST GENETICS IN LEISHMANIASIS...... 34 3.4 MECHANISMS OF PATHOGENESIS – DIFFERENCES AMONGST LEISHMANIA SPECIES ...... 37 3.5 CELLULAR IMMUNE RESPONSES (SUMMARY) ...... 40 3.6 VECTOR–PARASITE AND VECTOR–HOST INTERACTIONS IN LEISHMANIASIS ...... 42 Annex 4. IMPROVED TOOLS AND INTERVENTION METHODS ...... 45 4.1 VACCINES...... 46 4.2 MOLECULAR TOOLS FOR STUDYING THE EPIDEMIOLOGY OF LEISHMANIASIS ...... 54 4.3 DRUG TRIALS ...... 71 4.4 CONTROL OF ZOONOTIC VISCERAL LEISHMANIASIS ...... 74 4.5 POST KALA-AZAR DERMAL LEISHMANIASIS ...... 76 4.6 EPIDEMIOLOGY OF LEISHMANIA-HIV CO-INFECTIONS ...... 79

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 iii Annex 5. IMPROVED STRATEGIES AND POLICIES...... 81 5.1 ECONOMIC ASPECTS OF VISCERAL LEISHMANIASIS CONTROL...... 82 Figure 1. Number of kala azar cases reported in India (1986–2000) by State and total. . . 82 Table 1. Baseline value and range of efficacy estimates of currently available chemotherapy against visceral leishmaniasis in immunocompetent patients ...... 85 5.2 CLIMATE VARIABILITY AND VISCERAL LEISHMANIASIS IN EUROPE ...... 88 Table 1. Distribution in Europe of sandfly species that may act as vector for L. infantum, the cause of European visceral leishmaniasis ...... 88 Figure 1. Approximate distribution in Europe of visceral leishmaniasis and its vectors compared to cutaneous leishmaniasis and its vectors ...... 89 Figure 2. Cases of Leishmania/HIV co-infection per locality and population density, 1990–1998...... 89 Table 2. Number of reported cases of VL/HIV co-infection in southern Europe, 1990–2001 90 Table 3. Important Phlebotomus species in Europe and their climate thresholds ...... 91 5.3 TOOLS AND STRATEGIES FOR USE OF INSECTICIDE IMPREGNATED FABRICS IN THE CONTROL OF SANDFLIES AND LEISHMANIASIS...... 94 Annex 6. PARTNERSHIP AND CAPACITY BUILDING ...... 99 6.1 THE IMPORTANCE OF RISK FACTORS IN THE CONTROL OF LEISHMANIASIS...... 100 Table 1. Intervention targets in leishmaniasis...... 100 6.2 CHALLENGES AND OPPORTUNITIES IN RESEARCH AND CONTROL ...... 103 Table 1. Some specifications for clinical and epidemiological diagnostic methods . . . . . 105 Annex 7. COMMISSIONED PAPERS ...... 107 7.1 DRUG RESISTANCE IN LEISHMANIASIS...... 108 Leishmaniasis Figure 1. Changes in clinical visceral leishmaniasis response in India: Decline in efficacy of pentavalent antimony regimens in Bihar, India, in the 1980s and 1990s ...... 110 Figure 2. Proposed Sb metabolism in Leishmania ...... 112 7.2 FROM GENOME TO DISCOVERY IN LEISHMANIA: RECOMMENDATIONS FOR THE DEVELOPMENT OF DRUGS, VACCINES AND DIAGNOSTICS ...... 120 Table 1. Drugs used to treat visceral leishmaniasis and their principal limitations . . . . . 124 Table 2. Genetic tools in trypanosomatids ...... 125 7.3 CAN LEISHMANIASIS STILL BE CONSIDERED A TDR CATEGORY 1 DISEASE? IF MOVED, WHAT IS REQUIRED? ...... 130

iv Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 1. Rationale for convening the Scientific Working Group on Leishmaniasis

The goal of the Scientific Working Group (SWG) meeting was to identify leishmaniasis research priorities for the next five years. In addition, the group was to identify strategic emphases, based on TDR’s comparative advantage, to add to the current ones. The discussions were to be focused on how to cope with and prevent drug resistance, and how to optimize the use of genomic information to improve drug, diagnostics and vaccine discovery. Additionally, the group was requested to consider if leishmaniasis should remain on the list of TDR category I diseases (emerging and uncontrolled; worsening epidemiological situation; increasing incidence of infection and disease).

The meeting provided an opportunity to discuss the tools needed to control leishmaniasis, i.e. diagnostics, drugs, vector control, and vaccines, and to assess their adequacy as well as the new knowledge and new tools needed to improve the current tools and control measures.

The SWG reiterated that control of leishmaniasis is achievable, although current tools have proved insufficient for dealing with the biological complexity of the disease and with the unmanageable changes caused by environmental, societal, political and behavioural factors.

Currently available tools need to be improved and validated under diverse field realities. One of the best drugs available on the market for treatment of visceral leishmaniasis, Ambisome, is inaccessible to populations in need due to its high cost. Methods must be sought to advocate Leishmaniasis for reduced prices for such drugs. This highlights the inequities of access to available tools and healthcare, which plague people suffering from leishmaniasis. Despite the high effectiveness of some of the current drugs, development of new drugs must be pursued in face of the constant threat of resistance and the clear limitations of available drugs.

Preventing disease morbidity and mortality remains of prime strategic importance; this requires vector control, including evaluating behaviour changes and susceptibility to insecticides, as well as better and affordable diagnostics for early case detection coupled with adequate treatment. These measures constitute a key mechanism for limiting the human reservoir in anthroponotic foci. Development of a vaccine against leishmaniasis also remains a prime strategic goal, which requires improved knowledge of pathogenesis of the disease.

The SWG concluded that, while visceral leishmaniasis (VL) should remain TDR’s highest priority, research into other forms of leishmaniasis is necessary both because they are diseases in their own right and in order to gain knowledge to aid in the fight against VL. Specific recommendations are given for the development of new and improved intervention

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 1 methods, prevention strategies and policies. The recommendations for generating needed basic knowledge and new tools are less specific and point to areas where gaps exist.

It is evident that the needs for research aimed at control of leishmaniasis are numerous and that TDR can benefit from working with various partners. Leishmaniasis

2 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 2. Recommendations

The SWG recommended that the following areas should receive special attention: • Validation of existing tools for leishmaniasis control by a global network of clinical trial centres. • Operational research to ensure effective implementation of control strategies. • Use of innovative approaches including those generated from genomic information to improve upon existing and to develop new diagnostic, therapeutic and epidemiological tools as well as to develop vaccines. • Research for better understanding of the pathogenesis and the mechanisms of protection against the human disease. • Co-funding of research activities on leishmaniasis in cooperation with interested parties including those within endemic countries. • Research capacity strengthening in all these research areas in disease endemic countries. Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 3 Leishmaniasis

4 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 3. Introduction

Leishmaniasis is the collective name for a number of diseases caused by protozoan flagellates of the genus Leishmania, which have diverse clinical manifestations. The form that is deadly is visceral leishmaniais (VL), caused by Leishmania donovani in the Old World and L. infantum in both the Old and New World. If left untreated, full-blown VL invariably leads to death, thus making early diagnosis of paramount importance. A number of different species of Leishmania cause cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, which, if not fatal, are still responsible for considerable morbidity of a vast number of people in endemic foci. The estimated global burden of disease is believed to be inaccurate due in part to the passive case detection data used to estimate the disease prevalence in many endemic countries. The current global estimate of 350 million people in 88 countries at risk of leishmaniasis and of an incidence per year of 500 000 for VL and 1.5 million for CL belies higher burdens of disease in endemic foci.

A number of challenges exist for the prevention and control of leishmaniasis. Endemic leishmaniasis is perceived as a problem of low priority by governments, society, and in some instances by the patients themselves. Factors that contribute to this perception of low priority include: • the leishmaniases are diseases of poor people in peripheral areas far from the centre of government • other or urgent health problems are of greater magnitude or have greater impact

• resources are limited Leishmaniasis • there is a lack of information about the current magnitude or nature of the problem • there is no knowledge about the most effective points of intervention • affected communities are ignorant about the importance and availability of diagnosis and treatment.

Epidemics of leishmaniasis are known to occur frequently in many foci, but often the strategy of government and health authorities is to react rather than to anticipate and prevent.

The leishmaniases are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural host and the vector, HIV infection, and the consequences of conflict, all contribute to the changing leishmaniasis landscape. Regular epidemiological studies to address risk factors and transmission patterns are necessary in order to integrate information on the current situation into control strategies.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 5 The tools currently available for the control of leishmaniasis are limited in number and utility, and have not been completely validated. Hence intervention research is needed in order to validate the tools in the field, and new and improved tools are required. To achieve this, a better understanding of the human diseases is essential; this is best studied in human beings but would be facilitated by having improved animal models that more accurately reflect the human diseases.

Due to the dynamics of the disease and the substantial developments provided by ongoing innovative research in other disciplines, the recommendations presented in this document are not finite. An element of flexibility is needed in order to ensure that opportunities for advancing knowledge and developing means of controlling this disease are taken advantage of.

While TDR has the comparative advantage to take a number of research areas forward, the daunting need for more research requires that TDR partner with other organizations and with industry to achieve the recommendations set out in this report. Partners may include other funding agencies, national governments, and grassroots organizations found locally in disease endemic areas.

Epidemics occur, and we need to be ready for them. This involves being prepared with validated diagnostic tools as well as research results which aid the prediction of epidemics.

LeishmaniasisThe following sections describe the rationale behind the SWG recommendations on research priorities, the appropriate utilization of available tools, and the development of new tools to reduce the burden of leishmaniasis.

6 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 4. Generating evidence to improve the effectiveness of control programmes

To maximize the likely impact on global bur- Many of these problems need to be addressed den of disease, the priority for applied research by implementation research, including social related to leishmaniasis control policy should and economic studies (see below), and by strong be VL, the lethal form of leishmaniasis, but not advocacy for increasing resources to improve to the exclusion of CL/MCL, which is a major access to drugs and reduce prices. Significant health concern because of the higher morbidity progress in reducing the burden of VL can also of CL (localized and diffuse, DCL [diffuse cuta- be made by the speedy but regulated introduction neous leishmaniasis]) and the debilitating nature of new tools for diagnosing and treating patients. of MCL. Visceral leishmaniasis is largely a disease of the rural poor, with the possible excep- In recent years, with the support of TDR and tion of Brazil where there is increasing evidence other agencies, new tools for diagnosis and that zoonotic VL is becoming urbanized. Fur- treatment have been developed which should thermore, domestic transmission is becoming be urgently tested for effectiveness across all widespread in settlements on previously under- VL endemic regions; and new/improved diag- developed lands, while outbreaks at the periph- nostic tools and drugs are likely to emerge in eries of urban centres in smaller cities have been the near future. Standardized procedures and reported in Colombia and Venezuela. A major large sample sizes are vital to ensure that defin- reason for the continuing high burden of disease itive conclusions can be drawn from such stud- caused by VL worldwide is the relatively low ies, leading to immediate evidence-based policy proportion of patients who have access to, are decisions. Hence, the formation of an extensive provided with, or who complete, a full course of Leishmaniasis Clinical Trial Group (LCTG) for safe and efficacious drugs. The reasons for this VL, CL/MCL and DCL – a network of WHO- are many, but include: certified centres across endemic regions for • the limited access to effective diagnostic tests undertaking multicentric clinical trials of cur-

where they are needed rently available and future drugs, diagnostics, Leishmaniasis and (as they become available) vaccines – is • problems with the sensitivity and specificity strongly recommended. Existing networks of of currently available diagnostic tests clinical trial centres working on leishmania- • the limited availability of drugs for rural pop- sis should be further strengthened and consoli- ulations dated into the LCTG. In relation to the currently • poor drug adherence due in part to the rela- available tools, the priority areas for research on tively long course of parenteral treatment gen- diagnostics and therapeutics are described below. erally required • the frequently reported high toxicity of the IMPROVED DIAGNOSTIC principal first-line drugs in use worldwide TOOLS AND STRATEGIES FOR (pentavalent antimonials [Sb]) • the development of widespread resistance to VISCERAL LEISHMANIASIS first-line drugs, and its spread from the epi- Diagnostic tests for VL need to be both highly centre of the VL endemic region in India sensitive (as failure to treat a case is likely to be the high cost of the best available second-line • fatal) and specific (as the drugs are costly, rel- drug for treatment of VL (Ambisome). atively toxic, and typically involve a long and invasive course of treatment). While parasito-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 7 logical examination of spleen and bone mar- Reliable measurements of specificity, sensitivity row aspirates is the most specific test, it too is and predictive values (as generated by the trials a highly invasive procedure requiring specifi- recommended above) are necessary but not suf- cally trained clinical personnel. The direct agglu- ficient for developing a leishmaniasis test-treat- tination test (DAT) is increasingly used to assist ment strategy which optimizes the use of limited diagnosis and reduces the need for bone mar- resources. This requires the development and row aspiration. However, clinically suspected VL validation of diagnostic algorithms for making patients typically attend rural health posts, clin- clinical decisions related to suspected leishmani- ics or district hospitals where facilities for (and asis patients. Such algorithms will depend, self- expertise in) standard serological diagnostic evidently, on locality-specific clinical features testing (including DAT) are not always availa- (associated with both parasite and human pop- ble; there are also concerns about the variabil- ulation and disease heterogeneities), and also ity between batches of antigen. The clear need on the health system context – on the optimal for an inexpensive, rapid and effective diagnostic case management strategies that accord with the test for VL has led to the recent development of resources and facilities available where patients immuno-chromatographic tests (dipsticks), cur- attend. The benefits of this approach are illus- rently manufactured by two companies, as well trated by the policy in Sudan where, during epi- as novel antigen detection tests, notably a latex demics, clinics can be overwhelmed by patients agglutination test for diagnosing antigen in urine. requiring immediate attention. In such circumstances, suspected VL patients are first tested A TDR-supported multicentre trial is ongoing in by DAT. High-titre patients are given treatment, East Africa and the Indian subcontinent, com- and low-titre patients are examined for differen- paring the effectiveness of one of the commer- tial diagnosis. Only intermediate-titre patients cially available dipsticks with freeze-dried DAT are examined by bone marrow or spleen aspi-

Leishmaniasisand a urine latex agglutination test. The effec- ration. Analogous diagnostic algorithms should tiveness of the dipstick differs between the two be developed and validated for leishmaniases regions, and concerns over specificity remain – worldwide. Clinical management decisions especially in relation to the differential diagno- should also be optimized by the development sis between disease and asymptomatic or healed and validation of algorithms determining, for infections. Diagnosis of apparently relapsed VL example, whether to extend a treatment course patients is a particular challenge. Hence, the or switch to a second-line drug. need for a direct comparison of the two currently available dipsticks remains a priority. In Secondary priorities for research aiming to addition, such a multicentre trial should incorpo- improve Leishmania diagnostics include: rate regions endemic for VL caused by L. infan- • evaluating diagnostic tools for post kala azar tum, where sensitivity and specificity is likely dermal leishmaniasis (PKDL) to differ considerably. Improvement of the anti- • comparing currently available rapid tests for gens used in the urine dipstick test should also diagnosis of canine leishmaniasis be explored. As improved rapid diagnostic tests become available in the future, the LCTG • evaluating tools for the detection of asympto- should have the capacity to respond without matic infection. delay and carry out systematic multicentre trials across all regions endemic for VL.

8 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 IMPROVED THERAPIES AND studies, combination therapies should be tested in systematic multicentre trials on the Indian PATIENT MANAGEMENT subcontinent.

Miltefosine, an alkylphosphocholine, is the first Secondary priorities for research aiming to oral drug to be registered for VL. Due to its ter- improve treatment protocols include: atogenic potential, this drug cannot be provided evaluating combination treatments for VL out- to females of childbearing age, unless contracep- • side the Indian subcontinent tion can be assured. With these exceptions, trials on the Indian subcontinent have confirmed • evaluating treatments for PKDL and ML/CL the safety and efficacy of miltefosine for VL • evaluating the implications of antiretroviral patients in that region; and there is great promise therapy in the clinical management of HIV co- that the regulated provision of this drug, prefer- infected patients. ably as directly observed therapy (DOT), could significantly improve drug adherence rates. It is now a priority to test the impact of miltefo- IMPROVED PREVENTION sine in systematic multicentre trials in all VL- STRATEGIES endemic regions outside the Indian subcontinent. Insecticide-treated materials, in particular insec- Paromomycin, an aminoglycoside, is currently ticide-treated nets (ITNs), provide one of the in Phase III trials in India and also shows great most effective methods for reducing man-vec- promise, not least due to its low price and good tor contact and transmission of vector-borne dis- safety profile. This is one of the few drugs avail- eases. ITNs have replaced residual insecticide able that could potentially replace Sb and/or be spraying of houses as the malaria control strat- used in multidrug therapy. It is now a priority to egy of choice in much of sub-Saharan Africa test the impact of paromomycin on VL patients and Asia on the basis of comparative cost, logis- Leishmaniasis worldwide. tic effort, sustainability, and (to some extent) environmental impact. Pyrethroids are currently The development and spread of resistance to selected for treating the materials on the basis of Sb in India has led to widespread concern that safety, environmental considerations, and effi- the limited supply of second-line drugs needs cacy. It is important to monitor the develop- to be protected and used judiciously. One obvi- ment of pyrethroid resistance in vectors where ous approach to prevent the appearance or limit pyrethroid-treated materials are widely used, the spread of drug resistance (e.g. to miltefo- but fortunately pyrethroid resistance has not yet sine) is to provide multidrug therapy. The choice become an operational concern for the various of drug combination will depend on pharma- ITN malaria programmes worldwide. cokinetic properties and toxicity issues. The aim is to provide a combination with low toxic- ITNs will be most effective when the vector ity, high effectiveness (for example, due to syn- bites humans mostly inside houses during the ergism), low cost, low risk of developing drug night, when people are in bed. Hence, to deter- resistance (which may be related, for exam- mine the likelihood that an ITN programme ple, to half life) and shorter course of treatment could significantly reduce leishmaniasis trans- (to increase adherence rates). As soon as this mission in a given locality, it is essential first knowledge emanates from in vivo experimental to have sufficient evidence from the principal

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 9 site about the time of human transmission, for Secondary priorities for research aiming to vali- example by carrying out sandfly behavioural date prevention strategies include: studies. Where sandflies bite indoors but are sig- • comparing the effectiveness of competitor nificantly active from dusk onwards, insecticide insecticides applied to dogs for control of treatment of curtains may provide effective pro- zoonotic visceral leishmaniasis tection. Where people sleep outdoors, and are • evaluating the efficacy of insecticide treated at high risk of sandfly bites, protection can be materials in prevention of CL (except for L. achieved by using ITNs hung outdoors. tropica regions, where there is already proof of principle) Recent ITN trials in Syria and Afghanistan have demonstrated significant protection against CL • optimization of ITNs (materials, mesh size, (L. tropica) transmitted by Phlebotomus sergenti, coolness specifically for sandflies). and it can be assumed that this strategy would be effective throughout the range of P. sergenti Once a prevention strategy has been validated (dependent on local compliance). The same by field trials, there remain considerable bottle- trial in Afghanistan also showed that insecticide necks impeding the translation of available tools treated bed sheets provided protection against into more effective leishmaniasis control. Whilst CL, and there were some cost advantages of this efficacy trials can demonstrate the likely impact approach. Also, a field trial in Venezuela dem- of implementing an intervention under speci- onstrated the protective effect of insecticide fied conditions, further implementation research treated curtains (ITC) on transmission of CL (L. is required for policy-makers to decide where braziliensis) transmitted by Lutzomyia youngi. and when it is cost effective to do so. The first However, to date there is no other direct evi- requirement is to have reliable information on dence that insecticide treated materials reduce the leishmaniasis burden of disease at a local level (where decisions are ultimately made). The

Leishmaniasisleishmaniasis transmission. second requirement is for sufficient understand- The effectiveness of current residual insecticide ing of transmission ecology at the local level to spraying campaigns against VL on the Indian know which intervention (if any) is appropri- subcontinent and in Brazil is limited by the ate. Hence, in order that health delivery systems inherent difficulties of achieving widespread and can rationally allocate resources for leishmani- regular coverage. No such campaigns are ongo- asis control, it is essential that managers have ing in East African VL endemic areas (where ready access to reliable local data. In practice, sandfly vectors are largely exophilic). The pro- this can be most effectively achieved by devel- vision of ITNs (or ITCs) could, in contrast, lead oping geographic information systems (GIS) to a significant and sustainable reduction in VL into which surveillance data are regularly and morbidity. It is therefore a high priority to test systematically incorporated. Distribution maps the impact of ITNs (indoor or outdoor, as appro- of burden of disease and sandfly vectors, at a priate) or ITCs on VL incidence in large-scale resolution relevant for disease control decisions field trials where there is convincing entomolog- (e.g. municipality level), need to be generated ical evidence that these interventions are likely by the collation and analysis of available data. to be effective. Where such evidence is insuffi- Gaps can be filled in by producing risk maps fit- cient, further entomological studies should be ted by comparison of known distributions with undertaken to inform whether trials are justified. environmental correlates with known compre-

10 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 hensive distributions (such as from satellite With the development and spread of antimony images). Such an approach has been developed resistance on the Indian subcontinent, and the for a wide range of other infectious diseases, potential for introducing new drugs either sin- and should become an invaluable management gly or in combination to combat this threat, it is tool for leishmaniasis control programmes. The vital that a rational policy is devised to minimize approach should be tested by pilot studies, prob- the spread of resistance to new drugs. Experi- ably at sub-national level, in different endemic ence with antibiotics and antimalarials indicates regions to help develop a framework for best that, where drug adherence is poor and there are practice. The approach should take advantage high rates of transmission, the rate of spread of of existing risk maps for other diseases. The drug resistance could (in the absence of such development of low-technology instruments for a policy) exceed the rate at which new drugs epidemiological assessment would be an imme- become available. It is therefore a high prior- diately implementable strategy in many endemic ity to carry out analytical studies anchored by foci and could provide data for risk maps when empirical data to compare the likely impact of the infrastructure for GIS comes into place. different drug policies on the development and spread of Leishmania drug resistance (analo- The rapid assimilation of surveillance data gous to the considerable body of analytical work by health managers, combined with data on explaining the factors determining the spread of changes in climate, land cover or human popu- antibiotic resistance). lation movements, could provide a basis for predicting epidemics and so allow rapid responses As new drugs are introduced in accordance with to potential emergencies (such as in Southern rationally designed drug policies, it is vital to Sudan). This requires the development of leish- develop a systematic surveillance system for maniasis epidemic predictive models as well as drug resistance so that drug policy can respond

protocols for emergency response. rapidly to changing conditions, and to alert clin- Leishmaniasis ical practitioners. Currently, resistance sur- Although the number of Leishmania-HIV co- veillance must depend on reports of clinical infections has been drastically reduced in indus- outcome following patient treatment, validated trial countries since the introduction of highly by drug sensitivity assays of parasite isolates active anti-retroviral therapy (HAART), the from a sample of patients. But it would be ben- ruralization of HIV transmission and the urban- eficial to base a surveillance system on readily ization of leishmaniasis in many low-income identifiable molecular markers (as they become countries, where most HIV infection occurs, available). demand for alertness and surveillance. In addition, the benefit of HIV treatment does not apply Effective implementation of leishmaniasis con- to the 50–60% of co-infected patients for whom trol programmes, whether limited to diagnosis diagnosis of Leishmania infection precedes or and treatment or including prevention strategies concurs with the diagnosis of HIV. More impor- such as vector control, require an appreciation tantly, in Sub-Saharan African countries, which of the limits set by economic constraints and by account for the vast majority of HIV/AIDS glo- the structure of health systems. As a concerted bal cases, very few HIV-infected patients have attempt is made to exploit the battery of existing access to HAART. and new tools and information (as they become available) to reduce the global burden of leish-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 11 maniasis, it will be vital to invest in implementation research to ensure sustainability. This should include, for example, cost-effectiveness analyses of competing leishmaniasis test-treatment strategies and competing prevention strategies. Socioeconomic and behavioural studies are also required to evaluate factors determining drug adherence in order to aid the development of tools for increasing the proportion of patients receiving full treatment courses. The cost of not intervening with an integrated control strategy, or of interrupting such a strategy and facing the consequent resurgence of disease, is also an important consideration.

PARTNERSHIPS AND CAPACITY BUILDING

Capacity building in endemic countries should focus on the newly proposed Leishmaniasis Clinical Trial Group (LCTG), which needs to develop strategic partnerships with the Human African Trypanosomiasis Group and other clinical trial networks, such as the European and LeishmaniasisDeveloping Countries Clinical Trials Partner- ship (EDCTP) and Drugs for Neglected Dis- eases initiative (DNDi), and with industry, such as veterinary product companies manufacturing diagnostic tests for canine leishmaniasis. Clin- ical trials require capacity in epidemiological assessment and in communication to different stakeholders. In addition, there is an urgent need to increase capacity in health economics among the leishmaniasis research community.

12 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 2. Challenges and opportunities

Leishmaniasis remains a Category 1 TDR dis- sis, indicating the existence of inherited factors ease, signifying its status as emerging and in susceptibility in humans. The genes control- uncontrolled. While the reasons for this are ling these host response defects have not been many, the need for new and better tools, such as identified. Human genetic studies could pro- diagnostics, drugs and vaccines, and gaps in the vide critical information for the discovery of key basic knowledge needed to develop these tools, steps in the pathogenesis of Leishmania infec- remain serious barriers to the design and imple- tions and allow the identification of new targets mentation of effective control strategies. for chemotherapy and vaccination.

CHALLENGES POSED BY GAPS There is a need to develop standardized and effective vaccines for both prevention and treat- IN BASIC KNOWLEDGE AND ment of various forms of human and canine LIMITATIONS OF CURRENT TOOLS leishmaniasis. While a number of antigens have been identified that show promise as potential Despite a wealth of information about the candidate vaccines, none has so far been evalu- mechanisms underlying immunity to disease ated in clinical trials. There is a need to identify expression following Leishmania infection in additional antigens for VL vaccines, validate the experimental animal models, the correlates potential of promising antigens, and move for- of immunity in human leishmaniasis remain ward with selected recombinant protein or DNA- poorly defined. Understanding the mechanisms based vaccines into clinical development. The of innate or acquired resistance in patients with choice of target antigens for further development self-limiting forms of cutaneous leishmaniasis, should be based on a number of criteria, includ- or in individuals with asymptomatic infections, ing their expression by amastigotes and their especially involving visceralizing strains, is cru- ability to confer cross-protection against differ- cial to the development of effective vaccines

ent Leishmania species, especially L. donovani. Leishmaniasis and immunotherapies. Animal models that are Existing and new adjuvants, and antigen deliv- used to address these issues should more accu- ery systems need to be explored to elicit effec- rately reflect human disease. tive immune response of long duration. Given the failure of whole cell killed vaccine plus There is limited knowledge concerning the host BCG to protect in clinical trials, the advantages and parasite factors controlling the pathogen- and disadvantages of live vaccination with L. esis of non-healing and reactivating disease major (leishmanization) should be re-examined. forms in humans, especially visceral leishmania- The ability of live L. major vaccines to confer sis, PKDL, DCL, mucosal disease and co-infec- protection against VL needs to be investigated tion with HIV. This information could lead to but care must be taken not to introduce new spe- the design of novel treatments to promote cure cies of Leishmania into areas where they do not or to prevent dissemination or reactivation of currently exist. Meanwhile target populations latent infections, an emerging challenge in VL- should be identified, and the potential cost effec- endemic areas that are increasingly engulfed by tiveness of vaccination should be assessed. the HIV epidemic. The main problems associated with the current Epidemiologic studies have shown familial and treatments for VL are toxicity, cost, non-compli- ethnic clustering of certain forms of leishmania- ance, and resistance. There is a need to develop

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 13 novel oral, non-toxic, short-course combination RESEARCH OPPORTUNITIES therapies for visceral leishmaniasis. Combina- tion therapies with new and existing drugs need TO CLOSE THE GAPS IN to be explored in humans and in animal models. BASIC KNOWLEDGE AND TO The various forms of cutaneous leishmaniasis are DEVELOP NEW TOOLS commonly treated with SbV in a dose of 20 mg/ kg for 20 days, but there are few data on whether The completion of the L. major genome (and this regimen is appropriate for all species. A spe- the soon to be completed L. infantum genome) cies-based approach needs to be investigated. is a landmark that for the first time identifies all genes that encode proteins and processes that Current diagnostics succeed mainly in confirm- may be targets for drug discovery, vaccine can- ing the Leishmania genus in active cases of dis- didates, and useful for diagnostics. Compara- ease. New diagnostic tools are needed that will tive genomic studies at both the gene sequence and RNA expression levels, along with compar- detect infections in active as well as asympto- ative proteomic studies, can now be undertaken matic cases, and that will discriminate Leishma- to identify key differences among stages, strains nia parasites according to species, drug resistant and species of Leishmania that affect the estab- phenotype, and possible strain-specific clini- lishment of infection and the development of cal associations, especially PKDL, MCL, and different types of disease. Identification of anti- asymptomatic visceral infection. gens present and, ideally, conserved in amastigotes may permit the selection of antigens The incidence of the human leishmaniases is for potential use as vaccines. DNA microar- increasing due to the growth and redistribution rays can be useful to identify more species- and of populations, HIV co-infection, poor levels of strain-specific markers for diagnostic studies hygiene and nutrition, increasing poverty, plus and for identification of drug resistant isolates. The identified sequences in conjunction with

Leishmaniasischanges in climate, ecology and vector behav- polymerase chain reaction (PCR) techniques can iour. The socioeconomic, environmental, and be used to detect and discriminate Leishmania behavioural risk factors for infection and disease species and strains within suspected sandfly vec- need to be identified, especially in peri-urban tors, animal reservoirs, and in individuals with and refugee populations. latent or asymptomatic infections.

More knowledge about the impact of inequity Genome-wide analyses of host gene sequences of access to health care, and of health sector can be used to identify genes linked to disease reform, will be needed for interventions involv- susceptibility in human population studies, and ing use of even the best tools (drugs, vaccines, host RNA expression profiling can be used to diagnostics) to succeed. identify host factors associated with immunity or with particular disease outcomes. There is limited knowledge of the identity and behaviour of sandfly vectors, and of the role of A number of recombinant proteins and DNA animal reservoir hosts in many endemic regions. vaccines have already been shown to protect This information is crucial to the design and against leishmaniasis in animal models, and implementation of control strategies involving, some may be ready for clinical development. In for example, insecticide impregnated bednets or addition, a number of novel adjuvants that may dog collars. be able to potentiate the appropriate immune

14 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 response in humans are available, including Toll- like receptor (TLR) agonists like monophosphoryl lipid A (MPL), oliginucleotides (CpGs), imiquimod, and transcutaneous delivery systems. The only proven vaccine to date against human disease is live L. major (leishmanization), given on the arm to prevent severe and multiple lesions. The problems associated with live vaccination may be minimized by engineering strains with reduced pathogenicity, or by administer- ing the live vaccine with certain immune poten- tiating/modulating adjuvants. The use of live L. major challenge in validating vaccine efficacy in human should also be evaluated.

Satellite images can be exploited as a source of environmental data to explain the distribution of reservoirs, vectors, and disease. Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 15 Leishmaniasis

16 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 3. High priorities

INTERVENTION METHODS BASIC KNOWLEDGE

• Evaluate rapid diagnostic tests for visceral • Comparative genomics and proteomics to leishmaniasis. identify molecules with roles in disease • Develop and validate diagnostic algo- for development of new drugs, diagnostics, rithms for test-treatment strategies. and vaccines. • Evaluate new treatments for visceral • Immune correlates of protection against leishmaniasis: paromomycin, miltefo- human leishmaniasis. sine (outside the Indian subcontinent), and • Definition of host and parasite factors combination therapies on the Indian sub- involved in pathogenesis. continent and subsequently elsewhere. • Human population genetic studies to • Evaluate insecticide-treated nets for vis- determine genes controlling susceptibility ceral leishmaniasis control. to VL and response to drugs. • Identification of new vaccine candidates STRATEGIES & POLICIES for VL, and novel adjuvants and delivery systems to promote effective and long- • Economic, social, and health systems lived immune response. research to enhance implementation of • Epidemiologic studies to identify socioe- control strategies and drug adherence. conomic, environmental, and behavioural • Develop decision support systems to target risk factors for infection and disease. control activities and predict epidemics. • Analytical studies to develop drug pol- NEW AND IMPROVED TOOLS icy and design of surveillance protocol to limit and monitor the spread of drug • High-throughput technologies for identi-

resistance. fication of target molecules and develop- Leishmaniasis ment of novel assays. PARTNERSHIPS & • Selection and development of candidate vaccines and adjuvants for evaluation in CAPACITY BUILDING clinical trials, including live L. major as a vaccine against VL. • Capacity building of new global network: • Development and evaluation of immuno- Leishmaniasis Clinical Trial Group. therapeutics. • Strengthen input of health economics in • Novel oral, non-toxic, short-course treat- leishmaniasis control. ments for visceral leishmaniasis and com- • Develop links with clinical trial and bination therapy in VL involving new and surveillance networks, the Drugs for existing drugs to improve efficiency and Neglected Diseases initiative (DNDi), and reduce emergence of drug resistant strains. industry. • Diagnostics that will discriminate between Leishmania in active disease, asymptomatic or latent infections, drug resistant phenotypes, and disease associations. • Tools to identify sandflies at species level and to identify infected flies.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 17 Leishmaniasis

18 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 1 AGENDA: Scientific Working Group on Leishmaniasis Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 19 Day 1, Monday 2 February 2004

Time Item Speaker 08.30–09.00 Welcome address 1Dr Asamoa-Baah, ADG/CDS Dr R. Ridley, Director a.i./ TDR 09.00–09.15 TDR disease strategic plans and role of the Dr J. Lazdins, TDR Scientific Working Groups 09.00–09:45 Current strategic emphases for leishmaniasis research Dr P. Desjeux, TDR 09.45–10.00 Meeting objectives and process Dr P. Desjeux, TDR 10.00–10.30 Coffee break

STRATEGIC RESEARCH – Dr A. Oduola, STR 10.30–11.00 From genomics to discovery Drs Zilberstein, K. Stuart and A. Fairlamb 11.00–11.30 Drug resistance Dr S. Croft 11.30–12.00 Host genetics Dr A. Dessein 12.00–13.00 Lunch break

IMPROVED TOOLS – Dr R. Ridley, PRD 13.00–13.30 Vaccines Dr S. Reed 13.30–14.00 Molecular tools for epidemiology Dr I. Guizani 14.00–14.30 Drug trials Dr S. Sundar 14.30–15.00 Tools/strategies for canine leishmaniasis Dr C. Davies 15.00–15.30 Tools/strategies for insecticide-treated fabrics (Dr D. Elnaiem) Dr A. Warburg 15.30–16.00 Coffee break

IMPROVED STRATEGIES – Dr H. Remme, IR 16.00–16.30 Cost-effectiveness of diagnostic and therapeutic strategies Dr M. Boelaert 16.30–17.00 Leishmania/HIV co-infection Dr J. Alvar 17.00–17.30 (Impact of inequity of access to services) Dr N.K. Ganguly2 17.30 Reception (main cafeteria)

Day 2, Tuesday 3 February 2004

PARTNERSHIP & CAPACITY BUILDING – F. Zicker/RCS, A. Oduola/STR & H. Remme/IDE

Time Item Speaker 08.30–09.00 (Field diagnostic tests) Dr J. Seaman2 09.00–09.30 Risk factors Dr A. Llanos Cuentas 09.30–10.00 Can leishmaniasis still be considered as category 1?3 If moved, what is required? Dr N. Saravia 10.00–10.30 Working Groups I, II (jointly) Salle M.105 Working Groups III, IV & V (jointly) Salle C 10.30–11.00 Coffee break 11.00–12.30 Working Groups I, II (jointly) Salle M. 105 Working Groups III, IV & V (jointly) Salle C 12.30–13.30 Lunch break 13.30–18.00 Working Groups I, II (jointly) Salle M. 105 Working Groups III, IV & V (jointly) Salle C 15.30–16.00 Coffee break (Salle C)

1 Unable to attend 2 Shift in presentation period between Drs Seaman (section II) and Ganguly (section III) 3 TDR deﬁnition of category I disease: epidemiological situation getting worse, and incidence of infection and disease increasing.

20 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Day 3, Wednesday 4 February 2004

Time Item Speaker 08.30–09.30 Plenary report Working Groups I and II Discussion on overall prioritization with functional area coordinators 09.30–10.30 Plenary report Working Groups III, IV and V Discussion on overall prioritization with functional area coordinators 10.30–11.00 Coffee break 11.00–12.30 Rapporteurs to prepare draft SWG report Draft report distributed for plenary discussion 12.30–13.30 Lunch break 13.30–15.30 Plenary presentation, discussion & amendment of the SWG draft report 15.30–16.00 Coffee break 16.00–17.30 Discussion, amendment and concluding remarks Chairman Director, TDR

Day 4, Thursday 5 February 2004

Time Item Speaker 08.30–17.30 Finalization of the SWG report (chairman and rapporteurs only)

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 21 22 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 2 LIST OF PARTICIPANTS: Scientific Working Group on Leishmaniasis Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 23 Temporary Advisers

Dr Hannah Akuffo Dr Jenefer Blackwell Microbiology and Tumour Biology Center Glaxo Professor – Molecular Parasitology Karolinska Institute Cambridge Institute for Medical Research Box 280 Wellcome Trust/MRC Building 17177 Stockholm Addenbrooke’s Hospital SWEDEN Hills Road Tel.: (46) 8.698.52.63 Cambridge CB2 2XY Fax: (46) 8.698.56.56 UK E-mail: [email protected] Tel.: (44) 1.2233.36947 Fax: (44) 1.2233.31206 Dr Jorge Alvar Ezguerra E-mail: [email protected] Servicio de Parasitologia Centro Nacional de Microbiologia Dr Marleen Boelaert Instituto de Salud Carlos III Institute of Tropical Medicine Ctra. Majadahonda, Pozuelo Km 2 Department of Public Health 28220 Majadahonda, Madrid Nationalestraat 155 SPAIN 2000 Antwerpen Tel.: (34) 91.387.7761 BELGIUM Fax: (34) 91.509. 79 66 Tel.: (32) 3.247.6286 E-mail: [email protected] Fax: (32) 3.247.6258 E-mail: [email protected] Dr Abraham Aseffa Armauer Hansen Research Institute Dr Simon L Croft P.O. Box 1005 Department of Tropical and Infectious Diseases Addis Ababa London School of Hygiene and Tropical Medicine ETHIOPIA Keppel Street Tel.: (251) 1.710. 288 London WC1E 7HT Fax: (251) 1.711.390 UK E-mail: [email protected] Tel.: (44) 2.07927.2345 Fax: (44) 2.07323.5687 Dr Richard Ashford E-mail: [email protected] (Retired) Professor of Parasite and Vector Biology Liverpool School of Tropical Medicine Dr Clive Davies Liverpool L3 5QA Senior Lecturer UK Department of Tropical and Infectious Diseases Tel.: (44)1.51632.2714 London School of Hygiene and Tropical Medicine Fax: (44)1.51705.3371 Keppel Street E-mail: [email protected] London WC1E 7HT UK Dr Roberto Badaro Tel.: (44) 2.07927.2350 Professor of Medicine Fax: (44) 2.07636.8739 Chief of the Division of Infectious Diseases E-mail: [email protected] Prof. Edgard Santos University Hospital Federal University of Bahia Prof Jean-Pierre Dedet Rua Joao das Botas, s/n Canela Laboratoire de Parasitologie 40110-160 Salvador 163, rue Auguste Broussounet BRAZIL 34090 Montpellier Tel.: (55)71.332.4445 FRANCE Fax: (55)71.247.2756 Tel.: (33) 467.632.751 E-mail: [email protected] Fax: (33) 467.630.049 E-mail: [email protected]

24 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Prof Kusai Dellagi Dr Luigi Gradoni Directeur Head, Section of Protozoology Institut Pasteur de Tunis Istituto superiore di Sanita 13 Place Pasteur Laboratorio di Parassitologia B.P. 74, Belvédère Viale Regina Elena 299 1002 Tunis 00161 Rome TUNISIA ITALY Tel.: (216)71.845.415 Tel.: (39)06.4990.2957 Fax: (216)71.791.833 Fax: (39)06.4938.7065 E-mail: [email protected] E-mail: [email protected]

Dr Alain Dessein Dr Marina Gramiccia Laboratoire d’Immunologie et génétique des maladies Istituto superiore di Sanita parasitaires Laboratorio di Parassitologia Faculté de Médecine Viale Regina Elena 222 27 boulevard Jean Moulin 00161 Rome 13385 Marseille Cedex 5 ITALY FRANCE Tel.: (39) 06.4990.3015 Tel.: (33)4913.24452 Fax: (39)06.4938.7065 Fax: (33)4917.96063 E-mail: [email protected] E-mail: [email protected] Dr Nouzha Guessous-Idrissi Dr Dia-Eldin A Elnaiem (unable to attend) Faculté de Médicine et de Pharmacie Research Scientist Laboratoire de Parasitologie-Mycologie Vector Genetics Laboratory 19 rue Tarik Ibn Ziad, Casablanca Department of Entomology 20006 Maroc 367 Briggs Hall, UC-Davis MOROCCO 1 Shields Avenue Tel.: (212)22.271.630 Davis, CA 95616 Fax: (212)22.475.560 USA E-mail: [email protected] Tel.: 1(530)752.7333 Dr Ikram Guizani Fax: 1(530)752.7333 Scientist E-mail: [email protected] Institut Pasteur de Tunis Prof Alan Fairlamb Laboratoire d’Epidemiologie et d’Ecologie Parasitaire Wellcome Principal Research Fellow Boîte postale 74 Division of Biological Chemistry & Molecular Belvédère Microbiology 1002 Tunis Wellcome Trust Biocentre TUNISIA University of Dundee Tel.: (216)71.792429 Dundee DD1 5EH Fax: (216)71.791833 Scotland E-mail: [email protected]; UK [email protected] Tel.: (44) 1.38234.5155 Dr Barbara Herwaldt Fax: (44) 1.38234.5542 Centers for Disease Control and Prevention E-mail: [email protected] Division of Parasitic Diseases Prof Nirmal K Ganguly 4770 Buford Hwy NE, MS F-22 Director General Atlanta, GA 30341–3724 Indian Council of Medical Research USA Ansari Nagar Tel.: 1(770)488.7772 Postbox 4911 Fax: 1(770)488.7761 New Delhi 110 029 E-mail: [email protected] INDIA Tel.: (91)11.2658.8204 Fax: (91)11.2686.8662 E-mail: [email protected]

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 25 Dr R. Killick-Kendrick (unable to attend) Dr Maowia Mukhtar Department of Pure and Applied Biology Department of Molecular Biology Imperial College Field Station Institute of Endemic Diseases Ashurst Lodge, Silwood Park University of Khartoum Ascot, Berks S15 7PY P.O. Box 11463 UK Khartoum Tel.: (44) 1.34429.4267 SUDAN Fax: (44) 1.38429.4339 Tel.: (249)1177.9712 E-mail: [email protected] Fax: (249)1171.9712 E-mail: [email protected] Dr Alejandro Llanos Dean Dr Ana Rabello Facultad de Salud Publica y Administracion Centro de Pesquisas René Rachou “Carlos Vidal Layseca” Fundaçao Oswaldo Cruz Universidad Peruana Cayetano Heredia Av. Augusto de Lima 1715 Av. Honorio Delgado No. 430 30390-002 Barro Preto Urb. Ingenieria SMP Minas Gerais C.P. 4314, Lima 100 Belo Horizonte PERU BRAZIL Tel.: (511) 482.7739 Tel.: (55)313.295.3566 Fax: (511) 382.0338 Fax: (55)313.295.3115 E-mail: [email protected] E-mail: [email protected]

Dr Jacques Louis Dr Steven G. Reed Division of Parasitology Founder and CEO Pasteur Institute Infectious Disease Research Institute 28, Rue du Dr Roux Suite 600 75724 Paris Cedex 15 1124 Columbia Street FRANCE Seattle WA 98104 Tel.: (33) 1443.89125 USA Fax: (33) 1406.13169 Tel.: 1(206)754.5712 or 57 E-mail: [email protected] Fax: 1(206)754.5715 E-mail: [email protected] Dr Diane McMahon-Pratt Professor Dr Suman Rijal Department of Epidemiology and Public Health Associate Professor Yale University School of Medicine Department of Internal Medicine 60 College Street – LEPH 711 B.P. Koirala Institute of Health Sciences P.O. Box 208034, New Haven Dharan CT 06520-8034 NEPAL USA Tel.: (977)2552.5555/2052 Tel.: 1 (203) 785.4481 Fax: (977)2552.0251 Fax: 1 (203) 737.2921 E-mail: [email protected] E-mail: [email protected] Dr David Sacks Dr Farrokh Modabber Head Director Intracellular Parasite Biology Section Infectious Disease Research Institute Laboratory of Parasitic Diseases Suite 600 NIAID, Bldg 4, Room 126 1124 Columbia Street 4 Center Drive, MSC 0425 Seattle WA 98104 Bethesda MD 20892-0425 USA USA Tel.: 1(206)381.0883 Tel.: 1(301)496.0577 Fax: 1(206)381.3678 Fax: 1(301)480.3708 E-mail: [email protected] E-mail: [email protected]

26 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Dr Nancy G Saravia Dr Ivan Dario Velez Bernal Executive Director Director Centro Internacionale de Entrenamiento e Programa de Estudio y Control de Enfermedades Investigaciones Medicas (CIDEIM) Tropicales (PECET) Apartado Aereo 5390 Universidad de Antioquia Avenida la Norte No. 3-03 Carrera 50 A #63-85 Cali, Valle Medellin COLOMBIA COLOMBIA Tel.: (57)266.82164 Tel.: (574)263.5555 Fax: (57)266.72989 Fax: (574)516.2675 E-mail: [email protected] E-mail: [email protected]

Dr Jill Seaman Dr Alon Warburg MSF Holland Office Department of Parasitology Street 27, Amaraat Hadassah Medical School P.O. Box 3319, Khartoum 2 Hebrew University SUDAN Jerusalem 91120 Tel.: (249)11.463.800 ISRAEL Fax: (249)11.472.826 Tel.: (972)2.675.7080 E-mail: [email protected] Fax: (972)2.675.7425 E-mail: [email protected] Dr Ken Stuart Seattle BIOM.RES.INS Dr Dan Zilberstein 4 Nickerson Street Department of Biology Seattle WA 08109 Technion-Israel Institute of Technology USA Senate House Tel.: 1(206)284.8846 Haifa 32000 Fax: 1(206)284.0313 ISRAEL E-mail: [email protected] Tel.: (972)4.829.3647 Fax: (972)4.829.5670 Dr Shyam Sundar E-mail: [email protected] Professor Banaras Hindu University Institute of Medical Sciences 6 S.K. Gupta Nagar, Lanka Varanasi 221 005 INDIA Tel.: (91)5423.67795/2368895 Fax: (91)5423.68912 E-mail: [email protected]

Representatives of partner institutions

Dr François Chappuis Dr Koert Ritmeijer Unité de Médecine des voyages et migrations Health Adviser Hopital Cantonal Médecins Sans Frontières Polyclinique de Médecine MSF Holland 24 rue Michel Ducret Plantage Middenlaan 14 1211 Geneva 14 P.O. Box 10014 SWITZERLAND 1001EA Amsterdam Tel.: (022)372.9620 NETHERLANDS E-mail: [email protected] Tel.: (31)20.520.8767 Fax: (31)20.620.5170 E-mail: [email protected]

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 27 Dr Els Torreele Dr Catherine Royce Drugs for Neglected Diseases Initiative (DNDi) Drugs for Neglected Diseases Initiative (DNDi) 1, place St Gervais 1, place St Gervais 1201 Geneva 1201 Geneva SWITZERLAND SWITZERLAND Tel.: (22)906.9230 Tel.: (44)20.8992.8925 Fax: (22)906.9231 E-mail: [email protected] E-mail: [email protected] Direct line: (32)3.218.8256

WHO Regional Office staff

Dr Mikhail Nicolaevich Ejov (unable to attend) Dr Bettina Menne Medical Officer/EURO Global Change and Health WHO European Centre for Environment and Health Dr Zuhair S Hallaj (unable to attend) Via Francesco Crispi, 10 Director/EMRO 00187 Rome Dr Thomas Sukwa ITALY Medical Officer (CRD)/AFRO Tel.: (39)06.487.7546 Fax: (39)06.487.7599 Dr Derek Anthony C Lobo E-mail: [email protected] Scientist/SEARO

Dr Zaida Estela Yadon PAHO, Buenos Aires E-mail: [email protected]

WHO headquarters staff

Dr Anarfi Asamoa-Baah (unable to attend) Dr Ayoade Oduola Asst. Director-General/Communicable Diseases (CDS) Coordinator, Basic and Strategic Research (STR)/TDR

Dr Philippe MP Desjeux Dr Jan HF Remme Leishmaniasis Research Coordinator Coordinator, Intervention Development and Implementation Research (IDE)/TDR Dr Hiroyoshi Endo (unable to attend) Director, Communicable Diseases Control, Prevention Dr Robert Ridley and Eradication (CDS/CPE) Director a.i., TDR

Dr HW Ghalib Dr Fabienne Tacchini-Cottier Medical Officer WHO/Immunology Research and Training Centre (IRTC) Dr Pascal Launois Institute of Biochemistry Medical Officer 1066 Epalinges Tel.: (41)22.791.2659 Tel.: (021)692.5707 Fax: (41)22.791.4854 Fax: (021)692.5705 E-mail: [email protected] Dr Fabio Zicker Dr Janis Karlin Lazdins-Helds Coordinator, Research Capability Strengthening Acting Coordinator, Product Research and (RCS)/TDR Development (PRD) & Coordinator, Disease Research

Coordinators (DRCs)/Programme Management (PPM)/TDR

TDR Disease Research Coordinators

Dr Lester Chitsulo, Schistosomiasis Dr Janis Lazdins, Focal point for disease research Dr Philippe Desjeux, Leishmaniasis coordinators Dr Deborah Kioy, African trypanosomiasis Dr Philip Onyebujoh, Tuberculosis Dr Axel Kroeger, Dengue Dr Yeya Touré, Malaria

28 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 3 STRATEGIC RESEARCH

3.1 From genome to discovery in Leishmania: Recommendations for the development of drugs, vaccines and diagnostics ...... 30

3.2 Drug resistance in leishmaniasis (summary) ...... 33

3.3 Host genetics in leishmaniasis ...... 34

3.4 Mechanisms of pathogenesis – differences amongst Leishmania species

3.5 Cellular immune responses (summary) ...... 40

3.6 Vector–parasite and vector–host interactions in leishmaniasis ...... 42 Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 29 3.1 FROM GENOME TO • Gene expression profiling at the RNA and/or DISCOVERY IN LEISHMANIA: protein level among strains that differ in disease. • Studies that generate transgenic Leishmania with RECOMMENDATIONS FOR THE altered capabilities for producing pathogenesis. DEVELOPMENT OF DRUGS, • Studies of host factors that affect the develop- VACCINES AND DIAGNOSTICS ment of disease. • Genome-wide analyses that predict metabolic Kenneth Stuart,1 Alan Fairlamb2 and Dan pathways and cellular processes, especially in Zilberstein3 amastigotes. 1 Seattle BIOM:RES.INS, Seattle WA 08109, USA • Analysis of pathogenesis in skin lesions, liver, 2 Division of Biological Chemistry & Molecular Biology, spleen, and mucocutaneous lesions. Wellcome Trust Biocentre, University of Dundee, UK • Studies of macrophage expression profiles asso- 3 Department of Biology, Technion-Israel Institute of ciated with disease and protection. Technology, Haifa 32000, Israel • Identification of Leishmania proteins that are predicted to affect host functions.

VACCINES INTRODUCTION Substantial evidence exists for the ability to The completion of the Leishmania major genome develop a vaccine, although the specific correlates sequence is a landmark that, for the first time, identi- of acquired and induced immunity are yet to be fies all potential proteins that may be targets for drug defined. Therefore, we recommend support for vac- development, vaccine candidates, and/or useful for cine development with an emphasis on a genome- diagnostics. However, it is not known how these wide approach, and suggest: pathogens utilize the proteins to establish infection, • Protein-wide analyses (protein microarrays) to prevent elimination by the host, and cause disease. identify protein antigens that are predicted to protect against infection or disease. Recent technological advances in experimental and • Using the same approach to identify amastigote in silico analyses create a promising environment specific antigens. for generating the knowledge needed to combat the • Characterization of immune response specific spectrum of leishmanial diseases. These advances for natural or induced protection. can be exploited for comparative genomics at both • Development of live attenuated (recombinant) the gene sequence and RNA expression levels, for parasite vaccines. parasite and host cell expression profiling at the RNA • Studies that combine vaccine development with (DNA microarrays) and protein (mass spectrome- analysis of immuno-pathogenesis. try) levels, and for more comprehensive analyses of • Studies of epitopes that may cross react between host immune responses to infection or experimen- host and pathogen. tal immunization including detection of T-cell recep- • Assessment of protection in mouse model sys- tor associated peptide antigens (mass spectrometry) tems and in parallel human analyses. and reactivity to specific antigens by ELISPOT and protein arrays. Our recommendations are below. Mouse model systems have been very fruitful and should continue to be used in combination with genetic manipulation of the pathogens and in concert FUNDAMENTAL STUDIES with examination of human parallels to the exper- Eluciding aspects of Leishmania gene function, phys- imental mouse model systems. Such studies will iology, genetic diversity, and host/pathogen interac- advance our understanding of factors that are crit- tion have advanced the ability to develop measures ical for the development of disease and can lead to to prevent or treat disease. We suggest: insights that could be used to develop preventative • Genome-wide studies to identify key genetic or therapeutic measures or for patient management. differences among strains and species of Leishmania that affect the establishment of infec- DIAGNOSTICS tion and/or the development of disease. • Studies to determine sequence or expression dif- Current methods of diagnosis of leishmaniasis ferences that correlate with different types of involve pathology and serology, using patient sera leishmanial disease (visceral, cutaneous, muco- against specific parasite antigens. However, the cutaneous, diffuse cutaneous) and post-kala- emergence of genetic engineering allows molecular azar dermal leishmaniasis (PKDL). diagnosis, which includes amplification of species-

30 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 specific parasite sequences using the polymerase DRUGS chain reaction (PCR). We recommend that TDR sup- Current drug treatment of leishmaniasis is unsatis- ports the application of DNA and protein microar- factory due to drug resistance, lack of efficacy, tox- ray technologies for diagnosis of leishmaniasis as icity, route of administration, prolonged treatment well as for the study of parasite virulence, develop- schedules, high cost, etc. In particular, the clinical ment, and host-pathogen interactions, as follows: value of frontline pentavalent antimonial drugs (pen- • In using DNA microarrays for the identification tostam, glucantime) is being seriously eroded by the of better and more species-specific markers, the emergence of widespread resistance in parts of India following directions should be encouraged: and Bangladesh. New drugs are required primar- – Comparative genomics of the various patho- ily for treatment of the life-threatening visceral form genic Leishmania strains/species. These of leishmaniasis and secondarily for the disfiguring studies should elucidate all possible species- mucocutaneous and cutaneous forms of the disease. specific genes. In addition, their expression profiles during infection can be assessed. The genome sequence provides a substantial oppor- – Gene expression of host genes during infec- tunity for identifying novel potential drug tar- tion. This can be done using either DNA gets through bioinformatics and database mining. microarrays of infected vs. non-infected mac- Comparative genomic studies could identify bio- rophage cell lines, or using patient tissues. chemical targets that are common to Leishmania and The results of these studies should provide trypanosomes, thereby reducing the drug discov- important information on host-parasite inter- ery and drug development costs of finding a broad actions as well as on host reaction to parasite spectrum anti-trypanosomatid drug. Many genes virulence. The results will also be useful for that are unique to Leishmania (or better still, are com- improved diagnosis. mon with trypanosomes but absent from humans) • Proteomics is an additional tool to study gene have unknown functions that may be essential for expression. Since regulation of gene expres- parasite growth or survival. The essentiality/redun- sion in Leishmania is post transcriptional, the dancy of such genes needs to be investigated by gene appearance of specific proteins is critical for the disruption or gene knockout, and the biological or study of pathogenesis, drug development and biochemical function of their products established. diagnosis. The following directions should be encouraged: WHO/TDR could play a coordinating role in these – Identification and characterization of post-genomic activities. WHO/TDR could also iden- Leishmania proteins in all developmental stages, tify potential drug targets with suitable profiles for using state of the art methodology. This should drug discovery (i.e. those targets that can be readily include post translational modifications such produced in large quantities using a robust, cheap as phosphorylation and glycosylation. and reproducible assay method for high through- – Comparative proteomics of pathogenic species. put screening). – Comparative analyses of gene expression, e.g. mRNA and protein abundance, throughout The Leishmania genome also provides new opportu- the life stages of Leishmania, including devel- nities to further define the mode of action of existing oping bioinformatics tools to compare mRNA drugs, with a view to developing novel classes of with protein abundance in order to assess drugs that act against existing targets or to provid- whether it is necessary to use both for diag- ing a scientific rationale for combination chemother- nostics and drug development. apy. Drug resistance is a serious threat to current – Development of microarrays of Leishmania therapy and a more detailed understanding of resis- proteins. This can be done by preparing a tance mechanisms in clinical isolates may reveal Leishmania expression library of the whole novel strategies to circumvent drug resistance. The genome and expressing all in bacteria fol- development of improved diagnostic methods lowed by printing the proteins/peptides on could be useful in monitoring clinical response to a solid phase, as has been done recently in drug therapy and as a predictor of drug sensitivity yeast. This will be useful for studying phe- in clinical isolates. notypic expression in Leishmania and, more importantly, for diagnosis. The following activities are recommended: • Development of tools for genome-wide based • Target identification by database mining and diagnosis in the field, for genomic and pro- metabolic pathway reconstruction (identify teomic microarray analyses. novel or unique/different pathways or enzymes common to trypanosomatids).

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 31 • Target validation using reverse genetics (identify essential genes for amastigote growth/survival). • Target characterization (establish biochemical function, molecular mechanism and structure). • Assay development and high throughput screening (identify lead compounds). • Screening of lead compounds against whole cells (chemical validation of target; demonstration of selectivity for parasite vs. host). • Development of transgenic parasites with reporter systems for use in WHO/TDR screening centres (in vitro and in vivo). • Comparative studies at the DNA, RNA and protein levels in drug sensitive and drug resistant lines (modes of drug action and drug resistance).

32 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 3.2 DRUG RESISTANCE IN toring and surveillance of the spread of resistance LEISHMANIASIS (SUMMARY) in anthroponotic foci and species specific diagnostic markers in some zoonotic foci where species of different intrinsic sensitivity co-exist. For antimo- Simon L. Croft nials and most drugs, this is problematic due to the Department of Infectious and Tropical Diseases, London absence of suitable markers and the reliance on a School of Hygiene and Tropical Medicine, UK culture assay to adequately correlate clinical and in vitro resistance.

The efficacy of drugs used for the treatment of visceral and cutaneous leishmaniasis is influenced by many factors. These include host factors and pharmacokinetics as well as both intrinsic variation in the sensitivity of Leishmania species and acquired drug resistance due to selection.

Intrinsic variation in sensitivity (susceptibility) has been described in relation to pentavalent antimonials, paromomycin, miltefosine, azoles and other drugs that have reached clinical trials. For several of these drugs, rank orders of species sensitivity of promastigote and amastigote forms have been described from laboratory studies. This has potential impact on clinical treatment, as demonstrated with respect to antimonials, ketoconazole and miltefosine.

Acquired resistance, due to selection, has been reported from laboratory studies on both clinically used and experimental antileishmanial drugs. However, it is only in the past decade that the clinical importance of resistance has become evident as pentavalent antimonials have become increasingly ineffective for the treatment of visceral leishmaniasis in Bihar State, India. Clinical Leishmania donovani isolates from non-responding patients showed a lower sensitivity to pentavalent antimonials than those from responsive patients. The mechanism(s) of resistance to pentavalent antimonials are a subject of current research and are still not clearly understood. Mechanisms involved in resistance to pentamidine, miltefosine, amphotericin B and azoles have been more clearly defined.

The development of resistance to pentavalent antimonials in a clinical focus of visceral leishmaniasis, and the potential of resistance developing to other drugs, has several implications. It makes the search for new drugs more urgent, it focuses attention on our lack of knowledge about the molecular and biochemical mechanisms of action of antimonial drugs on Leishmania, it places emphasis on strategies and policies for use of antileishmanial drugs in endemic areas, and it brings into focus the need to consider drug combinations in antileishmanial therapy. There is a need to develop systems for moni-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 33 3.3 HOST GENETICS IN Early attempts to identify genes involved in suscep-

LEISHMANIASIS tibility to cutaneous leishmaniasis in humans have focused on candidate-gene testing (15). Associations have been reported between some HLA alleles (16– Alain Dessein 18) and TNF gene polymorphisms (19) and the risk Laboratoire d’Immunologie et génétique des maladies of cutaneous and mucocutaneous leishmaniasis, parasitaires though all attempts to demonstrate an association Faculté de Médecine between HLA and VL have failed (20–23). 13385 Marseille Cedex 5 FRANCE Two recent studies in Sudan have begun to study the genetic control of VL in endemic populations. One study was carried out by our group during an outbreak of L. donovani that caused infection of Infections by Leishmania cause a wide spectrum almost all inhabitants of a village on the Ethiopian- of disease ranging from the asymptomatic to the Sudanese border (24). Though >90% of the villag- severely clinically symptomatic (1). Experimental ers showed immunological evidence of infection, work and studies in endemic populations show that only 30% developed visceral disease. Certain fam- the variety of manifestations in leishmaniasis can be ilies were more affected than others, suggesting ascribed in part to parasite virulence/pathogenic- some familial components involved in disease (25). ity and in part to host factors. Immunological stud- Familial clustering was not, however, as clear as that ies in human populations have associated certain reported in populations affected by schistosomes clinical manifestations with qualitative or quantita- (14). An attempt to demonstrate that this familial tive changes in the host specific immune response to component was due to a major genetic effect seg- Leishmania. These studies, however, do not in gen- regating in populations as a mendelian trait was eral identify the primary defects that cause the com- unsuccessful, indicating that the hypothesis of a sin- plex immunological phenotypes observed. If the gle major locus is probably not tenable. However, genetic make-up of the host is critical in leishmani- a linkage study carried out on 120 affected sib- asis, genetic analysis will identify these defects and ling pairs, a strategy that accommodates a multi- demonstrate the causal link between immunological genic control, and scanning the entire genome of the phenotype and disease. patients, showed that one locus (Chr. 22q12) and probably two genetic loci (Chr.2q22-q23) were con- The genetics of infection by Leishmania was first trolling susceptibility to VL in that population (26). studied in mice, and mutations in the Nramp1 gene Given the size of the study sample, the 22q12 locus (2, 3)(Bradley et al. 1979; Vidal et al. 1995) and the H- makes an important contribution to the control of 2 locus (4) were shown to affect multiplication of L. VL. It is not known yet whether one single gene or donovani. The possibility that susceptibility to leish- several genes at this locus is (are) involved in the maniasis may be increased by mutations at certain control. It is particularly interesting that a strong genetic loci is supported by linkage studies in mice genetic effect exerted by a single locus was reported infected by Leishmania major; several genetic regions in the population infected by three Leishmania spe- have been linked to susceptibility to infection (5–8). cies (27) (L. donovani 2/3 of the infections, L. infantum [1/3] and L. archibaldi [1/3]), all of them able to Epidemiological studies have shown familial and cause visceral disease. This indicates that the genetic ethnic clustering of leishmaniasis phenotypes consis- control is acting across L. donovani complex strains. tent with the existence of inherited factors in suscep- This study (28) has also revealed that, as in other tibility to cutaneous (CL) and visceral leishmaniasis populations (29), certain ethnic groups are more (VL) in humans (9, 10). A few genetic-epidemiology affected than others; thus, it would be most interest- studies have indicated that the distributions of dis- ing to determine whether the frequency of deleteri- ease phenotypes in certain endemic populations are ous alleles differs in ethnic groups in a manner that best explained by the segregation of a few major loci parallels susceptibility to VL. (11–13). However, the genetic models proposed are not always convincing, certainly not as convincing The other study was carried out by Blackwell, El as those described for the control of other parasitic Hassan and collaborators, also in Eastern Sudan diseases (14). Nevertheless, data from experimental in an area situated 70 km from our study village. models and endemic populations strongly suggest In this study, higher susceptibility of certain eth- that human susceptibility to leishmaniasis is influ- nic groups was also observed (29). Only gene test- enced by host genetics; this control is complex and ing was reported. Candidate genes were chosen involves several loci of equal importance. based on observations made in experimental stud-

34 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 ies. Linkage of VL with IL4 (encoding IL-4) and of References PKDL with IFNGR1 (encoding a chain of the IFN- 1. Desjeux P. Leishmaniasis: public health aspects gamma receptor) was reported (30). Interestingly, and control. Clinics in Dermatology, 1996, the regions containing IL4 and IFNGR1 showed 14:417–423. no linkage with VL in our study, although we have 2. Bradley DJ et al. Regulation of Leishmania popu- not yet tested linkage with PKDL. This may reflect lations within the host. III. Mapping of the locus the fact that association studies are more powerful controlling susceptibility to visceral leishmaniasis than linkage studies for detecting genes with minor in the mouse. Clinical and Experimental Immunology, effects on the studied phenotype. The linkage of PKDL with IFNGR1 is interesting since the IFNGR1 1979, 37:7–14. locus has been linked to control of susceptibility to 3. Vidal S et al. The Ity/Lsh/Bcg locus: natural resist- severe schistosomiasis (31) and to atypical dissemi- ance to infection with intracellular parasites is nated infections by mycobacteria (32). abrogated by disruption of the Nramp1 gene. Journal of Experimental Medicine, 1995, 182:655–666. Both studies in Sudan tested the linkage with the 4. Blackwell J, Freeman J, Bradley D. Influence of H-2 SLC11A1 (formerly NRAMP1) region. We found complex on acquired resistance to Leishmania dono- suggestive linkage with polymorphisms in the vani infection in mice. Nature, 1980, 283:72–74. NRAMP1 gene promoter (28). This result was con- 5. Demant P, Lipoldova M, Svobodova M. Resistance firmed in the other study by transmission disequi- to Leishmania major in mice. Science, 1996, librium test analysis (33). Thus the gene encoding 274:1392–1393. NRAMP1, or a closely linked gene, is linked to VL. 6. Beebe AM et al. Serial backcross mapping of mul- Given the large body of literature on the role of tiple loci associated with resistance to Leishmania NRAMP1 in both mouse and human susceptibility major in mice. Immunity, 1997, 6:551–557. to intracellular pathogens (34, 35), these results are 7. Roberts LJ et al. Resistance to Leishmania major is consistent with a role, although minor, of NRAMP1 linked to the H2 region on chromosome 17 and in susceptibility to VL. to chromosome 9. Journal of Experimental Medicine, 1997, 185:1705–1710. Thus studies in animals and endemic populations 8. Lipoldova M et al. Susceptibility to Leishmania suggest that the risk of severe clinical leishmanaisis major infection in mice: multiple loci and hetero- is markedly increased by allelic variants at certain geneity of immunopathological phenotypes. Genes genetic loci. These are the HLA locus, the SLC11A1 and Immunity, 2000, 1:200–206. gene, and alleles of cytokine or cytokine receptor 9. Zijlstra EE et al. Endemic kala-azar in eastern genes. Additional work in distinct populations is Sudan: a longitudinal study on the incidence of however required to definitively establish the impli- clinical and subclinical infection and post-kala- cation of certain of these genes in susceptibility to azar dermal leishmaniasis. American Journal of leishmaniasis. Tropical Medicine and Hygiene, 1994, 51:826–836. 10. Cabello PH et al. Familial aggregation of In conclusion, genetics could provide critical infor- Leishmania chagasi infection in northeastern Brazil. mation for the discovery of key steps in the patho- American Journal of Tropical Medicine and Hygiene, genesis of Leishmania infections and allow the 1995, 52:364–365. identification of new targets for chemotherapy and 11. Shaw MA et al. Human genetic susceptibility vaccination. Drugs targeted at host genetic defects and infection with Leishmania peruviana. American are less likely than pathogen targeted drugs to gener- Journal of Human Genetics, 1995, 57:1159–1168. ate resistant parasites. Genetic studies will also allow 12. Alcais A et al. Evidence for a major gene control- the identification of subjects at high risk of severe ling susceptibility to tegumentary leishmaniasis in disease. Such subjects will benefit from targeted pro- a recently exposed Bolivian population. American phylactic measures; they will also be evaluated care- Journal of Human Genetics, 1997, 61:968–979. fully in drug and vaccine trials. Susceptibility alleles 13. Peacock CS et al. Genetic epidemiology of viscer- are likely to play the same role in various infectious al leishmaniasis in northeastern Brazil. Genetic diseases, including diseases prevalent in rich coun- Epidemiology, 2001, 20:383–396. tries; thus, exploitation of the results of genetics for 14. Abel L et al. Evidence for the segregation of a new therapeutic and prophylactic measures should major gene in human susceptibility/resistance to benefit from support and work in other fields. infection by Schistosoma mansoni. American Journal of Human Genetics, 1991, 48:959–970. 15. Blackwell JM. Genetic susceptibility to leishmanial infections: studies in mice and man. Parasitology, 1996, 112:S67–74.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 35 16. Barbier D et al. Susceptibility to human cutaneous 30. Mohamed HS et al. Genetic susceptibility to viscer- leishmaniasis and HLA, Gm, Km markers. Tissue al leishmaniasis in The Sudan: linkage and asso- Antigens, 1987, 30:63. ciation with IL4 and IFNGR1. Genes and Immunity, 17. Lara ML et al. Immunogenetics of human 2003, 4:351–355. American cutaneous leishmaniasis. Study of HLA 31. Dessein AJ et al. Severe hepatic fibrosis in haplotypes in 24 families from Venezuela. Human Schistosoma mansoni infection is controlled by a Immunology, 1991, 30:129–135. major locus that is closely linked to the interferon- 18. Petzl-Erler ML, Belich MP, Queiroz-Telles F. gamma receptor gene. American Journal of Human Association of mucosal leishmaniasis with HLA. Genetics, 1999, 65:709–721. Human Immunology, 1991, 32:254–260. 32. Casanova JL, Abel L. Genetic dissection of immu- 19. Cabrera M et al. Polymorphism in tumor necrosis nity to mycobacteria: The Human Model. Annual factor genes associated with mucocutaneous leish- Review of Immunology, 2002, 20:581–620. maniasis. Journal of Experimental Medicine, 1995, 33. Mohamed HS et al. SLC11A1 (formerly NRAMP1) 182:1259–1264. and susceptibility to visceral leishmaniasis in The 20. Blackwell JM et al. Immunogenetics of leish- Sudan. European Journal of Human Genetics, 2004, manial and mycobacterial infections: the Belem 12:66–74. Family Study. Philosophical transactions of the Royal 34. Searle S, Blackwell JM. Evidence for a function- Society of London. Series B, Biological sciences, 1997, al repeat polymorphism in the promoter of the 352:1331–1345. human NRAMP1 gene that correlates with autoim- 21. Singh N et al. Molecular typing of HLA class I mune versus infectious disease susceptibility. and class II antigens in Indian kala-azar patients. Journal of Medical Genetics, 1999, 36:295–299. Tropical Medicine & International Health, 1997, 35. Blackwell JM et al. SLC11A1 (formerly NRAMP1) 2:468–471. and disease resistance. Cellular Microbiology, 2001, 22. Meddeb-Garnaoui A et al. Association analysis of 3:773–784. HLA-class II and class III gene polymorphisms in the susceptibility to mediterranean visceral leishmaniasis. Human Immunology, 2001, 62:509–517. 23. Peacock CS et al. Genetic analysis of multicase families of visceral leishmaniasis in northeastern Brazil: no major role for class II or class III regions of HLA. Genes and Immunity, 2002, 3:350–358. 24. El-Safi SH et al. Epidemiology of visceral leishmaniasis in Atbara River area, eastern Sudan: the outbreak of Barbar El Fugara village (1996–1997). Microbes and Infection, 2002, 4:1439–1447. 25. Bucheton B et al. The interplay between environmental and host factors during an outbreak of visceral leishmaniasis in eastern Sudan. Microbes and Infection, 2002, 4:1449–1457. 26. Bucheton B et al. A major susceptibility locus on chromosome 22q12 plays a critical role in the control of kala-azar. American Journal of Human Genetics, 2003, 73:1052–1060. 27. Pratlong F et al. Sudan: the possible original focus of visceral leishmaniasis. Parasitology, 2001, 122:599–605. 28. Bucheton B et al. Genetic control of visceral leishmaniasis in a Sudanese population: candidate gene testing indicates a linkage to the NRAMP1 region. Genes and Immunity, 2003, 4:104–109. 29. Ibrahim ME et al. Kala-azar in a high transmission focus: an ethnic and geographic dimension. American Journal of Tropical Medicine and Hygiene, 1999, 61:941–944.

36 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 3.4 MECHANISMS vations of the immune response in the susceptible OF PATHOGENESIS – murine host indicated that a Th2 response was not, in fact, associated with susceptibility to infection [2]; DIFFERENCES AMONGST this suggested that the immune interactions for this LEISHMANIA SPECIES cutaneous parasite might in fact be distinct from L. major. Interestingly, in the case of L. amazonensis, Diane McMahon-Pratt most mouse strains examined to date have proven Yale University School of Medicine, susceptible to infection; there are, however, qual- Department of Epidemiology and Public Health, itative degrees of susceptibility. Further, CBA and New Haven, C57BL/6 mice, which are resistant to L. major infec- USA tion, are susceptible to L. amazonensis. The differences between these two cutaneous species have become more evident based on recent studies in immunodeficient mice (T, B and cytokine [IL-10/IL- Although members of the genus Leishmania share 12]) [1, 5, 8, 9, 12, 19]. (by definition) specific molecular and biologic characters, one of the hallmarks of this family of patho- In the case of L. major, as expected, mice geneti- genic protozoa is the diversity of tropism/disease cally deficient in T cells (or more specifically, CD4+ resulting from infection. With few notable excep- T cells) display an exacerbated infection in com- tions, the form (visceral, cutaneous, diffuse cuta- parison to wild type control mice. The mice geneti- neous, mucocutaneous) and severity of disease is a cally deficient in MHC class II expression (and thus, function of the infecting Leishmania species together CD4+ T cells) lack the capacity to develop a Th1 with host genetics and consequent immune response. (or Th2) response to infection [6, 10]. Without the We are just beginning to appreciate the complexi- capacity to develop a Th1 response, these geneti- ties of these interactions, yet they have import for cally deficient mice are incapable of controlling L. the development of a vaccine against leishmania- major infection. In contrast, L. amazonensis infec- sis. Given the fact that Leishmania species groups tion in mice genetically deficient in T cells (athy- differ significantly at the DNA sequence level (esti- mic nu/nu or RAG-2 -/- mice deficient in T and B mated to be approximately 15%), it perhaps should cells), or more specifically CD4+ T cells (MHC class not be surprising that their interactions with the II deficient mice), fail to develop disease pathology mammalian host differ. However, the literature has or to allow the in vivo multiplication of parasites been focused upon unifying immunologic mecha- [19]. Analyses have demonstrated that this appar- nisms of control, which would be important for the ent parasite containment was not due to the devel- development of vaccine control measures. Genetic opment of a non-T cell immune response nor due studies utilizing the murine model, though, clearly to the metastasis of parasites to alternate (non-cuta- indicate that the genes controlling innate suscep- neous) anatomic sites. Histochemical and immuno- tibility to infection differ amongst the species [4, histochemical analyses indicated the presence in the 7, 20]; these data indicate that different immuno- wild type mice (C57BL/6) of infiltrating MAC-1+ logic control mechanisms might regulate infection macrophages packed with intracellular parasites, as by distinct Leishmania species, even at identical tis- well as the presence of CD4+ and CD8+ T cells and sue sites. Further, multiple genes apparently con- the up-regulation of ICAM-1 at the site of cutane- trol infection in susceptible murine hosts (acquired ous infection. These results are consistent with an immunity phase). Although some of these genes are ongoing Th1-like immune response. Further, these commonly involved in the control of infection to L. infected wild type mice produced IFN-γ and TNF- major, L. donovani and L. mexicana (i.e. H-2 and H-11), α/lymphotoxin (LT) but not IL-4 in response to par- not all genes appear to contribute equally to the con- asite antigen. In short, the cytokine responses were trol of infection by all Leishmania species [3, 14, 15]. typical of an ongoing Th1-like response; yet, these mice were susceptible to infection. Obviously, other Infection caused by L. amazonensis appears to be ongoing immune events could modulate the effec- readily distinguished from infection due to L. major tiveness of the Th1 response. An evident immune in a number of significant immunologic features. response or similar Th1-like immune response was Analyses of the contributions and effects of such not observed in the MHC class II deficient mice. In cytokines as IL-10 and IL-12 suggest distinct pat- fact, immunohistochemical analyses revealed that terns of immunologic response in L. mexicana and the low numbers of parasites present were found L. major parasites [1, 5, 8, 12]. For L. amazonensis, the either within non-MAC-1+ cells or were located mechanisms involved in pathogenesis appear to sin- extracellularly within the tissue matrix. Disease/ gularly involve immune subversion. Initial obser- pathology was able to be reconstituted in RAG-2

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 37 deficient mice (deficient in both T and B cells) using 18]. Although important for the activation of mac- either splenic cells or enriched populations of CD4+ rophages to kill intracellular pathogens, IFN-γ has T cells; the results indicated that these cells, indeed, also been demonstrated in numerous immunologic were responsible for the pathology observed. systems to be responsible for inflammation and the ability of monocytic and other cells to reach the site Although IFN-γ producing CD4 T cells appeared to of antigen presentation. In addition, earlier work reconstitute disease, it was apparent that splenic cells in vitro suggests that L. amazonensis-infected mac- were more effective [19]. Consequently, it appeared rophages may be more resistant to activation with that another cell population could be contributing to IFN-γ [16] than macrophages similarly infected with the pathogenic response. Further studies indicated, L. major. Consequently, the balance between macro- in the case of L. amazonensis and another related L. phage recruitment and level of activation is critical mexicana complex parasite, L. pifanoi, that B-cell defi- for control of infection; IFN-γ is critical to both pro- cient mice appeared to be refractory to infection cesses and the “balance” point (i.e. level of IFN-γ [10]. Interestingly, similar observations have been produced) determines control versus pathogenesis. made for L. donovani and L. mexicana in B cell defi- Recent observations in IFN-γ deficient mice further cient mice [13, 17], but not for L. major. B cells are strengthen this view. IFN-γ deficient mice are com- known to function as antigen presenting cells and parable to wild type control mice in their suscepti- to produce immunoglobulin. B cell deficient mice, bility to L. amazonensis infection; this is in contrast to transgenically reconstituted [10] to express mem- observations for L. major and L. donovani, where IFN- brane (function as antigen-presenting cells [APCs]) γ-/- mice show a very significant increase in sus- and/or secreted IgM, were found to be phenotyp- ceptibility to infection. However, IFN-γ is critical for ically identical to the parental B cell deficient mice. protection in vaccinated mice. Consequently, the bal- However, reconstitution of the B cell deficient mice ance of activation versus recruitment of “safe” tar- with immunoglobulin (IgG) specific for Leishmania get monocytic cells in wild type immunocompetent membrane components made the mice susceptible naive mice clearly favours the parasite. The ques- to L. amazonensis infection. Further FcRγ-deficient tion remains though, will exacerbation (defined as mice also were refractory to infection. Hence, it is greater parasite burdens than those seen in non-vac- the production of antibody (specifically IgG) and cinated mice) be observed as protection wanes in a the uptake of the parasite through the host Fc recep- vaccine model. If IFN-γ at low levels favours mono- tor that is critical to the development of disease. cytic recruitment, and at higher levels results in mac- This result is in contrast to observations of infection rophage activation and parasite killing, a waning with L. donovani, where passive transfer of antibody immune response could be problematic. This has in B cell deficient mice failed to reconstitute their implications for vaccine strategies, where the time susceptibility to infection; however, antibody appar- between immunization and subsequent boosts may ently reversed the influx of neutrophils into the liver be important for the overall efficacy of the vaccine. observed in the B cell deficient mice [17]. CONCLUSIONS The interaction between the T cell and B cell com- partments in terms of the mechanisms involved In summary, the critical importance of immunoglob- in pathogenesis in murine L. amazonensis infection ulin (B cells) and T cells in the development of disease remains to be explored. Obviously, CD4+ T cells are in the case of Leishmania mexicana complex parasites important in providing “help” for the development appears to distinguish this group from other mem- of a B cell response and the production of antibody. bers of the genus Leishmania. The requirement for an In addition, macrophage cell uptake of opsonized immune response of the host is obviously a reflec- (antibody coated) organisms is known to enhance tion of the fact that evolutionarily the parasite has oxidative burst and killing; this could presumably only encountered the immunocompetent mamma- result in enhanced parasite antigen presentation, and lian “host” (unlike in the immunologist’s laboratory) therefore in an enhanced T cell response. Further and hence has developed and adapted to surviving work needs to be done to explore these possibili- in response to an ongoing immune onslaught by the ties and to understand the nature of the immune host. However, this appears to be an extreme case of response facilitating infection by L. mexicana com- immune subversion, as without the host’s immune plex parasites. The role of IFN-γ in infection may be a response the parasite fails to thrive. Consequently, double-edged sword, especially in the case of L. mex- without the host immune response it appears that icana complex organisms. Indeed, in vaccine studies the transmission of these parasites would be seri- the up-regulation of IFN-γ has been observed to cor- ously hampered. relate with protection against infection with L. mexicana complex parasites, including L. amazonensis [11, In terms of development of a vaccine against New

38 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 World cutaneous leishmaniasis caused by members 9. Kima PE et al. Internalization of Leishmania mex- of the L. mexicana complex however, we need to fur- icana complex amastigotes via the Fc receptor is ther understand the mechanisms of pathogenesis as required to sustain infection in murine cutane- these may have important implications for vaccine ous leishmaniasis. Journal of Experimental Medicine, development. Antibody and IFN-γ, when high lev- 2000, 191:1063–1067. els are achieved, may represent mechanisms for par- 10. Locksley RM et al. The development of effector T asite destruction; however, as the immune response cell subsets in murine Leishmania major infection. wanes (with time post-vaccination) these could con- Ciba Foundation Symposium, 1995, 195:117; discus- tribute to enhanced susceptibility of the vaccinated sion 117–122. host to infection. Further work is required to exam- 11. McMahon-Pratt D et al. Recombinant vaccin- ine these possibilities. ia viruses expressing GP46/M-2 protect against Leishmania infection. Infection and Immunity, 1993, Acknowledgements 61:3351–3359. 12. Padigel UM, Alexander J, Farrell JP. The role This work was supported through grants from the of interleukin-10 in susceptibility of BALB/c National Institutes of Health (USA), AI27811 and mice to infection with Leishmania mexicana and AI45044. Leishmania amazonensis. Journal of Immunology, 2003, 171:3705–3710. References 13. Peters C et al. The role of macrophage receptors in adhesion and uptake of Leishmania mexicana amas- 1. Afonso LC, Scott P. Immune responses associ- tigotes. Journal of Cell Science, 1995, 108:3715–3724. ated with susceptibility of C57BL/10 mice to 14. Roberts LJ et al. Resistance to Leishmania major is Leishmania amazonensis. Infection and Immunity, linked to the H2 region on chromosome 17 and 1993, 61:2952–2959. to chromosome 9. Journal of Experimental Medicine, 2. Aguilar TF et al. Endogenous interleukin-12 is crit- 1997, 185:1705–1710. ical for controlling the late but not early phase of 15. Roberts M, Mock BA, Blackwell JM. Mapping of Leishmania mexicana infection in C57BL/6 mice. genes controlling Leishmania major infection in Infection & Immunity, 2002, 70:5075–5080. CXS recombinant inbred mice. European Journal of 3. Beebe AM et al. Serial backcross mapping of mul- Immunogenetics, 1993, 20:349–362. tiple loci associated with resistance to Leishmania 16. Scott P, Sacks D, Sher A. Resistance to macrophage- major infection. Immunity, 1997, 6:551–557. mediated killing as a factor influencing the patho- 4. Blackwell JM. Genetic control of recovery from vis- genesis of chronic cutaneous leishmaniasis. Journal ceral leishmaniasis. Transactions of the Royal Society of Immunology, 1983, 131:966–971. of Tropical Medicine & Hygiene, 1982, 76:147–157. 17. Smelt SC et al. B cell deficient mice are high- 5. Buxbaum LU et al. Control of New World cutane- ly resistant to Leishmania donovani infection, but ous leishmaniasis is IL-12 independent but STAT4 develop neutrophil-mediated tissue pathology. dependent. European Journal of Immunology, 2002, Journal of Immunology, 2000, 164:3681–3688. 32:3206–3215. 18. Soong L et al. Leishmania pifanoi amastigote anti- 6. Chakkalath HR et al. Class II major histocom- gens protect mice against cutaneous leishmaniasis. patibility complex-deficient mice initially control Infection and Immunity, 1995, 63:3559–3566. an infection with Leishmania major but succumb 19. Soong L et al. Role of CD4+ T cells in pathogenesis to the disease. Journal of Infectious Disease, 1995, associated with Leishmania amazonensis infection. 171:1302–1308. Journal of Immunology, 1997, 158:5374–5383. 7. Davies EV, Singleton AM, Blackwell JM. 20. Vidal S et al. The Ity/Lsh/Bcg locus: natural resist- Differences in Lsh gene control over system- ance to infection with intracellular parasites is ic Leishmania major and Leishmania donovani or abrogated by disruption of the Nramp1 gene. Leishmania mexicana mexicana infections are caused Journal of Experimental Medicine, 1995, 182:655–666. by differential targeting to infiltrating and resi- dent liver macrophage populations. Infection and Immunity, 1988, 56:1128–1134. 8. Jones DE et al. Early enhanced Th1 response after Leishmania amazonensis infection of C57BL/6 interleukin-10-deficient mice does not lead to resolution of infection. Infection & Immunity, 2002, 70:2152–2158.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 39 3.5 CELLULAR IMMUNE infection with L. major result from the development RESPONSES (SUMMARY) of Th1 and Th2 responses, respectively. This murine model of infection with L. major has Pascal Launois1 and Jacques Louis2 been used to study the mechanisms underlying 1 WHO Immunology Research and Training Center, genetic differences in Th subset maturation. Many Lausanne, Switzerland results from several laboratories strongly suggest 2 Department of Parasitology, Institut Pasteur, Paris that IL-4, during the early phase of infection with L. major, plays an important role in the development of specific Th2 CD4+ T cells. In this context, it has been demonstrated that a specific subpopulation of Human infections with Leishmania species induce Vβ4 Vα8 CD4+ T cells provides the IL-4 necessary a spectrum of clinical manifestations ranging from for Th2 maturation. Furthermore, a single antigen cutaneous lesions which spontaneously heal to vis- (LACK) from this complex microorganism drives ceral leishmaniasis which is fatal unless treated. The this early IL-4 response that underlies subsequent clinical outcome depends on the strain of parasite Th2 cell maturation resulting in progressive disease. but also, largely, on the specific immune response However, recent results have shown that the pre- to Leishmania antigens. Murine models of infection cursor frequency of these IL-4-producing cells and have been used to characterize the types of immune their expansion during the first days of infection are response influencing both resistance and suscepti- identical in resistant and susceptible mice, suggest- bility to infection with Leishmania. ing that IL-4 is not the only signal necessary for Th2 differentiation. Since other Th2 cytokines such as IL- THE MURINE MODEL OF INFECTION 10, IL-13 and TGF-β have been implicated in some WITH LEISHMANIA MAJOR conditions (particularly when IL-4 is lacking) in the development of Th2 cells during infection with L. Upon experimental infection with Leishmania major, major, new aspects accounting for Th2 differentia- distinct features of the spectrum of clinical manifes- tion and consequently for the susceptibility to infec- tations seen in patients with cutaneous leishmani- tion should be assessed. In this context, it has been asis can be reproduced in inbred mice of different recently shown that a minor population of CD4+ T genetic backgrounds. Mice from the majority of cells constitutively expressing CD25, the α chain of inbred strains (C3H/He, CBA, C57BL/6, 129Sv/Ev) the IL-2 receptor, controls the importance of the IL- develop locally cutaneous lesions, which spontane- 4 produced by susceptible mice early after infection ously resolve. These mice do not develop lesions with L. major. Furthermore, in resistant mice, these after a second inoculation of L. major and belong CD25 regulatory T cells appear to allow parasites to to the resistant phenotype. Mice from a few strains persist and effector memory responses in immune (BALB/c, DBA/2) develop severe and uncontrolled mice to be maintained. lesions without becoming immune to reinfection and are representative of the susceptible phenotype. Although the role of IL-12 in Th1 cell development is now well documented, the importance of IFN-γ Two functionally distinct CD4+ T cell subsets, T in Th1 development is still debated. In addition to helper (Th)1 and Th2, distinguishable by the pattern these two cytokines, other cytokines such as IL-18, of cytokines they produce upon stimulation in vitro, IL-23 and IL-27 favour IFN-γ production by CD4+ T were described in the late 80s. Th1 cells are charac- cells, but their exact role in Th1 differentiation and terized by secretion of IFN-γ and lymphotoxin (LT) resistance to infection with L. major is still not com- that are known to activate host defences against pletely understood. intracellular pathogens, while Th2 cells produce IL- 4, IL-5, and IL-13 that favour the development of humoral responses protecting against extracellular THE MURINE MODEL OF INFECTION pathogens. The murine model of infection with L. WITH LEISHMANIA DONOVANI major provided the first correlation in vivo between In murine models of infection with L. donovani, the 1) the development of protective immunity and an relationship between Th1/Th2 cell development expansion of Th1 CD4+ T cells in resistant mice, and and resistance/susceptibility to infection is not clear. 2) the expression of progressive disease and the Effective defences towards visceralizing strains development of a CD4+ Th2 cell response in suscep- depend strictly upon Th1 cells, and acquired resis- tible mice. It is now generally accepted that genet- tance is governed by T-cell and macrophage-acti- ically determined resistance and susceptibility to vating cytokines including IL-12 and IFN-γ, which play a predominant role. Down-regulation mecha-

40 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 nisms including Th2 cell responses have also been demonstrated during experimental infection with L. donovani. Although TGF-β, IL-4 and IL-13 may exert limited effects in visceral leishmaniasis, available evidence support a central role for IL-10. Indeed, IL-10-/- mice are resistant to infection with L. donovani, and the enhanced resistance is accompanied by increased production of IFN-γ and nitric oxide, a radical that is toxic for the parasites. Thus, IL-10 is a critical component of the immune response that inhibits resistance to infection with L. donovani.

HUMAN INFECTION WITH LEISHMANIA In humans, the roles of Th1 and Th2 CD4+ T cells and the cytokines they produce are not yet well understood. The role of IFN-γ in protective immunity has been documented but the role of regulatory Th2 cytokines including IL-4, IL-10, IL-13 in the disease susceptibility is still under debate. Although in mice IL-4 is a potent inducer of Th2 cell development, IL- 13 has recently been shown to predominate over IL- 4 in T cell functions in humans. Indeed, IL-13 was demonstrated to be the predominant Th2 cytokine in localized cutaneous leishmaniasis (LCL) lesions which renders specific CD4+ T cells unresponsive to IL-12, a mechanism that could possibly maintain Th2 cell development and disease susceptibility. In addition, high levels of IL-10 have been shown to be associated with active visceral and mucosal leishmaniasis. Some IL-10 production has also been demonstrated in human LCL due to L. braziliensis, and its down- regulation of IFN-γ production has been proposed to explain disease. Futhermore, IL-10 expression was significantly higher in the more slowly healing lesions on patients with LCL caused by L. major and was a promoter of disease prolongation in patients infected with L. mexicana. In human LCL due to L. guyanensis, IL-10 was poorly produced in spite of the association of intralesional IL-10 mRNA expression with unresponsiveness to chemotherapy. Another well known regulatory cytokine with anti-proliferative and antigen-presenting cell deactivating properties, TGF-β, has also been implicated in human leishmaniasis. The expression of TGF-β in the lesions of active and chronic human cutaneous leishmaniasis has been clearly demonstrated, and its potential effect on the development of the disease has been proposed. However, recent data have demonstrated that TGF-β can only poorly inhibit IFN-γ production by peripheral blood mononuclear cells (PBMC) from cutaneous or mucosal leishmaniasis patients stimu- lated with soluble antigens from Leishmania, suggesting that TGF-β is not able to reverse IFN-γ- dependent anti-parasite effector mechanisms. Thus, its exact role in disease susceptibility remains unclear.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 41 3.6 VECTOR–PARASITE AND The barriers to complete development that have VECTOR–HOST INTERACTIONS been identified in refractory flies include the digestive enzymes that are induced by bloodfeeding and IN LEISHMANIASIS can inhibit the early growth of parasites in the bloodmeal; the peritrophic membrane, that can behave David Sacks as a physical barrier to parasite migration out of Laboratory of Parasitic Diseases, National Institute of the abdominal midgut; and the excretion of mid- Allergy and Infectious Diseases, National Institutes of gut contents following bloodmeal digestion that can Health, USA result in the removal of parasites from the gut. The majority of studies that have followed the development of various Leishmania species within inappropriate vectors have not observed an early inhibition Current research involving the vector biology of of parasite survival and growth. Instead, the loss leishmaniasis is severely limited because few lab- of infection is associated with the excretion of the oratories maintain sandflies for study; fewer still digested bloodmeal, suggesting that the inability address molecular aspects of vector–parasite or vec- of Leishmania strains to persist in an inappropriate tor–host interactions. Key gaps in our knowledge sandfly is related mainly to their failure to remain concerning these interactions include the molecu- anchored to the gut wall via specific attachment sites. lar basis for hindgut vs. midgut development that The role for the predominant promastigote surface distinguishes the Viannia and Leishmania subgenera; glycoconjugate, lipophosphoglycan (LPG), in medi- the role of digestive enzymes in controlling differ- ating attachment to the midgut epithelium has been ential survival of Leishmania species in the blood- supported by a number of findings: 1) purified LPG fed midgut; the importance of parasite and sandfly binds to midguts in vitro; 2) LPG completely inhib- chitinases in escape from the peritrophic matrix its the binding of promastigotes to the gut in in vitro and in transmission by bite; identification of mid- attachment assays; and 3) LPG deficient mutants gut receptors controlling species- and stage-specific fail to attach to the midgut in vitro or to persist in binding to midgut epithelial cells; factors that pro- the sandfly following bloodmeal excretion in vivo. mote anterior migration of promastigotes and their The evidence that the polymorphic structures of the stage differentiation, especially metacyclogenesis; phosphoglycan domains of LPG might control spe- the constituents and importance of the biological cies-specific midgut attachment, and by extension plug in transmission by bite; the number of meta- species-specific vector competence, is also strong, cyclic promastigotes egested by transmitting flies; based on studies in both P. papatasi and P. sergenti. the importance of salivary secretions in promoting The ability of P. papatasi to transmit only the species infections in the mammalian host; and the relevance L. major has been attributed to the unique, highly of pre-exposure to sandfly bites to the epidemiology substituted nature of L. major LPG that provides for of leishmanial disease. The following is a brief over- multiple terminally exposed ß-linked galactose res- view of those studies that have described parasite- idues for binding. The LPGs of Leishmania species or vector-derived molecules that play discernable or of L. major mutants that lack side-chain substitu- roles in the development of transmissible infections tions, or that express side chains not terminating in in the fly; as virtually all of the studies have been ß-linked galactose residues, fail to bind to P. papatasi cited in a number recent reviews, specific references midguts in vitro, and the parasites that bear these can be obtained from the list of reviews included at surface structures fail to persist in P. papatasi follow- the end of this report. ing bloodmeal excretion.

As far as is known, Lutzomyia and Phlebotomus spp. These findings suggest that gut-associated lectins account for virtually all transmissions of Leishmania or lectin-like molecules, which have been described to humans. Vector-competent species often display for sandflies, serve as parasite attachment sites, and remarkable specificity for the Leishmania species furthermore that these receptors can vary between they transmit in nature; but many, if not most, phle- different phlebotomine species. A gene encoding for botomine species appear to be inherently refractory mammalian-like galectin, a lectin family with affin- to the full development of Leishmania. Specific asso- ity for β-galactoside sugars, was found only in the ciations have been reproduced in the laboratory; midgut cDNA library of P. papatasi, but was absent for example, P. papatasi, fed on either experimental from libraries constructed from three other refrac- lesions or through a membrane, will support the full tory vector species (Kamhawi et al., unpublished). growth and development of L. major, but not of any Recombinant P. papatasi midgut galectin (Ppgal) other Leishmania species. bound specifically to L. major strains bearing ß-gal- containing branching sugars on their LPGs, and anti-

42 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 bodies against Ppgal inhibited binding of L. major malian host. Because infected sandflies also inoc- LPG to the midgut, confirming its role in parasite ulate small amounts of saliva, recent studies have attachment. The data indicate that midgut galectin examined how modification of the inoculation provides the molecular basis for species restricted site by salivary components can influence the out- vector competence, as well as the evolutionary pres- come of infection. For several different species of sure for the LPG structural polymorphisms that are Leishmania, the co-injection of parasites with sali- unique to L. major. vary gland homogenates of either Lu. Longipalpis or P. papatasi produced a substantial increase in lesion So long as Leishmania can generate and maintain size and/or parasite burden compared to controls high parasite loads in the midgut during and fol- injected with parasites alone. The molecules pres- lowing bloodmeal digestion and excretion, there is ent in salivary gland homogenates that are respon- little evidence that the final phase of development sible for exacerbation of Leishmania infection have in the fly, including anterior migration, accumula- not been identified, with one exception: the disease tion behind the stomodeal valve, and differentiation enhancing effects of maxadilan, a powerful vaso- to infective-stage metacyclic promastigotes, varies dilatory peptide found in the salivary glands of Lu. according to species or strain. Following passage of Longipalpis, have been attributed to its immunomod- the bloodmeal, maturation of the infection involves ulatory properties, including inhibition of T-cell the release of large numbers of parasites from the activation and delayed-type hypersensitivity (DTH) midgut, preceded or not by their differentiation to response. metacyclic promastigotes. While further replica- tion and attachment of some promastigotes occurs To the extent that salivary secretions also contain within the anterior midgut, metacyclic promasti- molecules that are immunogenic, the transmission gotes themselves have never been seen in division of Leishmania by bite into a host previously sensi- or attachment. This behaviour might be explained, tized to sandfly saliva, including by an uninfected at least in part, by their loss of intrinsic binding sandfly, will elicit an immune response at the site potential, controlled by the developmentally reg- of the bite and potentially modify the outcome of ulated loss of ß-gal side chains in favour of side- infection. The exacerbative effect of saliva on infec- chains terminating in arabinose. The detachment tion, seen when mice were co-inoculated with L. of parasites from the midgut during development major and a sonicate of P. papatasi salivary glands, might also be explained by saturation of binding was completely abrogated in mice pre-exposed to sites due to released phosphoglycans. The anterior the salivary sonicate. This protection was repro- migration of unattached promastigotes to the tho- duced following transmission of L. major by the racic midgut and stomodeal valve has generally bite of infective P. papatasi flies: compared to naive been attributed to promastigotes following a sugar mice, mice pre-exposed to the bites of uninfected concentration gradient, formed as the sugar meal flies showed a reduction in lesion pathology and a is gradually spilled from the crop into the anterior reduction in parasite load. The protection conferred gut. The accumulation of large numbers of meta- by pre-exposure of mice to saliva was associated cyclic promastigotes in the anterior regions of the with a strong DTH response as well as up-regula- gut, including their presence in the proboscis, may tion of INF-γ and IL-12 at the site of bite, suggest- not in themselves be sufficient conditions for trans- ing that within this inflammatory setting, infected mission by bite. The prevailing view is that, in addi- macrophages might be activated for early killing of tion to the presence of infective stage promastigotes the parasites. The induction of a Leishmania-specific in the anterior gut, efficient transmission involves Th1 response might also be accelerated. The power- the formation of a biological plug that impairs the ful protection against cutaneous leishmaniasis that intake of blood. This is thought to promote regur- results from pre-exposure to saliva indicates that the gitation of infective promastigotes from the foregut immunogenic salivary molecules might be used as or behind the stomodeal valve as the fly attempts to components of an anti-leishmanial vaccine. A DNA dislodge the plug from the feeding apparatus. One vaccine encoding one such protein from P. papatasi element of the plug, aside from the mass of parasites saliva conferred strong protection in mice against themselves, is a gel-like matrix that is morphologi- L. major plus saliva. Protection against Leishmania cally and immunologically identical to the filamen- infections conferred by pre-exposure to sandfly bites tous LPG-related structure, peptidophosphoglycan might explain why, in areas that are endemic for (PPG), that is secreted by these parasites in vitro. cutaneous leishmaniasis, the indigenous inhabitants, who are mostly bitten by uninfected flies, generally The relationship of vector sandflies to the leishman- show attenuated infections compared to newcom- ial diseases they transmit does not end with the ers such as tourists or immigrants. The hypothesis deposition of parasites into the skin of the mam- that induction of an immune response against com-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 43 ponents in vector saliva can facilitate induction of a protective response against leishmaniasis is supported by field observations in an area endemic for visceral leishmaniasis in Brazil, where individuals who experienced seroconversion against L. chagasi antigens did not have an increased anti-saliva antibody response, while those who developed a positive anti-L. chagasi DTH response, a correlate of immunity, had increased anti-saliva antibody levels.

References Killick-Kendrick R. Phlebotomine vectors of the leishmaniases: a review. Medical and Veterinary Entomology, 1990, 4:1–24. Sacks DL. Leishmania–sand fly interactions controlling species-specific vector competence. Cellular Microbiology, 2001, 3:189–186. Sacks DL, Kamhawi S. Molecular aspects of parasite-vector-host interactions in leishmaniasis. Annual Reviews in Microbiology, 2001, 55:453–83. Gillespie RD, Mbow ML, Titus RG. The immunomodulatory factors of bloodfeeding arthropod saliva. Parasite Immunology, 2000, 22(7):319–31.

44 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 4 IMPROVED TOOLS AND INTERVENTION METHODS

4.1 Vaccines ...... 46

4.2 Molecular tools for studying the epidemiology of leishmaniasis ...... 54

4.3 Drug trials ...... 71

4.4 Control of zoonotic visceral leishmaniasis ...... 74

4.5 Post kala-azar dermal leishmaniasis ...... 76

4.6 Epidemiology of Leishmania-HIV co-infections ...... 79 Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 45 4.1 VACCINES Because dogs are a natural host for L. infantum (L. chagasi), they represent a special case. Dogs can be Steven Reed used as models for human visceral leishmaniasis Infectious Disease Research Institute (VL) as well, but are also legitimate vaccine targets Seattle WA 98104 in their own right. In southern Europe, pet owners USA are faced with the problem of incurable VL, and the potential for this to be a significant problem in dogs in the US was recently highlighted. More importantly, dogs play an important role as reservoirs of VL in southern Europe and Latin America, where INTRODUCTION control of infection in dogs would lead to significant Vaccination against human cutaneous leishmania- reduction in human infection. sis has been practiced for centuries. Deliberate inoculation of virulent organisms from the pus of an Leishmania parasites exist in two principal forms: the active lesion is an ancient practice. Promastigotes promastigote, a flagellated form in the invertebrate of Leishmania major grown in culture were first used host that can be grown in cell-free tissue culture in Russia in 1937 by Lawrow and Dubowokoj as a media; and the amastigote, a round intracellular means to effectively induce protection against natu- form found in the vertebrate host’s macrophages. ral infection. More recently, standardized inoculums There is some differential antigen expression in the of culture promastigotes were developed by Israeli two forms, and most vaccine candidates are selected scientists and used in several trials. This process, based on their presence at least in the amastigote, known as leishmanization, is still used in some coun- though their presence also in the promastigote is tries, notably Uzbekistan. Leishmanization has been an added advantage. Antigens associated with dif- shown to be efficacious against Old World cutane- ferentiation of the parasite in the insect vector, par- ous leishmaniasis. However, several basic and logis- ticularly lipophosphoglycans of the promastigote tic problems have precluded the widespread use surface, are considered as possible antigens for of this procedure to prevent cutaneous leishmani- transmission-blocking vaccines, as are components asis, including difficulty in standardizing the viru- of sandfly guts. Specific antibodies to these anti- lence of the vaccine and the occasional occurrence gens transferred to the sandfly during the blood- of severe and persistent lesions resulting from the meal could, in principle, prevent normal maturation inoculum. Vaccination using crude antigen prepa- of the parasite within the vector. ration obtained from promastigote forms of various species of Leishmania has been tested in clinical tri- ANTIGENS FOR THE DEVELOPMENT als in humans in both the Old and New Worlds. The OF SECOND GENERATION VACCINES results vary from 0–75% efficacy against cutaneous leishmaniasis, with only modest protection afforded One of the first antigens to be explored extensively against visceral leishmaniasis. Although none of in animal models is the promastigote surface pro- these crude vaccine approaches is ideal, they do tease gp63. The native or recombinant protein and offer some support for animal data which indicate peptides have been used in several vaccine stud- that induction of protection against leishmaniasis ies with varying degrees of success, perhaps due is feasible and can be achieved with either a viable to differences in expression of gp63 on amastigotes. vaccine or with parasite components. More recently, LACK, a molecule associated with Th2 responses in BALB/c mice, was shown to have Many Leishmania species infect mice, hamsters, and excellent vaccine potential against L. major, but not non-human primates. For vaccine studies, most against L. donovani. The potential of using LACK as investigators have used BALB/c mice and L. major a vaccine to inhibit development of Th2 responses infection. Laboratory animals are usually chal- is an intriguing possibility. Other antigens with vac- lenged by cultured promastigotes without the com- cine potential include the GP anchored PSA-2, P-8, ponents of sandflies, which have a profound impact gp46/M-2, cysteine proteases (CP), glucose regu- on the fate of the infection. Sometimes amastigotes lated protein (GRP78), HASPB1 (K26), and the are injected i.v., as in infection with L. donovani (or fucose-mannose ligand (FML). All of these antigens L. infantum) in dogs, mice, and hamsters. Infection have shown potential in animal models of one or using exudates of sandfly salivary glands mixed more forms of leishmaniasis. A systematic approach with cultured promastigotes has been employed in to determine which of these is/are most appropriate mice and monkeys, and Belkaid et al. have devel- for clinical development would be useful. oped a model by which infected sandflies deliver the parasite to the ears of mice. Another intriguing target for vaccine development

46 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 against leishmaniasis is sandfly saliva. Recent obser- nation with LACK-DNA induced long-term protec- vations in mouse models point to a protective effect tion. In addition, these studies suggested that the of the phlebotomine’s saliva components against short-term protection could not be overcome by cre- challenge with L. major. The mechanism of this ating a depot condition after the mice were immu- protection has not yet been completely elucidated. nized with LACK plus IL-12. Thus, mice immunized However, it has been suggested that immunization with a mixture of LACK + IL-12 + alum developed of mice with the saliva of P. papatasi induces a strong a strong Th1 response to LACK but short-term pro- delayed type hypersensitivity (DTH) to the saliva tection could not be overcome by the generation components. When parasites are delivered with sal- of depot. These results question the utility of IL-12 ivary gland excretions during a bloodmeal, a local for general use in vaccine development. Moreover, delayed type hypersensitivity reaction is induced at the present time, it is difficult and expensive to which could mediate killing of parasites. Inclusion manufacture IL-12 on a large scale, imposing seri- of sandfly components that may induce hypersensi- ous logistics restrictions to its use for mass vaccina- tivity in a vaccine given to normal individuals who tion in humans. are exposed to the bites of sandflies (mostly uninfected) may not be without risks. Although DNA vaccines are still in the relatively early stages of development, experimentally they have been shown to induce excellent protection ADJUVANTS IN LEISHMANIASIS against several intracellular pathogens includ- VACCINE DEVELOPMENT ing Leishmania. Recent studies have demonstrated Most vaccine studies aim to limit parasite replica- that DNA encoding the leishmanial proteins LACK, tion in the vertebrate host. Several investigators, LmSTI1, and TSA could effectively immunize sus- including us, have, over the past decade, searched ceptible BALB/c mice against L. major by inducing for genes encoding leishmanial proteins that could CD4+ and cytotoxic lymphocyte (CTL) responses. So induce protection against cutaneous and visceral DNA immunization may provide an effective way leishmaniasis in experimental models of the diseases. to deliver leishmanial vaccines. However, the DNA However, identification of candidate antigens is not vectors currently available are less effective in stim- enough. Appropriate antigen delivery to induce the ulating appropriate immune responses in humans, right type of immune response against leishman- even when very large amounts are used, and there iasis is another critical component of an effective is need for significant optimization before this deliv- vaccine, i.e., the induction of a strong antigen spe- ery system can be considered for a Leishmania vac- cific Th1 response. The two adjuvants approved for cine. CpG ODN alone has been shown to induce a human use, alum and squalene, induce potent anti- state of partial resistance in BALB/c mice for up to body responses but are poor inducers of antigen- five weeks against challenge with L. major. If CpG specific Th1 responses. Several different strategies, ODN is injected in conjunction with leishmanial sol- including IL-12, live vectors, naked DNA, oligonu- uble antigen (SLA), significant protection is obtained cleotides (CpG sequences), and monophosphoryl in these animals that is maintained for as long as lipid A as a stable emulsion (MPL®-SE) have been six months. In these experiments, the immuno-stim- evaluated. MPL® has been used as an adjuvant in ulatory properties of the CpG ODN were associ- several safety and immunogenicity clinical trials ated with stimulation of IL-12 and the emergence in humans, including in vaccines for malaria, hep- of strong Th1 response to SLA. Further studies are atitis B, genital herpes, and allergy desensitization. required to optimize CpG ODN as an adjuvant for These studies all found that MPL® was well toler- humans, largely for safety concerns. Several other ated and there was no evidence of systemic toxicity. adjuvant and delivery systems have emerged during We thus evaluated and compared the immunogenic the past decade that deserve attention also. These and protective efficacies of formulating Leish-111f include vectored (adenovirus, pox virus) DNA, (a promising recombinant vaccine candidate) with peptides targeting antigen-presenting cells (APCs), either the naturally derived disaccharide adjuvant water-in-oil emulsions, oil-in-water emulsions, and of Salmonella minnesota, monophosphoryl lipid A others. Because this area is one of such active inter- (MPL®) or with squalene (MPL®-SE). est, we will focus on adjuvant/delivery systems that may applicable to VL vaccine development, both in One of the drawbacks of IL-12 is its inability to stim- terms of efficacy and practicality/affordability. ulate strong immunological memory to the immunizing antigen. Thus, vaccination of BALB/c mice with the leishmanial antigen LACK mixed with IL- 12 as adjuvant resulted in short-term protection against challenge with L. major. In contrast, vacci-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 47 CLINICAL DEVELOPMENT OF A 12 has been successfully used as a Th1 adjuvant SECOND GENERATION VACCINE for a variety of antigens in both the murine and non-human primate models of several infec- Based on the protection seen in mice and non-human tious diseases including leishmaniasis. primates, we selected three leishmanial antigens to be included as a single poly-protein in a recombi- We have performed extensive protection studies nant vaccine. The vaccine will be developed both for with these antigens, individually, combined, and prophylaxis as well as for therapy of different forms as fusion proteins. BALB/c mice were immunized of leishmaniasis. The three antigens are: twice (three weeks apart), subcutaneously, with • TSA: This novel protein of L. major, which has LmSTI1 and TSA mixed with murine recombinant sequence homology to eukaryotic thiol-specific- IL-12. Mice were infected with L. major and devel- antioxidant (TSA), was discovered in experi- opment of disease was monitored for the next three ments performed to characterize the immune months. Mice immunized with either LmSTI1 or responses elicited by L. major promastigote cul- TSA with IL-12 are protected against disease. In con- ture filtrate proteins (CFPs). Southern blot trast, mice immunized with saline or IL-12 alone hybridization analyses indicated that there are developed severe lesions, illustrating the need for multiple copies of the TSA gene in all species an antigen and adjuvant formulation in achieving of Leishmania analysed (L. tropica, L. donovani, L. protection in this study. LmSTI1 as a single antigen infantum, L. amazonensis, L. braziliensis, L. guya- induced protection, while a combination of LmSTI1 nensis). Northern blot analyses indicated that the and TSA was also effective. An antigen combina- TSA gene is constitutively expressed in L. major tion is likely to be a more effective vaccine, consist- promastigotes and amastigotes. Immunization ing as it does of a broad range of different protective of BALB/c mice with recombinant TSA pro- epitopes, and thus is less likely to suffer from MHC- tein resulted in the development of strong cellu- related unresponsiveness in outbred human and lar immune responses and conferred protective dog populations. immune responses against infection with L. major when the protein was combined with IL-12. The efficacy of LmSTI1 and TSA was also tested in • LmSTI1: Screening of an L. major amastigote Macaca mulatta (rhesus monkeys). This model, obvi- cDNA library with sera from L. major infected ously used to a lesser extent than the mouse, has BALB/c mice identified one clone with strong been accepted as a system that more closely mir- homology with eukaryotic stress-inducible pro- rors human immunity for vaccine development. tein-1, designated as LmSTI1. LmSTI1 contains Monkeys immunized with a preparation contain- six copies of the tetratricopeptide consensus ing LmSTI1 and TSA, with the recombinant human motif that is common to stress-inducible pro- IL-12 and alum as adjuvant, mounted excellent proteins. Recombinant LmSTI1 protein plus IL- tection against challenge with 107 metacyclic pro- 12 elicited a mixed cellular response that was mastigotes of L. major. skewed towards a Th1 phenotype and protected susceptible BALB/c mice. A practical vaccine for use in underdeveloped coun- • LeIF: Leishmania elongation and initiation fac- tries should be safe, effective, long lasting, and as tor (LeIF) was identified by expression cloning inexpensive to produce as possible. A poly-protein using sera from a patient with mucosal leish- vaccine consisting of a single fusion with multiple maniasis to screen an L. braziliensis genomic antigenic epitopes would be less costly to manu- library. Immunoreactive antigens were purified facture than a vaccine consisting of several recom- and analysed, in patient T cell assays, for ability binant proteins. For this reason, a poly-protein to stimulate proliferative responses and prefer- comprised of the three priority candidate antigens ential Th1 cytokine production. Several cDNAs TSA, LmSTI1 and LeIF, fused in tandem, was made, were identified, one of which was LeIF. An L. and referred to as Leish-111f. Recombinant Leish- braziliensis homologue of the LeIF was selected 111f with the adjuvant rIL-12 was evaluated in the because this unique molecule has two important mouse model of L. major and shown to be protective. properties: 1) LeIF is a powerful stimulator of The most effective combination was Leish-111f for- the innate immune system for the production of mulated with 20 µg of MPL®-SE, where protection IL-12, IL-18, and IFN-γ, therefore a Th1 inducer; was observed for at least 14 weeks. Mice immunized and 2) LeIF has immuno-therapeutic properties with both 2 µg and 10 µg of Leish-111f plus MPL®-SE in mice. It appears that IL-12, or the induction were protected against L. major challenge for at least of IL-12, together with specific antigen, provides 10 weeks, at which point all the mice in the saline the necessary stimulus that causes the respond- and adjuvant control groups had to be euthanized. ing T cells to differentiate to Th1 phenotype. IL-

48 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Further studies involved testing the protective application to the FDA, and completed a Phase I dose response of rLeish-111f (2 µg versus 10 µg) clinical trial in the US. with 20 µg of MPL®-SE, and delaying challenge with Leishmania parasites for 3 months. Our stud- Selected references ies found that, in combination with MPL®-SE, Leish- 111f induces a very potent immune response that Abath FG, Montenegro SM, Gomes YM. 1998. protects mice against cutaneous leishmaniasis (CL) Vaccines against human parasitic diseases: an over- when challenged 3 or 12 weeks post immunization. view. Acta Tropica, 1998, 71:237–254. In addition to the delayed challenge studies, the Aebischer T et al. Subunit vaccination of mice against combination of rLeish-111f and MPL®-SE has been new world cutaneous leishmaniasis: comparison of tested for its ability to protect against another spe- three proteins expressed in amastigotes and six adju- cies (L. amazonensis). vants. Infection and Immunity, 2000, 68:1328–1336.

Because the antigens comprising Leish 111f are con- Afonso LC et al. The adjuvant effect of interleukin-12 served in species causing VL, including L. infan- in a vaccine against Leishmania major. Science, 1994, tum and L. donovani, we asked whether the same 263:235–237. antigen/adjuvant formulation shown to be effec- Akuffo H et al. Leishmania aethiopica derived from tive in models of CL would also protect against VL. diffuse leishmaniasis patients preferentially induce Both mouse and hamster studies were performed. mRNA for interleukin-10 while those from local- Overall, we observed partial protection follow- ized leishmaniasis patients induce interferon-gamma. ing immunization with Leish-111f formulated with Journal of Infectious Diseases, 1997, 175:737–741. MPL®-SE. In four of eight mice immunized with Leish-111f plus MPL®-SE, partial protection against Amaral VF et al. Leishmania amazonensis: the Asian L. infantum challenge was observed. A 75% reduc- rhesus macaques (Macaca mulatta) as an experi- tion in mean parasite burden was measured at day mental model for study of cutaneous leishmaniasis. 42 after challenge in the spleens of hamsters receiv- Experimental Parasitology, 1996, 82:34–44. ing the Leish-111f vaccine compared with hamsters Antunes CM et al. Controlled field trials of a vac- receiving saline or MPL®-SE alone. However, the dif- cine against New World cutaneous leishmaniasis. ference in Leishman-Donovan units (LDU) between International Journal of Epidemiology, 1986, 15:572–580. those receiving the Leish-111f vaccine and the con- Armijos RX et al. Field trial of a vaccine against New trol groups was not statistically significant. In con- World cutaneous leishmaniasis in an at-risk child pop- clusion, it appears that our current vaccine construct ulation: safety, immunogenicity, and efficacy during is not optimal as a prophylactic vaccine against VL. the first 12 months of follow-up. Journal of Infectious Diseases, 1998, 177:1352–1357. We have demonstrated that our vaccine formulation containing the Leish-111f protein with MPL®-SE Badaro R et al. Successful use of a defined anti- was able to confer protection against leishmania- gen/GM-CSF adjuvant vaccine to treat mucos- sis caused by L. major or L. amazonensis in BALB/ al Leishmaniasis refractory to antimony: A case c or C57BL/6 mice, respectively. We have shown report. The Brazilian journal of infectious diseases, 2001, that MPL®-SE (20 µg/dose) is the most potent of 5:223–232. the nine different adjuvant formulations tested in Belkaid Y et al. Development of a natural model of eliciting protection against leishmaniasis in BALB/ cutaneous leishmaniasis: powerful effects of vector c mice. Whether the vaccine contained 2 or 10 µg saliva and saliva preexposure on the long-term out- of rLeish-111f, or whether the challenge dose was come of Leishmania major infection in the mouse increased from 2 x 105 to 4 x 105, was irrelevant with ear dermis. Journal of Experimental Medicine, 1998, regard to the protective effect. Of particular impor- 188:1941–1953. tance is the finding that susceptible BALB/c mice immunized with the Leish-111f vaccine were pro- Borges MM et al. Potent stimulation of the innate tected whether they were challenged 3 weeks or 12 immune system by a Leishmania braziliensis weeks post-vaccination. This is the only example recombinant protein. Infection and Immunity, 2001, that we know of in which long-term protection in 69:5270–5277. this CL model has been achieved using a defined Bretscher PA et al. Establishment of stable, cell-medi- protein antigen together with an adjuvant suit- ated immunity that makes “susceptible” mice. Science, able for human use. With support from the Gates 1992, 257(5069):539–42. Foundation, we have produced good manufacturing practice (GMP) grade Leish-111f, filed an IND

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Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 53 4.2 MOLECULAR TOOLS FOR Angarano et al., 1998; Ibrahim et al., 1997; Morsy et STUDYING THE EPIDEMIOLOGY al., 1997; Mebrahtu et al., 1993; Ghalib et al., 1992; Naik et al., 1978.). Clinical prognosis differs accord- OF LEISHMANIASIS ing to the parasite species, and even for the same species according to the clinical form. Treatment is Ikram Guizani necessary to stop evolution of the disease or to save Institut Pasteur de Tunis, Laboratoire d’Epidémiologie life, and the mainstay therapy is pentavalent anti- et d’Ecologie Parasitaire mony (WHO, 1990). Variable sensitivity of isolates Tunis or species to some of the drugs available has been Tunisia documented (Croft et al., 1997), and treatment of the disease does not provide sterile cure (Mendonca et al., 2004; Dereure et al., 2003; Schubach et al., 1998). For all these reasons, it is important to be able to ECO-EPIDEMIOLOGY OF THE LEISH- identify/differentiate Leishmania parasites accord- MANIASES: FEATURES AND FACTS ing to the clinical form they induce or according to their eco-epidemiological features (Guerbouj et al., The leishmaniases constitute a group of widely dis- 2001a; Cupolillo et al., 2003), and also to differenti- tributed infectious, parasitic diseases, which can ate a relapse of a latent infection from a re-infection be cutaneous, mucocutaneous or visceral. They are (Saravia et al., 1990; Morales et al., 2002). considered to be public health problems because of the severity of the diseases, their prevalence, and/or Co-existence of Leishmania parasites with other the burdens they create. Mucocutaneous and some Leishmania species or flagellates (known or unknown) cutaneous lesions are severely debilitating while in the same endemic areas, even within the same visceral leishmaniasis is fatal. Although reported in vector or host, has been documented (Martinez et al., 88 countries worldwide, only 33 countries consider 2002; Noyes et al., 2002; Strelkova et al., 2001; Cortes notification of leishmaniasis obligatory. The distri- et al., 1997; Barrios et al., 1994; Mebrahtu et al., 1991; bution of transmission foci follows the distribution Pratlong et al., 1989; Morsy et al., 1988; Evans et al., of the transmitting vector. The leishmaniases can be 1987). Some of the known Leishmania species have classified into two epidemiological entities accord- been isolated only from animals in endemic areas, ing to type of transmission: anthroponotic, when and are considered non-pathogenic to humans (eg. humans are the sole reservoir involved in the trans- L. turanica, L. gerbilli, L. arabica). The impact of this mission (and the sole source for vector infection); co-existence on the clinical or eco-epidemiologi- zoonotic, when at least one mammalian reservoir cal features of the diseases is not fully understood. host is involved (WHO, 1990; Alvar, 1997). Complete elucidation of the transmission cycle requires identifying the same organism in the dif- The leishmaniases are caused by parasites belong- ferent hosts (mammals and vectors). All this justifies ing to the genus Leishmania, which has two subgen- the need for high-resolution tools. era: Leishmania and Viannia. The epidemiology of the leishmaniases is extremely diverse and far from Molecular tools are needed to identify the parasites being fully elucidated. because all species share similar morphology at the different stages of their life cycle (WHO, 1990). For So far, at least 20 species of Leishmania are identi- a long time, identification has been conditional on fied as being pathogenic to humans (Rioux et al., in vitro/in vivo isolation of parasites. Success of 1990). The differentiation of the parasites and their in vitro isolation depends on transformation of the accurate identification is relevant to eco-epidemiol- amastigotes into promastigotes, then on their ability ogy, clinical diagnosis, and management of patients. to grow in the medium used; frequently, bacterial or Indeed, their clinical presentations can be pleomor- fungal contaminants are a problem (WHO, 1990). In phic, and one species/complex (e.g. L. braziliensis, vivo maintenance of Leishmania parasites in appro- L. infantum, L. donovani, L. aethiopica) can be respon- priate animal models is only documented for some sible for more than one clinical form. Most spe- species (Hommel et al., 1995). cies cause cutaneous disease, but some species are responsible for mucocutaneous and visceral disease Molecular identification of Leishmania parasites (WHO, 1990; Alvar, 1997). The number of reports on has been addressed using different kinds of tech- atypical clinical presentations of the leishmaniases nique (e.g. isoenzyme typing, monoclonal antibod- in immunocompetent and immunocompromised ies, hybridization with DNA probes, PCR assays). patients is increasing (Nigro et al., 2003; Marshall DNA techniques offer multiple possibilities: distinc- et al., 2000; Rosenthal et al., 2000; Desjeux, 1999; tion between the different parasite isolates and their

54 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 assignment to taxa, detection of Leishmania par- more than 30 sandfly species have been incrimi- asites and their concomitant identification, detec- nated as vectors in transmission of the parasites, but tion of intra-specific diversity, investigation of the the list is far from closed (WHO, 1990; Alvar, 1997). parasites within their hosts or vectors, for example. In many foci, the vectors still need to be identified. Different taxonomical levels of resolution (isolate, Three major aspects hamper such tasks: species, complex, genus) have been achieved using • The number of criteria necessary to demonstrate the assays developed, some of which are sensitive the vector role of a sandfly, which are difficult enough to be considered for diagnosis of disease to fulfil: congruent geographical distribution of and infection, and for eco-epidemiological studies the sandfly and the parasite, trophic preferences (cycles, infection rates, etc.). However, such tools are of the sandfly for humans and the reservoirs, not available for all known Leishmania species, and natural infection of the sandfly with the same it is difficult to differentiate among closely related parasites encountered in humans and the reser- species. voirs, persistence of the infection in the gut after digestion of the bloodmeal, ability of the sandfly Leishmania parasites represent a biologically diverse to transmit the parasites upon biting. group of organisms, whose origins and evolution are • The low infection rates, which necessitate catch- still under speculation (Momen & Cupolillo, 2000; ing and dissecting a large number of flies, and Cupolillo et al., 2000). The genetics of these organ- the difficulty of isolating the parasites without isms is still poorly understood, and the genome can contamination. be very plastic (Cruz et al., 1993). Leishmania para- • The difficulty of identifying the sandfly spe- sites are supposedly diploid; however, ploidy was cies or of differentiating among closely related shown to vary within a karyotype or between iso- organisms (sibling species, subspecies); it is eas- lates (Pages et al.,1989; Bastien et al., 1992). Different ier to identify the males, but the females are of studies have confirmed that Leishmania have epidemiological relevance. diverged mainly due to mutations without involvement of large inter-chromosomal rearrangements Molecular studies are obviously important to the (Beverley et al., 1987; Wincker et al., 1996). There is design and development of appropriate tools, to the also evidence that an active mutational mechanism elucidation of sandfly taxonomy, and to establish- for loss of heterozygosity (Gueiros Filho & Beverley, ing the epidemiological relevance of sandfly spp. In 1996), and recombination machinery which allows some instances, molecular Leishmania typing/detec- gene replacement (Cruz et al., 1991), exist. Inter- tion assays have been used to estimate infection rates, clonal variations within isolates are also docu- to incriminate vectors, or to address epidemiological mented (Bastien et al., 1990; Macedo et al., 1992). issues. Development of sandfly markers and stud- The occurrence of such mechanisms and/or oth- ies of sandfly populations are gaining momentum, ers (to be identified) in natural conditions, and their but still relate to only a few of the known vectors. involvement in the emergence of “new genetic make- Establishment of laboratory colonies of sandflies, ups”, are poorly appreciated. A major consequence when successful, provides an alternative way of for epidemiology is that the correlation between addressing their biology and role in transmission, the genetic determinants of the parasite and the and has also contributed greatly to molecular stud- pathology or any other biologically important phe- ies of vectors, which have helped in our understand- notype, e.g. drug resistance, is not well established. ing of vector–parasite and vector–host interactions. Leishmania parasites show considerable intra-spe- It has principally been shown and is well established cific variability, and the existence of geographical that the restriction of some Old World parasite spe- populations has been illustrated in some instances cies to their vectors is driven by molecular inter- (L. braziliensis, L. infantum, L. donovani) (Ishikawa actions mediated by parasite lipophosphoglycan et al., 2002; Cupolillo et al., 2003; Guerbouj et al., and lectin-like receptor structures in the midgut of 2001[b]; Mauricio et al., 2001; Schonian et al., 2000). the sandfly (Sacks et al., 2000, 1994; Kamhawi et al., There are few descriptions of population structure in 2000[a]; Pimenta et al., 1992, 1994), most probably relation to clinical form or eco-epidemiological fea- reflecting co-association and/or co-evolution rather tures (Guerbouj et al., 2001[a]; Cupolillo et al., 2003). than co-speciation (Ready, 2000). The effects of sali- Genetic definition of clinically and epidemiologi- vary components on the infectivity of the parasites, cally relevant strains still needs to be established. on modulation of immune responses, and on protection, have been demonstrated and investigated Leishmaniasis vectors are female sandflies that (Titus & Ribeiro, 1988; Belkaid et al., 1998; Donelly et belong to two genera: Phlebotomus and Lutzomya. al., 1998; Mbow et al., 1998; Kamhawi et al., 2000[b]; Transmission of the parasites occurs when they take Valenzuela et al., 2001). Host and environmental the bloodmeal necessary for oviposition. To date, factors have also been shown to interfere with the

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 55 development of Leishmania promastigotes in the vec- and vectors. On the other hand, changes in epidemi- tor and with transmission (Warburg & Schlein, 1986; ological pattern are registered as a consequence of Schlein, 1993; Jacobson & Schlein, 1999; Schlein & an increase in risk factors (Desjeux, 2001), atypical Jacobson, 1994[a], 1994[b], 1996, 2001, 2002; Schlein, clinical manifestations (Nigro et., 2003; Marshall et Jacobson & Muller, 2001). The importance of these al., 2000; Rosenthal et al., 2000; Angarano et al., 1998; different kinds of interaction in the eco-epidemiol- Ibrahim et al., 1997; Morsy et al., 1997; Mebrahtu et ogy of leishmaniasis (e.g. importance of bloodmeal al., 1993; Ghalib et al., 1992; Naik et al., 1978.), drug source or plant diet in successful establishment of resistance (Lira et al., 1999; Sundar, 2001; Rijal et the intra-vectorial cycle), the impact of vectors on al., 2003), outbreaks (Marlet et al., 2003; Bhutto et shaping parasite diversity (e.g. selective pressures al., 2003; Gontijo et al., 2002; Rowland et al., 1999; on the parasites, recombination, genetic exchange, Tayeh et al., 1997; Seaman et al., 1996; Jeronimo multiple infections), and the impact of each of these et al., 1994), HIV co-infection (Wolday et al., 2001; interactions on the pattern of leishmaniasis, still Desjeux & Alvar, 2003), multi-Leishmania infections need to be fully understood and appreciated. (Martinez et al., 2002; Strelkova et al., 2001; Cortes et al., 1997; Mebrahtu et al., 1991), or, as recently The reservoir hosts of leishmaniasis are very diverse; reported, of mixed Leishmania/Trypanosoma cruzi, or they vary according to the parasite species and bio- multi-Leishmania/T. cruzi infections (Chiaramonte tope. The reservoirs of infection include rodents, et al., 1999; Bastrenta et al., 2003). In many instances, canids, marsupials, and primates. The concept of knowledge about a specific epidemiological sit- ‘reservoir’ involves the ability of this host to main- uation in an endemic area/country is obsolete or tain the parasite, spatially and temporally, within incomplete, emphasizing the necessity for regular the transmission area; this infers that the reservoir updating using powerful and appropriate molecu- host is present in sufficient numbers to increase lar methods. The control of leishmaniasis is primar- the chances of a sandfly meeting an infected ani- ily based on diagnosis and treatment of cases and, mal. The reservoir host should preferably be gregar- whenever possible, on interventions against vectors ious, and live in close relationship with the vector. (insecticide spraying) and/or reservoirs (culling, Furthermore, to ensure the parasite is present peren- poisoning) (WHO, 1990). Vaccination constitutes nially in the transmission area, its course of infection a rational alternative to the control of leishmania- should include a chronic component. For human sis. However, in spite of the different generations of cases to appear, the level of enzooticity should vaccines developed, this alternative remains under be sufficient and contact between sandflies and investigation (Modabber, 2000; Mauel, 2002). The humans should be possible. Secondary reservoirs leishmaniases are the result of complex interplay respond only partially to these criteria, indicating between the environment, the parasite, the host(s) an unstable, still evolving or more recent interac- and the vector(s), and the current trend is to con- tion with the parasite. In general, a sylvatic cycle sider holistic approaches in improving the control is maintained between the wild reservoir and the of leishmaniasis. This has allowed new concepts of sandfly; if either settles in a peridomestic area, this prediction and prevention of epidemics to be estab- facilitates the development of a domestic transmis- lished, based on warning systems involving ecolog- sion cycle, which can include infection of humans. ical data (climate, vegetation, population dynamics), Alternatively, human activities can interfere with monitoring of emerging epidemiological patterns the sylvatic cycle and so facilitate the development (e.g. emerging foci, atypical clinical manifestations, of a domestic or peridomestic cycle (WHO, 1990; drug resistance, co-infections), risk factor studies Alvar, 1997). The population biology of the reservoir (urbanization, migration, big projects), case map- hosts awaits to be well appreciated, and this knowl- pings, etc. edge to be translated and integrated into the control of leishmaniasis (Fichet-Calvet et al., 2003). In this MOLECULAR TOOLS AND regard, it is important to understand: the patterns of infection; how the parasite is maintained over the ECO-EPIDEMIOLOGY OF THE lifespan and within the populations of the animal LEISHMANIASES: TOWARDS host; how migration would contribute to emergence MOLECULAR ECO-EPIDEMIOLOGY of the disease; the relationship between the reservoir Identification of organisms constitutes a key issue in hosts and the vector; the correlation between prev- the eco-epidemiology of parasitic diseases like the alence and transmission; how the reservoir hosts leishmaniases. Parasitological, clinical or epidemi- interfere with the parasite’s biology and diversity. ological features are by themselves insufficient for identification, which has to be based on a molec- In fact, each transmission focus should be considered ular approach. However, use of molecular tools is unique because of the diversity of biotopes, hosts, not optimal in disease endemic countries. Current

56 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 issues in identification for eco-epidemiological pur- building, enhanced resource utilization, promoting poses can be classified into three major categories: mechanisms of scientific interaction among inves- 1. Establishing a diagnosis in order to identify the tigators in DECs, training and sharing of resources, etiological agent of the disease, to assess relapse complementary expertise and knowledge. or reinfection, and/or to confirm if a disease is emerging or its clinical pattern is changing. This PROGRESS IN MOLECULAR includes identifying the parasite at the species or variant/strain level, and identifying novel IDENTIFICATION OF LEISHMANIA species/strains. PARASITES: A REVIEW 2. Defining/assessing roles within the transmis- Over the past 20 years, three major categories of sion cycle, which includes: establishing the molecule have been considered for use in the identi- cycle(s), confirming the epidemiological rele- fication of Leishmania parasites: vance of a species/subspecies/population/variant (of host, vector and/or parasite), and studies 1. Isoenzymes. Isoenzyme analysis gathers parasites on vector, host or parasite populations. into zymodemes, which are considered as opera- 3. Control and surveillance of endemic or epi- tional taxonomic units (OTU). The experimental demic foci, which includes: eco-epidemiolog- protocols used, which are easily adapted, can be ical follow-up of foci, making an inventory of considered an advantage. The large-scale use of iso- species/populations/variants, monitoring and enzyme analysis over a wide range of organisms understanding changes in epidemiological pat- for a long time has made it a standard technique in tern, and predicting or preventing epidemics. genetic studies (Pasteur et al., 1987). Its use in leishmaniasis, although limited to certain centres and to These issues address the parasite, the vector and/ different or overlapping sets of isoenzymes, largely or the hosts. Lots of unknowns remain and the supports cycle elucidation (Aljeboori & Evans, new concepts of surveillance are being established. 1980[a], [b]; Rioux, 1986; Ben Ismail et al., 1986, 1987; Many tools are still needed. In this respect, recent Al Zahrani et al., 1989[a], [b]; Mebrahtu et al., 1992; advances in science and technology are very promis- Bonfonte-Garrido et al., 1992; Campino et al., 1994; ing. Molecular tools should no longer be considered Gramiccia et al., 1991; Jimenez et al., 1995; Pratlong as mere supports to eco-epidemiology but as driv- et al., 1995; Harrat et al., 1996; Campino et al., 1997) ing tools to monitor and understand the biological and has allowed description of many parasite spe- complexity of parasites, their hosts and vectors, and cies (Peters et al., 1986; Strelkova et al., 1990; Yoshida ultimately to provide rational ways of controlling et al., 1993), and tentative establishment of a classi- and managing the leishmaniases. Besides assigning fication system for Leishmania parasites (Le Blancq, a species to the infecting organism, incriminating Cibulskis & Peters, 1986; Rioux et al., 1990; Cupolillo, the host(s) or vector(s), or elucidating the taxonomic Grimaldi, & Momen, 1994; Thomaz-Soccol et al., status of a vector or host, molecular eco-epidemiol- 2003). However, with the growing and systematic ogy would open up the possibilities for establish- use of molecular typing, assignment to zymodeme ing working hypotheses to address and understand: becomes less circumstantial and the biological sig- the intricate relationships between the parasites nificance of zymodemes has come under question. A infecting a host and between the host and the par- disadvantage of the technique is that it can only be asites; the consequences of such interactions on the applied to large numbers of isolated promastigotes. outcome of the infection; the impact of host/vector It is also time-consuming, and interpretation of the interactions on parasite populations and emergence data (numerical analysis, cladistic analysis, popula- of virulent/pathogenic variants; the importance of tion genetics) requires qualified, competent person- environmental change to host and vector popula- nel. Because the technique addresses the analysis tions and to transmission of the disease; the genesis of proteins, and indirectly the DNA mutations that of epidemics, etc. affect the charge on these proteins, the use of isoenzyme data underestimates the level of genetic vari- The concept of using molecular tools in disease ability. The resolution power of electrophoresis has endemic countries (DECs) is often still associated also been questioned. with preconceived ideas about cost, conducting experiments in the field, and leaving it to sophisti- 2. Monoclonal antibodies. The development of mono- cated laboratories in the North. Molecular tools are clonal antibodies progressed laboriously and needed and are necessary. They call for various lev- steadily. Although monoclonal antibodies have els of expertise. Potential and capacity already exist been acknowledged by several authors as useful for in DECs; this needs to be strengthened and utilized. studying parasite taxonomy and evolution (Rioux, All this underscores the need for enhanced capacity 1986; Shaw, 1994; Momen & Cupollilo, 2000), it is

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 57 well known that they can target antigens/epitopes 1985; Pacheco et al., 1986), or for describing para- which are common to many parasite species or are site species (Yoshida et al., 1993; Peters et al., 1986). unique to certain developmental stages. Different Different species-specific probes (L. major, L. aethi- species-specific (eg. L. tropica, L. major, L. braziliensis, opica, L. donovani complex, L. mexicana) were devel- L. naiffi) monoclonal antibodies have been produced oped based principally on minicircle fragments and described as able to differentiate among sets of (Lopes & Wirth, 1986; Smith et al., 1989; Laskay et al., different Leishmania species, but are only used in cer- 1991[a]; Gramiccia et al., 1992; Ben Hamouda et al., tain laboratories (Grimaldi & McMahon-Pratt, 1996; 2000); some of these probes were evaluated for con- Jaffe et al., 1984; de Ibarra, Howard & Snary, 1982; comitant detection and identification of parasites Handman & Hocking, 1982). The formats of the tests within touch-blots of dog viscera (e.g. L. infantum) developed for Leishmania identification correspond (Guizani et al., 2001) or squashes of sandflies (e.g. L. to the indirect fluorescent antibody test (IFAT), radio- major, L. aethiopica) (Laskay et al., 1991[b]). It is inter- immunoassay (RIA), or enzyme linked immunosor- esting that, in spite of the limited sensitivity of the bent assay (ELISA); they all allow assessment of minicircle fragment probes in detecting 103–105 pro- reactivity to different antibodies in one experiment mastigotes within a dot-blot, the probes are able to (Anthony et al., 1987; Shaw et al., 1987; Grimaldi, detect as few as 50–150 organisms within sandflies David & McMahon-Pratt, 1987; Adini et al., 1998). or bone marrow, respectively (Laskay et al., 1991[b]; Different kinds of results can be obtained accord- Guizani et al, 2001). It is also interesting to note that ing to the antibodies and test format used, which are the performance of one of the probes, specific to L. scored as: presence vs. absence of signals, proportion aethiopica, was shown to be equivalent to the whole of reactive promastigotes, and optical density (OD) kDNA probe and to dissection of sandflies (Laskay units or counts retained. The profiles of reactivity et al., 1991[b]). The savings in time are obvious. identify the species (by reference to control sets) and gather the parasites into serodemes. Application Extension of the use of probes to genomic DNA frag- in epidemiological investigations is largely associ- ments was initially done to confirm the evolutionary ated with in vitro or intra-vectorial promastigotes relationships between different taxa of Leishmania, (Grimaldi, David & McMahon-Pratt, 1987; Barral et to estimate the divergence rate, and to infer that al., 1991; Bonfonte–Garrido et al., 1992; Barrios et al., Leishmania diversity relies principally on mutational 1994; Canto-Lara et al., 1999). A number of limita- mechanisms (Beverley et al.,1987). The scarcity of tions have been identified, including: antigenicity is large inter-chromosomal rearrangements was later not always well defined; limiting amounts of vector confirmed by studies on karyotypes and physical material can interfere with the testing of large bat- mapping of markers on sets of Leishmania species teries of antibodies; false negatives may result from representatives (Wincker et al., 1996). Genomic DNA inhibitory factors in the sandfly gut; interpretation probes were developed and validated for the iden- of results is very dependent on the antibodies tested tification and discrimination of all Old World spe- and, in particular, a positive or negative reaction cies and/or species complexes (Van Eys et al., 1989, with a species-specific antibody does not allow the 1991; Guizani et al., 1994). The power of such probes presence of another parasite species in the sample to is based on the invariant hybridization patterns at be ruled out (Shaw et al., 1987). Also, the possibil- the intra-specific level, and on the immediate dis- ity that taxonomic specificity might be questioned tinction made between the different species; their by extending the use to larger sets of parasite spe- potential in detecting hybrids between clearly dis- cies or genera cannot be ruled out (Shaw, 1994). For criminated species has also been proven (Kelly et these reasons, it is important to always confirm any al., 1991). Genomic restriction fragment length poly- results obtained by using other molecular assays. morphism (RFLP) analysis requires lesser amounts of parasites than typing a whole set of isoenzymes, 3. DNA targets. The application of DNA probes to and offers the possibility to de-hybridize the mem- the detection and identification of Leishmania within branes and test other probes when there is an inter- biopsies and vectors has been promoted since the est in analyzing parasite variability. RFLP markers early nineteen eighties (Wirth & Pratt, 1982; Rogers, are co-dominant, so that hybridization profile can Burnheim & Wirth, 1988). Because they are unique be assimilated to genotype and can serve for pop- to the kinetoplastida, minicircles of kinetoplast DNA ulation genetics and population structure analyses. (kDNA) constituted the main target in the initial and DNA extraction, generation of restriction digests, many ensuing studies (Arnot & Barker, 1981; Barker, transfer of gels, use of radioactive labels, and com- 1987). Restriction profiles of the kDNA minicircles parison of hybridization patterns to reference sets were used to define schizodemes and to compare for parasite identification are all techniques that parasites obtained from humans and other mam- require the services of trained personnel; population malian hosts (Lopes et al., 1984; Cuba Cuba et al., analyses require competency also.

58 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 The polymerase chain reaction (PCR) and its versa- PCR assays followed by liquid phase hybridization tile applications entered the world of leishmaniasis coupled with immuno-enzymatic detection have in the early nineties (Rodgers, Popper & Wirth, 1990). been developed (Qiao, Miles & Wilson, 1995; Costa PCR assays aimed at characterizing Leishmania par- et al., 1996). Not all the assays described refer to an asites can be of different discriminatory power, dif- extensive set of Leishmania species representatives; ferentiating the parasites at species level or isolate however, whenever tested, there has been good cor- level. However, in many instances, only a limited relation between assignment to taxa using isoen- set of species has been referred to while developing zyme- or PCR-based assays. In many instances, the the assays. purpose has been to differentiate and identify only a subset of the Leishmania species/complex of rel- Besides the classical kinetoplast minicircle targets evance to a particular clinical or epidemiological (Smyth et al., 1992; Lopez et al., 1993; Rodriguez situation. Hence, to be considered as valid for the et al., 1994; Nuzum et al., 1995; Bhattacharyya et identification and differentiation of Leishmania spe- al., 1996; Belli et al., 1998; Salotra et al., 2001), the cies, some of these assays still need to be appropri- PCR targets so far addressed can be divided into ately validated. five major genomic categories including repetitive sequences (Piarroux et al., 1993; Qiao, Miles Potential increase in the sensitivity of detection of & Wilson, 1995; Fu, Perona-Wright & Barker, 1998), infecting organisms constitutes one major advan- arbitrarily defined targets (Mimori et al., 1998; tage of the PCR technique, allowing the difficulties Hanafi et al., 2001) and gene family clusters coding associated with in vitro parasite isolation to be over- for rRNA (Guevara et al., 1992; Van Eys et al., 1992; come and rendering possible identification of para- Uliana et al., 1994; Cupollilo et al., 1995; El Tai et al., sites within the patient, host or vector. Only small 2000), and miniexons (Fernandes et al., 1994; Harris amounts of DNA or parasite are needed. When et al., 1998; Marfurt et al., 2003) or gp63 (Victoir et al., tested in defined laboratory conditions, PCR assays 1998; Mauricio et al., 2001; Guerbouj et al., 2001[b]). can detect less than one parasite. However, the PCR has inherent limitations associated with the detec- The main purpose of the different PCR assays devel- tion of low amounts of target DNA against excess oped is to identify and differentiate Leishmania spe- background DNA, or with the presence of PCR cies. Some PCR products are unique to only one or inhibitors. Therefore, it is important to assess per- a group of parasite species (eg. L. donovani complex, formance of the assay in terms of sensitivity, speci- L. braziliensis complex) as a result of amplification of ficity and predictive value for negative and positive species-specific sequences or use of polymorphism- results, as well as in terms of discriminatory power. specific primers (Guevara et al., 1992; Lopez et al., For instance, if using a species-specific PCR assay 1993; Qiao et al., 1995; Fu, Perona-Wright & Barker, in a ‘presence vs. absence of amplification’ format, 1998; Mimori et al., 1998; Hanafi et al., 2001; Salotra it is important to remember that a negative PCR et al., 2001). In other cases, distinction between dif- does not necessarily mean the absence of amplifi- ferent species rests on the size difference in prod- cation, or that the organism does not belong to the ucts of the simple (Smyth et al., 1992; Piarroux et al., targeted species; also, it does not rule out the pres- 1993, Breniere et al., 1999) or multiplex (Belli et al., ence of other Leishmania species in the sample. It is 1998; Harris et al., 1998) PCR assays, on the size and important therefore to contemplate using other consequence of the amplicons (Fernandes et al., 1994), firmatory tests. or on additional steps such as detection of changes in single strand conformations (Van Eys et al., 1992), The performance of some of the PCR assays devel- restriction analysis (Marfurt et al., 2003), or hybrid- oped has been defined for the diagnosis of Leishmania ization of the products using different kinds of probe: infections in blood, cutaneous biopsy, or aspirate total kDNA probes (Rodgers et al., 1990), species- of bone marrow, spleen or lymph node of human specific probes (Rodriguez et al., 1994; Hernandez- patients (Rodriguez et al., 1994; Costa et al., 1996; Montes et al., 1998; Breniere et al., 1999), or a set of Andresen et al., 1996, 1997; Aviles et al., 1999) or dogs allele-specific probes (Uliana et al., 1994; Ramos et (Ashford et al., 1995; Lachaud et al., 2002; Reithinger al., 1996). Alternatively, a nested PCR using prim- et al., 2002, 2003). Compared to other diagnostic tests, ers defined within the amplicon could differentiate e.g. direct examination, culture and/or serology, the species according to the specificity of the primers PCR has proved more sensitive in most cases; spec- (Meredith et al., 1993; Aransay, Scoulica & Tselentis, ificity is variable, according to DNA target. Use of 2000) or the size of the products (Noyes et al., 1998). the PCR for mass screening of Leishmania infections The hybridization steps, when included in these pro- has been questioned (Reithinger et al., 2003). cesses, have principally been achieved on the classical nylon membrane support. In a few instances, The application of assays already developed requires

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 59 trained personnel and a special set-up to avoid PCR lished zymodeme types and the ‘DNA types’ iden- cross-contamination. It should be borne in mind tified using various kinds of assay, bringing more that it is sometimes necessary to readjust the assay fuel to debate about phylogenetic status of such to laboratory conditions, and to introduce the nec- zymodemes (Banuls, Hide & Tibayrenc, 1999; Hide, essary controls (e.g. internal controls, exhaustive set Banuls & Tibayrenc, 2001). Many of these studies of reference isolates, assessment of PCR inhibition) have discussed the polymorphism observed in nat- in the experimental design for correct interpretation ural populations of Leishmania species but still aim of the results. Competency is required to interpret at understanding the genetic diversity of the par- original results and to troubleshoot. asite and the correlation with eco-epidemiological and/or clinical features of the disease. Geographical Arbitrarily defined targets have shown poten- structuring has been illustrated for various species tial in the identification of and differentiation (Guerbouj et al., 2001[a],[b]; Mauricio et al., 1999, between Leishmania species (Tibayrenc et al., 1993; 2001; Cupolillo et al., 2003; Schonian et al., 2000). Motazedian et al., 1996; Schönian et al., 1996; Noyes, The importance of host/vector–parasite interac- Belli & Maingon, 1996; Guizani, Dellagi & Ben tions in shaping parasite population structure has Ismail, 2002), and on a few occasions have been con- been highlighted (Guerbouj et al., 2001[b]; Ishikawa sidered as phylogenetic markers. However, it has et al., 2002; Cupolillo et al., 2003); it has even been been demonstrated that such phylogenetic interpre- demonstrated that the level of L. braziliensis parasite tation should be cautious and based on molecular variability is correlated with the number of vectors characterization of the markers used (Hanafi et al., and/or animal reservoirs involved in the trans- 2001). Using RAPD for parasite identification con- mission cycle (Cupolillo et al., 2003). Leishmania stitutes a good alternative because it can be associ- genome projects are expected to bring invaluable ated with simple interpretation of data; however, it information and to shed new light on the parasites can only be applied to purified or isolated parasites and their biology (Myler et al., 1999; McDonagh, and requires protocols to be well standardized to Myler & Stuart, 2000; Martinez-Calvillo et al., 2003; ensure reproducibility of results (Guizani, Dellagi & Saxena et al., 2003; Worthey et al., 2003). Similarly, Ben Ismail, 2002). genome projects will improve knowledge about the parasite’s vertebrate hosts and its hematophagous Besides identifying the species, or assigning a par- insect vectors (Tabashnick, 2003; Justice et al., 2003; asite to a species, DNA techniques can be used to O’Brien et al., 1999). As a consequence, better design classify the parasite and/or analyse its variability, of tools, enhanced appreciation of parasite diver- populations and genetics. The PCR-based assays sity and biology, and new working hypotheses are developed for such purposes refer mainly to tar- anticipated. gets such as kDNA (Breniere et al., 1999; Noyes et al., 1998), intergenic sequences of the rRNA gene clus- It is difficult to recommend one technique over ter (Cupollilo et al., 1995; El Tai et al., 2000), gp63 another. Each has advantages and drawbacks. coding or intergenic sequences (Victoir et al., 1998; Appropriate assessment of the needs, expectations Mauricio et al., 2001; Guerbouj et al., 2001[b]), repet- and limits should help in deciding which assay to itive sequences e.g. microsatellites (Rossi et al., 1994; select or whether to tailor a new one. In some sit- Bulle et al., 2002), or arbitrarily defined sequences uations, any of the techniques would appear to be (Mauricio et al., 1999). The polymorphisms observed appropriate, while in other cases, the complementary have been based on amplification profiles (Breniere data that each will provide are needed. Development et al., 1999), allelic differences evaluated at the level of PCR assays, although powerful, should not pre- of the size of the fragments amplified (Rossi et al., vent us from attempting to improve culture condi- 1994), and the sequence of such products (Mauricio tions and parasite isolation, since in vitro isolates et al., 2001; Bulle et al., 2002), their conformation (El are still an invaluable resource (Belkaid et al., 1996; Tai et al., 2000) or restriction analysis (Cupolillo et al., Hendricks &Wright, 1979). It is also important to 1995; Victoir et al., 1998; Noyes et al., 1998; Guerbouj recall that knowledge about the parasite in relation et al., 2001[b]). Genomic RFLP or chromosome size to its transmission cycle has been circumstantial for variabilities in relation to copy number of target a long time, and that only in those areas where the genes have also been analysed (Victoir et al., 1995, team includes a molecular biologist does it tend to 1998; Inga et al., 1998; Kebede et al., 1999; Guerbouj become more systematic. et al., 2001[a]). All these studies have demonstrated intra-specific and even intra-zymodemic variability. In some instances, the genetic heterogeneity of parasites belonging to the same species resulted in a lack of correlation between its well-estab-

60 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 EXAMPLES OF MOLECULAR BIOLOGY for P. papatasi were used to appreciate how few were APPLICATIONS TO THE STUDY OF the population differences between peridomestic VECTORS AND RESERVOIRS sites and gerbil burrows in endemic areas in Iran (Parvizi, Benlarbi & Ready, 2003). Genes involved Sandflies constitute a large group of insects, with in courtship songs and in saliva content have also some 500 species. At least 30 species belonging to been studied, and show potential for differentiat- the genera Lutzomya or Phlebotomus are consid- ing among populations of Lu. longipalpis (Oliveira et ered as proven vectors of leishmaniasis, but the list al., 2001; Bauzer et al., 2002; Yin, Norris & Lanzaro, is far from closed and a lot remains to be known 2000). The study of these markers is relevant to pop- (WHO, 1990; Alvar, 1997). Lutzomya longipalpis is by ulation isolation, speciation, and mating behaviour far the most studied sandfly (Soares & Turco, 2003). of sandflies, or to infection outcome; they could be Different molecular tools (e.g. monoclonal antibod- promising for developing rational ways to manip- ies, DNA probes, PCR) for detecting Leishmania par- ulate sandfly populations and therefore to control asites have been designed, assessed and adapted to leishmaniasis. meet the criteria necessary for sandfly studies. In other cases, sandfly markers have been developed Similarly, application of molecular techniques to to address the issues. the study of reservoirs, and to their incrimination, has been well documented. Principally, isoenzymes Eco-epidemiological application of molecular tech- have been used to type the infecting Leishmania par- niques has included detection of L. aethiopica within asites. Schizodeme analysis and PCR tools have naturally infected sandflies in Ethiopia (Laskay been used to incriminate some Leishmania reservoirs et al., 1991[b]), and sequential hybridization of (Lopes et al., 1984; Cuba Cuba et al., 1985; Pacheco DNA probes specific to P. papatasi and L. major to et al., 1986; Llanos-Cuentas et al., 1999; Rodriguez et assess infection rates and their fluctuations in natal., 2002; Reithinger et al., 2003). Infection in the dog ural sandfly populations in Tunisia (Esseghir et al., and the dog’s role in transmission of Leishmania are 1993). Monoclonal antibodies have demonstrated by far the most documented; fewer studies relate the co-existence of different parasite species (L. mex- to rodents or other reservoir types. Leishmania PCR icana, L. braziliensis) within the digestive tract of the assays have been used to assess the presence of L. same vector, Lu. ovallesi (Barrios et al., 1994), while chagasi in the rodent Proechymis canicollis (Travi et a Leishmania-specific PCR assay has allowed Lu. ver- al., 1998), or to follow experimental infections in rucarum and Lu. peruensis to be incriminated in the the spiny rat (Proechimys semispinosus) as an animal transmission of leishmaniasis in Peru (Perez et al., model (Travi et al., 2002). Detection of Leishmania 1994). An L. braziliensis-specific PCR-dot blot assay using the PCR technique has been applied to pro- was used to confirm, in an endemic area of Brazil, posing a role for the Egyptian mongoose (Herpestes the hypothesis that infected vectors are clustered, ichneumon) as a reservoir of VL in eastern Sudan allowing the understanding that the low vector (Elnaim et al., 2001), and to appreciating the infec- infection rates recorded are indeed the consequence tion patterns in the reservoir host in a study of the of non-homogeneous distribution of infected sand- ecology of cutaneous leishmaniasis in an endemic flies (Miranda et al., 2002). area of Israel (Wasserberg et al., 2002). Similarly, the prevalence and seasonal transmission of L. mexi- Taxonomical differentiation of sandflies and vec- cana in populations of woodrats, Neotoma micropus, tors has also been addressed using morphometry in an endemic area of Texas were evaluated using and molecular tools: isoenzymes, DNA markers a Leishmania-specific PCR assay (Kerr, McHugh & (Hodgkinson et al., 2003; Arrivillaga et al., 2002, Dronen, 1995); this assay was also used to study 2003; Dujardin et al., 1999, 1997; Esseghir et al., 1997). the temporal and spatial distribution of L. mexi- Among the molecular markers showing potential for cana infections in a population of Neotoma micropus elucidating the taxonomic status of sandflies are iso- (Raymond et al., 2003). Finally, morphometry, isoenzymes and mitochondrial genes. Isoenzymes have enzyme typing and DNA haplotyping were used proved useful in differentiating between the sandfly to study the geographical distribution and analyse vectors encountered in Egypt and in analysing the populations of the rodents Psammomys and Meriones differences between P. papatasi, P. bergeroti, P. lange- in Tunisia (Ben Abderrazak et al., personal commu- roni and P. perniciosus (Kassem Fryauff & Hanafi, nication). 1999). Isoenzymes and mitochondrial haplotypes were found particularly efficient at separating genetically divergent populations of Lu. longipalpis and in Acknowledgements identifying four different clades (Arrivillaga et al., To Dr. S. Ben Abderrazak, M. Belkaid, R. Ben Ismail, 2003). Mitochondrial and endosymbiotic markers E. Cupolillo, K. Dellagi, M. Mukhtar for discussions.

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Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 69 Titus RG, Ribeiro JM. Salivary gland lysates from the Wincker P et al. The Leishmania genome compris- sand fly Lutzomyia longipalpis enhance Leishmania es 36 chromosomes conserved across widely diver- infectivity. Science, 1988, 239:1306–1308. gent human pathogenic species. Nucleic Acids Research, 1996, 24:1688–1694. Travi BL et al. Dynamics of Leishmania chagasi infection in small mammals of the undisturbed and Wirth DF, Pratt DM. Rapid identification of Leishmania degraded tropical dry forests of northern Colombia. species by specific hybridization of kinetoplast Transactions of the Royal Society of Tropical Medicine and DNA in cutaneous lesions. Proceedings of the National Hygiene, 1998, 92:275–278. Academy of Sciences of the United States of America, 1982, 79:6999–7003. Travi BL et al. Susceptibility of spiny rats (Proechimys semispinosus) to Leishmania (Viannia) panamensis and Wolday D et l. Emerging Leishmania/HIV co-infection Leishmania (Leishmania) chagasi. Memorias do Instituto in Africa. Medical Microbiology and Immunology, 2001, Oswaldo Cruz, 2002, 97:887–892. 190:65–67. Uliana SR et al. Discrimination amongst Leishmania Worthey EA et al. Leishmania major chromosome 3 con- by polymerase chain reaction and hybridization with tains two long convergent polycistronic gene clus- small subunit ribosomal DNA derived oligonucle- ters separated by a tRNA gene. Nucleic Acids Research, otides. The Journal of Eukaryotic Microbiology, 1994, 2003, 31:4201–4210. 41:324–330. Yin H, Norris DE, Lanzaro GC. Sibling species in Valenzuela JG et al. Toward a defined anti-Leishmania the Llutzomyia longipalpis complex differ in levels of vaccine targeting vector antigens: characterization of mRNA expression for the salivary peptide, maxadi- a protective salivary protein. Journal of Experimental lan. Insect Biochemistry and Molecular Biology, 2000, Medicine, 2001, 194:331–342. 9:309–314. van Eys GJ et al. Identification of ‘Old World’ Yoshida ELA et al. Description of Leishmania Leishmania by DNA recombinant probes. Molecular and (Leishmania) forattinii sp. n., a new parasite infecting Biochemical Parasitology, 1989, 34:53–62. opossums and rodents in Brazil. Memorias do Instituto Oswaldo Cruz, 1993, 88:397–406. van Eys GJ et al. A nuclear DNA probe for the identification of strains within the Leishmania donovani complex. Experimental Parasitology, 1991, 72:459–463. van Eys GJ et al. Sequence analysis of small subunit ribosomal RNA genes and its use for detection and identification of Leishmania parasites. Molecular and Biochemical Parasitology, 1992, 51:133–142. Victoir K et al. Plasticity of gp63 gene organization in Leishmania (Viannia) braziliensis and Leishmania (Viannia) peruviana. Parasitology, 1995, 111:265–273. Victoir K et al. The gp63 gene locus, a target for genetic characterization of Leishmania belonging to subgenus Viannia. Parasitology, 1998, 117:1–13. Warburg A, Schlein Y. The effect of post-bloodmeal nutrition of Phlebotomus papatasi on the transmission of Leishmania major. American Journal of Tropical Medicine and Hygiene, 1986, 35:926–930. Wasserberg G et al. The ecology of cutaneous leishmaniasis in Nizzana, Israel: infection patterns in the reservoir host, and epidemiological implications. International Journal for Parasitology, 2002, 32:133–143. World Health Organization. Control of the leishmaniases. Report of a WHO expert committee. Geneva, World Health Organization, 1990 (WHO Technical Report Series, No. 793).

70 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 4.3 DRUG TRIALS Pentavalent antimony in cutaneous leishmaniasis Shyam Sundar Cutaneous Old World (L major, L tropica, L. infan- Banaras Hindu University tum) and New World (L. mexicana and L. braziliensis Institute of Medical Sciences complexes) leishmaniasis is commonly treated with Varanasi Sb at a dose of 20 mg/kg for 20 days, but there are India few data on whether this regimen is appropriate for all species. A species-based approach to treatment has been advocated, especially in countries where several species may cause cutaneous leishmaniasis PENTAVALENT ANTIMONY (CL). Intra-lesional Sb has been used with encourag- COMPOUNDS ing results in Old World self-healing CL. On the Indian subcontinent, pentavalent antimonials (Sb) are fast losing ground as antileishmanial PENTAMIDINE agents; branded products and generic products are This is the first drug used in refractory patients. similar in safety and efficacy. Drugs from reputed However: manufacturers should only be used as bad lots can • cure rates have declined from an initial ~100% cause serious and fatal toxicity; even with standard to 70% in recent years; products, serious and fatal antimony toxicity is not • it has serious toxicity, e.g. insulin dependent infrequent (3–6% deaths). diabetes mellitus or occasional death, and this is unacceptable; Drug resistance • limited experience in Sudan suggests that pent- Resistance to antimony is a serious problem in amidine may not be very effective in refractory most parts of the State of Bihar, India, and is slowly cases. spreading to neighbouring areas of Uttar Pradesh and to the Terai regions of Nepal. There is evidence Use of the drug has now been abandoned in India, that resistant strains of Leishmania are emerging in although short courses are effective, without adverse Bihar, and a strategy is needed to combat and pre- events, in several forms of cutaneous disease. vent resistance developing to other antileishmanial drugs. This should include: AMPHOTERICIN B • molecular probes/in vitro drug sensitivity assays In India, this is the most extensively used drug in Sb • simple, cheap and easily applicable probes refractory patients; its efficacy is nearly 100%, with • multidrug therapy. no resistance noted, though occasional relapses do occur. However: In other parts of the world, pentavalent antimoni- • prolonged hospitalization for infusion of the als remain the most important antileishmanial com- drug is a severe limiting factor; pounds for all forms of leishmaniasis, but: • it is a toxic drug and infusion reactions are com- • in both immunocompetent and HIV co-infected mon; serious toxicity e.g. hypokalemia, neph- patients, chemical pancreatitis has been reported, rotoxicity, mycarditis and death occur not as has cardiotoxicity; infrequently; • the availability of branded products has become • cardiac and electrolyte monitoring are needed, difficult; there are not many manufacturers of and the patient has to be kept in hospital; safe antimony products. • most endemic countries do not have the capacity (hospital beds and qualified personnel) to Is there a need to replace antimony with cater for the needs of visceral leishmaniasis (VL) patients; safer antileishmanial drugs for treatment of • the cost of treatment is several times more than visceral leishmaniasis? with generic Sb. In view of the serious and fatal toxicity and declin- ing efficacy there is a need to replace Sb with safer The drug appears to be effective in other forms of and more efficacious compounds. leishmaniasis besides VL, although experience is limited is this respect.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 71 Lipid formulations of amphotericin B PAROMOMYCIN There has been remarkable improvement in the tol- Paromomycin, an aminoglycoside, is well tolerated erability of these drugs, and there is no organ tox- and effective for VL, but less so for CL. The drug has icity. AmBisome has the best safety profile. Short the potential to replace Sb as the primary drug for courses are possible with good effect. all forms of leishmaniasis. The safety profile seems to be excellent, and the drug could be used for com- However, the cost of the drugs is prohibitive and they bination chemotherapy. are not affordable for most patients. Cheap indigenous lipid preparations are needed, and the prices At the moment there is no industrial production, but of existing preparations, especially AmBisome, need an Indian company has started trial production; the to be brought down. cost is likely to be affordable (in the range of US$ 20–30). These lipid preparations have not been tested in a controlled manner in the case of cutaneous disease. In India, a dose of 16 mg/kg/day for 21 days cured 93% of VL patients. A Phase III trial has already Three preparations are available: started in India; results will be available by the end of 2004. Liposomal amphotericin B Results of parenteral use of paromomycin for treat- (AmBisome; Gilead Sciences, Foster city, CA, USA) ment of CL have so far been unsatisfactory. In Belize, cure rates of L. braziliensis sores were around 50%, For immunosuppressed patients, AmBisome in a and were lower than this in Colombia. In Brazil, total dose of 40 mg/kg spread over 38 days is recom- 16mg/kg intramuscular daily for 20 days cured only mended, but has not been formally compared with 33% patients with mucocutaneous leishmaniasis. shorter regimens. All co-infected patients relapse.

Three preparations of paromomycin ointment have In immunocompetent patients in Europe and South been used for CL: America, a total dose of 18–24 mg/kg, and in Kenya • paromomycin 15% plus methylbenzethonium a total dose of 14–18 mg/kg, given over 10 days, chloride 12%. In placebo controlled trials in cured 90%-100% of patients. Israel and Ecuador, this produced cure rates of 74% vs. 26% and 72% vs. 9%, respectively, at 50 In Indian VL, 6mg/kg (2mg/kg x 3) cured 100% days. In a double blind, randomized placebo patients, and 3.75 mg/kg cured 89% patients. In a controlled trial in Guatemala, 86% were cured subsequent study employing a single dose of 5 mg/ with the drug vs. 39% with placebo. kg of AmBisome, 91% patients were cured with min- • 15% paromomycin with 10% urea, a less toxic imal adverse events. Effective single-dose treatment formulation, had no effect on CL in Iran or makes it possible to treat a large number of patients Tunisia. in a very short time. In India, the cost of a single • WR279396, containing 15% paromomycin plus dose of 5 mg/kg of AmBisome for a 30 kg patient is 0.5% gentamicin, accelerated cure time in CL US$ 390, compared with ~US$ 60 for a typical treat- due to L. panamensis, but the cure rate was no ment regimen of conventional amphotericin B. higher than with placebo. Amphotericin B lipid complex (Abelect [ABLC]; Intermune, USA) MILTEFOSINE Miltefosine is an alkyllysophospholipid originally Similarly, a total dose of 10–15 mg/kg ABLC deliv- developed as an anti-cancer agent; it was found to ered over 5–10 days cured 90%-100% of patients. be leishmanicidal in the late 1980s. Several trials of miltefosine in VL have been carried out in India, Amphotericin B colloidal dispersion where the drug is now approved for the treatment (Amphocil [ABCD]; Sequus Phrmaceutical; Menlo of VL in adults at a dose of 100 mg (~2.5 mg/kg) Park, USA). daily for four weeks.

In Brazil, five and seven doses of Amphocil at 2 mg/ The drug has been shown to be safe and well toler- kg cured 90% and 100% of patients respectively, but ated. It has consistently cured ~95% patients, which side effects were a limiting factor. is comparable with the more toxic reference drug amphotericin. The principal adverse events are mild

72 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 (in the majority of patients) to moderate gastroin- testinal symptoms, notably vomiting (40%) and diarrhoea, reversible asymptomatic transient elevation of hepatic enzymes, and nephrotoxicity at high doses.

Tolerability and efficacy in children are similar to those seen in adults. Phase IV trials in adults and children have just been completed in India to see whether the promise holds good in the field.

As miltefosine causes abortions and congenital defects in animals, it is not used in women of childbearing age unless contraception can be secured.

Results from trials in CL in Colombia and Guatemala suggest the drug has variable efficacy in this type of leishmaniasis.

It has been suggested that directly observed therapy (DOT) would improve compliance with the drug.

Market prices in India are very high and the drug is not affordable to most of those who need it.

SITAMAQUINE The development of sitamaquine (GlaxoSmithKline), a primaquine analogue whose activity against L. donovani was demonstrated in the 1970s, has been slow. In 1994, a dose ranging study in Kenyan VL showed moderate activity. In 2002, a Brazilian study reported a surprising lack of linear correlation between dos- age and pharmacokinetics and cure rates.

Methaemoglobinemia is a common side effect, and some patients have developed nephropathy. The drug appears to have a good efficacy in Indian VL; however, there are some safety concerns.

MULTIDRUG THERAPY Experience in India suggests that the few antileishmanial drugs available need to be protected, and used judiciously and discretely.

To improve compliance, the duration of treatment needs to be shortened. The cost of therapy needs to brought down.

A few combinations, involving two parenteral drugs, are being tried out in Sudan and Brazil. After in vitro, animal safety and toxicity studies have demonstrated synergism, clinical trials, if well controlled, could pave the way for multidrug therapy of VL.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 73 4.4 CONTROL OF ZOONOTIC lived. A novel method – use of deltamethrin-treated VISCERAL LEISHMANIASIS dog collars – for topical application of insecticide on dogs has recently been developed, which enables the insecticidal effect to persist for up to 8 months. Clive Davies Experimental trials have consistently demonstrated Department of Tropical and Infectious Diseases that deltamethrin-treated collars reduce the propor- London School of Hygiene and Tropical Medicine tion of sandflies that bloodfeed and survive by up London, UK to 90%. In experimental trials, widespread implementation of these collars to domestic dogs in Italy and Brazil reduced their risk of being infected with L. infantum by 46–58% (in the first year of the inter- INTRODUCTION vention). Collared dogs clearly receive personal protection from canine leishmaniasis, but is there a Control strategies against zoonotic visceral leish- sufficient “mass effect” to cut transmission to chil- maniasis (ZVL) tend to rely on early diagnosis and dren living in villages whose dogs have been col- treatment of patients with expensive and potentially lared (i.e. by reducing the entomological inoculation toxic pentavalent antimonial drugs. In some endemic rate)? countries, control programmes are designed to prevent infections in humans by vector control (house spraying with residual insecticides) and reservoir DELTAMETHRIN-TREATED DOG control (culling of infected dogs). Infected dogs are COLLARS identified either by their clinical symptoms (such This paper reviews the evidence that widespread as hair loss, weight loss, extended nails) or by sero- provision of deltamethrin collars for domestic dogs logical diagnosis. Although this practice appears to could significantly reduce the burden of zoonotic have been effective in China, it is unclear whether it visceral leishmaniasis. Particular attention is paid has worked elsewhere. In Brazil, for example, ZVL to the results of a recent matched cluster random- has increased steadily during the last 10–20 years ized trial in North-West Iran (funded by the World despite the spraying of 200 000 houses and killing of Health Organization Regional Office for the Eastern 20 000 dogs per year. Following annual surveys in Mediterranean), which not only demonstrated an endemic regions, dogs are culled if their blood sam- effect on L. infantum incidence rates in collared dogs, ples are diagnosed positive by immunofluorescence. but also in the children from those villages where Although experimental trials indicate that dog con- dogs were collared. trol may reduce Leishmania incidence in both dogs and children, concerns over the delays between Eighteen villages were ordered into pairs matched sampling, diagnosis and culling, and a failure to by pre-intervention child prevalence of L. infantum reduce the number of notified cases, have led to infection. Within pairs, villages were assigned ran- scepticism of the effectiveness of the Brazilian con- domly as control or intervention. All domestic dogs trol programme. More effective diagnostic tools may in intervention villages were provided with col- allow culling without delay, but a recent trial of the lars throughout two transmission seasons. Children rK39 dipstick test showed poor specificity (< 75%) and dogs were surveyed on three consecutive occa- for diagnosing infected dogs. Given the seriousness sions at yearly intervals, i.e. pre-intervention in of the disease, the unwillingness of dog owners to 2000, 1 year post-intervention in 2001, and 2 years permit their dogs to be culled, and the inconsistent post-intervention in 2002. Incidence of L. infantum results of trials designed to test the effectiveness of infection was measured by seroconversion and culling, the need to identify alternative sustainable leishmanin skin test (LST) conversion in children, strategies to reduce the burden of ZVL is clear. and by seroconversion in dogs. Dipping dogs in insecticide or applying topical insec- During the two-year trial, a total of 67 direct agglu- ticide lotions can significantly reduce sandfly bites tination test (DAT) conversions were detected on dogs and so protect them from infection. The epi- amongst the 1622 follow-up tests in initially DAT demiological impact of topical insecticide treatments negative dogs. After adjusting for age, and pre- on dogs depends not only on the reduction in num- intervention village seroprevalence, we found a sig- ber of sandflies feeding on them (i.e. the anti-feed- nificant effect of collars in both years: in year 1, odds ing effect) but also on the reduction in survival of ratio = 0.45 (95% CI 0.30–0.68), p < 0.001; and in year those flies that do feed so that they are less likely to 2, odds ratio = 0.29 (95% CI 0.13–0.65). Although transmit Leishmania. But this strategy requires reg- there was some suggestion that protection increased ular re-treatment as the insecticidal effect is short in year 2 (from 54% to 71%), we found no significant

74 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 interaction between treatment and year (p = 0.187). provide a great logistic challenge for health work- But incidence did vary significantly with year (p ers to maintain high dog collar coverage rates dur- < 0.001), and so we controlled for year in the final ing a transmission season in Iran (which only lasts model. This final logistic regression model (clus- 2–3 months). However, it appears that transmission tered on village) detected a significant effect of treat- to children was reduced by less than the protection ment on the risk of DAT conversion per year: odds received by collared dogs, and even two years of ratio for DAT conversion in treated villages as com- collaring dogs failed to completely eliminate trans- pared with control villages = 0.39 (95% CI 0.24–0.63), mission in the villages. This could be because (1) p < 0.001. Hence, on average, the odds of DAT con- transmission continues in sylvatic reservoirs which version in dogs from intervention villages each year visit villages frequently (e.g. foxes and jackals); (2) was reduced by 61%. the culling rate of stray dogs is inadequate; or (3) some transmission takes place outside villages. During the two-year trial, a total of 115 DAT conversions were detected amongst the 4324 follow-up CONCLUSION tests in initially DAT negative children. After adjusting for age, LST status and pre-intervention village The duration of the sandfly transmission season, the seroprevalence, we found a significant effect of col- dog population turnover rate, and the rate of collars in both years: in year 1, odds ratio = 0.56 (95%CI lar loss will all impact on the logistics of adopting 0.36-0.89), p = 0.013; and in year 2, odds ratio = 0.47 this approach elsewhere, e.g. in Brazil. Nevertheless, (95% CI 0.26–0.87). Although, as for seroconver- we remain hopeful that community-wide use of sion rates in dogs, there was some suggestion that treated collars could provide an effective alternative protection increased from 44% to 53% in year 2, we to dog culling. Prior to making recommendations found no significant interaction between treatment that insecticide treated collars should be introduced and year (p = 0.518). But incidence did vary signifi- as a control measure in Iran or elsewhere, it is par- cantly with year (p < 0.001), and so we controlled for amount that their impact on disease (visceral leish- year in the final model. This final logistic regression maniasis in children) is measured, in order to judge model (clustered on village) detected a significant the cost-effectiveness of this intervention. The tri- effect of treatment on the risk of DAT conversion per als described in this presentation only measured the year: odds ratio for DAT conversion in treated vil- impact on infection. In order to detect an effect on lages as compared with control villages = 0.47 (95% disease, a much larger study population is required. CI 0.33–0.67), p < 0.001. Hence, on average, the odds With the support of WHO (the TDR Director’s of DAT conversion in children from intervention vil- Initiative Fund), such a trial is currently ongoing. lages each year was reduced by 53%. The entomological explanation for these epidemiological effects was confirmed by experimental field trials testing the impact of collars on both bloodfeeding and mortality rates of local sandfly vectors.

Following a pilot study using a series of focus group discussions, a questionnaire was designed and given out by local health workers to 1872 householders from a total of 50 villages throughout the endemic region. The answers to the questionnaires provided a measure of the local’s knowledge and attitude to visceral leishmaniasis, the function of domestic dogs in the region, the effort and expense that dog owners provide for their dogs (e.g. in relation to health), and the attitude of dog owners to the collars provided in the trial.

The overall conclusion is that the collars appear to be popular with dog owners, and they clearly reduce the risk of L. infantum infection in dogs and hence transmission to children. Collar loss rates averaged 5% during each transmission season, and the turnover rate of the dog population was about 13% per year. These rates are reasonably low, and should not

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 75 4.5 POST KALA-AZAR profile (RFLP), suggesting that PKDL is caused by 15 DERMAL LEISHMANIASIS the same parasite isolate that causes VL . Typing of unpaired PKDL parasite isolates collected from the same VL endemic area in eastern Sudan showed Maowia Muhktar that the majority of the isolates (13/15) belonged Institute of Endemic Diseases to L. archibaldi based on their isoenzyme profile University of Khartoum (MON 82) and gp63 PCR RFLP. The remaining two Khartoum PKDL isolates were typed as L. infantum (MON 30)14. Sudan Interestingly, in a separate study conducted in the same endemic area, four out of six (4/6) PKDL isolates were identified as L. donovani (MON18) and two as L. infantum (MON 30, MON 267)13. The ratio INTRODUCTION of the different Leishmania species in PKDL isolates was similar to their ratio in VL13,15. Post kala-azar dermal leishmaniasis (PKDL) is a der- matological form of leishmaniasis that develops in visceral leishmaniasis (VL) patients during or after PATHOGENESIS treatment with pentavalent antimony1,2. PKDL pre- Development of PKDL seems to be a complex mech- vails in VL endemic areas, and has been reported in anism that involves several host and parasite factors. India, Nepal, Bangladesh, Sudan, Ethiopia, Kenya, Osman et al (1998) showed that the presence of PCR the Mediterranean Basin, and sporadically in South detectable parasite DNA (persistence of infection) America3,4,5,6. in VL patients after the completion of a full course of pentostam treatment increased the risk for devel- The incidence of PKDL varies from 5–10% in treated oping PKDL16. Our recent study on typing paired VL patients in India to 30–60% in treated patients VL/PKDL isolates from eastern Sudan showed that in Sudan2,7,8. A few patients with no history of VL the parasite that causes VL is also capable of induc- develop PKDL10. PKDL lesions develop 1–13 months ing PKDL, a finding which suggested the tendency post antimony treatment in Sudan, and 1–3 years of these parasites to cause skin pathology possi- post antimony treatment in India7,11. The lesions are bly through differential gene expression15. The host confined to the skin and range from macular-macu- immune response in VL patients (TH2) is character- papular, to papular, and nodular; they usually start ized by increased secretion of IL-10, identified as an around the mouth then extend to the face (grade 1). indicator for increased risk for developing PKDL17,18. Lesions can extend from the face to the chest, back, It is known that following the treatment of VL upper arms and legs (grade 2) and can become gen- patients with pentostam, the peripheral blood lym- eralized (grade 111); they affect areas of the body phocytes and lymph node homing cells proliferate directly exposed to sunlight 8,9,12. in response to mitogens and Leishmania specific antigens, with increased production of IFN- gamma16. The age group of 4–8 years is more affected by PKDL Recently it was shown that variation in the IFNGR1 in eastern Sudan, with younger children developing receptor is significantly associated with the risk of more severe lesions while, in India, young adults developing PKDL19. are more affected. PKDL age ranges are similar to those of VL7,11. DIAGNOSIS THE PARASITE Diagnosis of PKDL is based on clinical presentation and history of VL and pentostam treatment. It was previously thought that PKDL was caused by Microscopic demonstration of the parasite in lesions specific Leishmania parasite isolates. However, later has low sensitivity (20–30%), but this increases to reports showed that parasite isolates from PKDL 80–90% using molecular biology and immuno-his- patients belonged to different zymodemes (L. don- tochemistry techniques14,16. Serological diagnosis is ovani MON 18; L. infantum MON 30, MON 267; L. of limited value in endemic areas, however IgG1 is archibaldi MON 82) which were also isolated from elevated in the sera of PKDL patients. PKDL sera VL patients 13,14. have been found to consistently react with antigens of 110 and 65 KDa in Leishmania lysate20,21. Isolation Furthermore, characterization of seven paired iso- of the parasites from PKDL lesions is rather tedious lates from the same VL/PKDL patients showed and prone to contamination, but can be achieved by they were identical in their isoenzyme profile and primary culture of skin slits in NNN media with fre- gp63 polymerase chain reaction (PCR) restriction quent subculture in the same medium, even when

76 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 apparent parasite growth is not observed15. Parasite 4. Karki P et al. Post kala-azar dermal leishmaniasis culture allows isoenzyme typing and more detailed (PKDL): the first case report from Nepal. Southeast epidemiological analysis. Asian Journal of Tropical Medicine and Public Health, 2003, 34(1):22–23. TREATMENT 5. Muigai R et al. Post kala-azar dermal leishmaniasis: the Kenyan experience. East African Medical Most PKDL patients in eastern Sudan heal spontane- Journal, 1991, 68:801–806. ously and don’t need medical interference. However, 6. Girgla HS et al. Post kala-azar dermal leishma- PKDL patients with severe or persistent skin lesions niasis. The British Journal of Dermatology, 1997, might need treatment with a prolonged course of 97:307–311. pentostam at 20mg/kg for 2–3 months. Alternative 7. Thakur CP. Epidemiological, clinical and thera- treatment with pentamidine and ketoconazole has peutic features of Bihar kala-azar (including post been used in areas that report parasite resistance kala-azar dermal leishmaniasis). Transactions of the to pentostam22,23,24,25. A trial for immunotherapeutic Royal Society of Tropical Medicine and Hygiene, 1984, treatment of persistent PKDL lesions with alum-pre- 78:391–398. cipitated Leishmania major plus BCG has started in 8. Zijlstra EE, Elhassan AM. Leishmaniasis in the Sudan (personal communication). Sudan 4: Post kala-azar dermal Leishmaniasis. Transactions of the Royal Society of Tropical Medicine CONTROL and Hygiene, 2001, 95 (suppl). S59–S75. 9. Zijlstra EE et al. Post kala-azar dermal leishmania- PKDL has been thought to contribute to the spread sis. The Lancet Infectious Diseases, 2003, 3:87–98. of VL infection by serving as a source of parasites; 10. Elhassan AM et al. Post kala-azar dermal leishma- the presence of PKDL patients has been associated niasis in the absence of active visceral leishmania- with the occurrence of VL epidemics in India, where sis. Lancet, 1990, 336:750. 7 VL transmission is thought to be anthroponotic . 11. Musa AM et al. The natural history of post kala- However, though it has been suggested that PKDL azar dermal leishmaniasis: clinical, immunological plays a role in transmission of Leishmania parasites and prognostic features. Annals of Tropical Medicine 25 in Sudan, this has not yet been proved . Since the and Parasitology, 2002, 96(8):265–272. same parasite zymodemes (L. donovani MON 18; L. 12. Zijlstra EE et al. Post kala-azar dermal leishmani- infantum MON30, MON 267; L. archibaldi MON 82) asis in the Sudan: clinical presentation and differ- were reported in PKDL patients and animal reser- ential diagnosis. The British Journal of Dermatology, voirs, the transmission of these isolates could be zoo- 2000, 143:136–143. 13 notic , while other zymodemes that were reported 13. Dereure J et al. Visceral leishmaniasis in eastern only in humans and not in animal reservoirs could Sudan: parasite identification in humans and dogs; be transmitted anthroponotically, with PKDL possi- host-parasite relationships. Microbes and Infection, bly playing the role of parasite source. 2003, 5:1103–1108. 14. Ismael A et al. Detection and characterization of Possible variation in the sensitivity to pentostam Leishmania in tissues of patients with post kala- treatment of some isolates in eastern Sudan might azar dermal leishmaniasis using a specific mono- contribute to the persistence of the parasite after clonal antibody. Transactions of the Royal Society of completion of the treatment course. Tropical Medicine and Hygiene, 1997, 91:283–285. 15. Elamin EM. Leishmaniasis of the Sudan: Parasite char- References acterization and phylogeny [Ph.D. thesis]. Khartoum, University of Khartoum, 2003. 1. Singh NKP, Agrawal SK, Jha TK. Visceral leishma- 16. Osman OF et al. Use of PCR for diagnosis of post niasis associated with post kala-azar dermal leish- kala-azar dermal leishmaniasis. The Journal of maniasis. The Journal of the Association of Physicians Clinical Microbiology, 1998, 36:1621–1624. of India, 1989, 37:191–196. 17. Ghalib HW et al. Interleukin-10 production corre- 2. Napier LE, Das Gupta CR. A clinical study of post lates with pathology in human Leishmania infec- kala-azar dermal leishmaniasis. Indian Medical tion. The Journal of Clinical Investigation, 1993, Gazette, 1930, 63:249–257. 92:324–329. 3. El-Masum MA, Evans DA. Characterization 18. Gassim S et al. High levels of plasma IL-10 and of Leishmania isolates from patients with kala- expression of IL-10 by keratinocytes during viscer- azar and post kala-azar dermal leishmaniasis al leishmaniasis predict subsequent development in Bangladesh. Transactions of the Royal Society of of post kala-azar dermal leishmaniasis. Clinical and Tropical Medicine and Hygiene, 1995, 89:331–332. Experimental Immunology, 1998, 111:64–69.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 77 19. Mohamed HS et al. Genetic susceptibility to visceral leishmaniasis in the Sudan: Linkage and association with IL-4 and IFNGR1. Genes and Immunity, 2003, 4:351–355. 20. Ghosh AK, Das Cupta S, Ghose AC. Immunoglobulin subclass-specific antileishmanial antibody response in Indian kala-azar and post kala-azar dermal leishmaniasis. Clinical and Diagnostic Laboratory Immunology, 1995, 2:291–296. 21. Salotra P, Raina A, Ramesh V. Western blot analysis of humoral immune response to Leishmania donovani antigens in patients with post kala-azar dermal leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1999, 93:98–101. 22. Thakur CP et al. Treatment of post kala-azar dermal leishmaniasis with sodium stibogluconate. British Medical Journal, 1987, 295:886–887. 23. Thakur CP et al. Amphotericin B is superior to sodium antimony gluconate in the treatment of Indian post kala-azar dermal leishmaniasis. Annals of Tropical Medicine and Parasitology, 1997, 91:611–616. 24. Mishra M et al. Amphotericin versus pentamidine in antimony unresponsive kala-azar. Lancet, 1991, 337:926. 25. Hashim FA et al. Apparently successful treatment of two cases of post kala-azar dermal leishmaniasis with liposomal amphotericin B. Transactions of the Royal Society of Tropical Medicine and Hygiene, 1995, 89:440. 26. Elhassan AM, Khalil EAG. Post kala-azar dermal leishmaniasis: does it play a role in the transmission of Leishmania donovani in the Sudan? Tropical Medicine & International Health, 2001, 6:743–744.

78 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 4.6 EPIDEMIOLOGY In southern European countries, the mean age of co- OF LEISHMANIA-HIV infected patients is 29–33 years, very similar to the age at which HIV infection is more frequent; 80–85% CO-INFECTIONS of co-infected patients are male, as occurs in HIV infection also. There are two main concepts about Jorge Alvar the HIV infection pattern, which is quite similar in WHO Collaborating Center for Leishmaniasis most South European countries where co-infection Centro Nacional de Microbiología occurs: the AIDS incidence in South European coun- Instituto de Salud Carlos III tries is higher than the European average, being Madrid almost four-fold higher in Spain; and the number Spain of drug addicts per million inhabitants in Spain is 5–7 times greater than the mean for European countries, and even 1.6 times greater than Italy. When risk groups for HIV infection are analysed in Spain, Visceral leishmaniasis (VL) in Mediterranean coun- intravenous drug addicts (IVDU) represent 66% tries is caused by Leishmania infantum, which appears of the total, with homosexuals (15%), heterosexu- as a hypoendemic disease with 0.2–0.3 cases every als (7%), vertical transmission (2%) and hemoderiv- 100 000 inhabitants. VL cases associated with HIV atives and unknown (10%) being far behind them. infection were increasingly reported between 1985 The cases of co-infection are distributed as follows: and 1997, when the introduction of highly active IVDU represent 50–92%, sexual transmission (homo- antiretroviral therapy (HAART) drastically reduced , bi- or heterosexual) 5–40%, hemoderivatives 4–13%, the number of co-infection cases seen. The dog acts and other unknown causes 3%. The frequency of co- as a reservoir, and the prevalence of canine leishman- infection among IVDU is thus 15–25% greater than iasis is 5–20% or even more. The sandflies responsi- the HIV frequency for the same risk group. ble for transmission are: Phlebotomus perniciosus, P. ariasi, P. perfiliewi and P. neglectus, from west to east The question always raised in co-infection is respectively. whether it represents a latent infection reactivated by immune depression or whether it is a primary Possible overlap in transmission of any Leishmania Leishmania infection taking advantage of the lym- species (which normally is rural and periurban) phocyte reduction caused by HIV infection. It is with HIV transmission (which is urban and peri- estimated that only one in every five or ten immu- urban) is a problem in those countries where both nocompetent persons infected by Leishmania sub- infections are highly endemic, such as India, Sudan, sequently suffers from clinical VL. The Leishmania and Brazil. This situation becomes more acute with infection/disease ratio in HIV+ patients is unknown, the now-established ruralization of HIV transmis- but it seems evident that all parasitized and severely sion and the urbanization of Leishmania due to pop- immunodepressed patients will develop symptom- ulation movements. However, the great majority of atic disease. Leishmania/HIV co-infection cases are still reported in southern Europe; Spain has the great majority of There are two hypotheses to explain co-infection. cases, followed by Italy and France. So far, few cases The first is based on the fact that intradermal skin- are reported from the above-mentioned countries in positive subjects may be asymptomatic carriers of L. which 90% of all VL normally occurs. infantum which, during a later immunodepression, would develop into clinical VL. In this way, the HIV Visceral leishmaniasis in immunocompetent individ- infection would have an unmasking effect on the uals in Mediterranean countries was basically found true endemicity of Leishmania infection. In the case in children until the mid eighties, but recently the of HIV infection, it is to be supposed that reactiva- age pattern has changed. The AIDS pandemic does tion is also a matter of fact. not seem to be the only reason for this; in southern France, fewer than half the adult VL patients The second possibility is that, since the need for a are currently HIV+, although in Spain the propor- T lymphocyte-mediated immune response to con- tion is higher. The prevalence of co-infection has trol Leishmania infection is known, the parasite been established: 7–17% of febrile HIV+ and 4% of takes advantage of the existing immunodepression non-febrile HIV+ subjects (121 patients in total) had at the time of primary infection to establish itself amastigotes in bone marrow. In fact, the WHO esti- and develop into a clinical syndrome. This might mates that 2–9% of all AIDS patients in southern also explain the presence of strains which are poorly Europe had developed VL before the HAART era. pathogenic in immunocompetent hosts and even the appearance of non-pathogenic flagellates. This sec-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 79 ond hypothesis takes into account transmission by primary infection. Leishmania transmission in HIV+ patients occurs naturally, by infection through the bite of parasitized sandflies, as in the general population, in the rural or periurban environment, which is commonly where intravenous drug addicts share drugs and syringes. However, in light of the epidemiological and microbiological data, we suggest a second cycle complementary to the conventional one, maintained among intravenous drug addicts who share syringes, a risk group which includes up to 66% of all those infected by HIV. This cycle has been defined as artificial, since syringes substitute for sandflies and metacyclogenesis is unnecessary as amastigotes are transmitted; as epidemic, because of the form of presentation and number of cases; and as anthroponotic, since drug addicts act as parasite reservoirs.

The ease with which sandflies can be infected in the laboratory with the blood of co-infected patients suggests that these immunodepressed subjects serve as true secondary leishmaniasis reservoirs in the natural environment. The epidemiological risk which co- infected individuals pose for the general population is remote, even though we were able to demonstrate by xenodiagnosis that all co-infected patients were infective to sandflies in inverse correlation with the CD4+ count.

80 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 5 IMPROVED STRATEGIES AND POLICIES

5.1 Economic aspects of visceral leishmaniasis control ...... 82

5.2 Climate variability and visceral leishmaniasis in Europe ...... 88

5.3 Tools and strategies for use of insecticide impregnated fabrics in the control of sandflies and leishmaniasis ...... 94 Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 81 5.1 ECONOMIC ASPECTS OF their emergence as a co-infection of HIV/AIDS, they VISCERAL LEISHMANIASIS were not perceived as a direct threat to industrialized countries. The importance of the disease as a CONTROL hindrance for economic development in the South is still not fully recognized, though insiders have Marleen Boelaert little doubt that leishmaniasis is a poverty-related Prince Leopold Institute of Tropical Medicine disease. Antwerp Belgium So far, the field of leishmaniasis control has had very little input from health economists. A few clinical trial reports include pharmaco-epidemiological data (on drug and care costs) and a few cost-effectiveness Leishmaniasis has recently earned public attention studies of competing control strategies have been as one of the “most neglected diseases” in the field published. This paper reviews the available infor- of drug research and development. It is indeed one mation on economic aspects of leishmaniasis con- of those diseases for which we lack effective, afford- trol, with an emphasis on the visceral form of the able, and easy to use drugs, and the pharmaceutical disease, and gives a brief outline of priority research industry has few incentives to engage in their devel- questions in the economic field. opment (Yamey & Torreele, 2002).

The term “neglected” can easily be extended from BURDEN OF DISEASE research to control. At country level, the leishman- Leishmaniasis patients often have poor access to iases are often a hidden problem; patients live in the health system, with gross underreporting of remote areas with poor access to services and case cases as a result. Therefore, it is a hazardous exer- loads are poorly documented. Desjeux (UNDP/ cise to estimate the current number of new cases World Bank/WHO Special Programme for Research and the proportion of all cases who get proper treat- and Training in Tropical Diseases, 1997) puts it this ment. WHO/TDR estimates the burden of disease way “Affected classes of population are those of lowest caused by the leishmaniases at 2 357 000 DALYs1 socio-economic status, who have minimal political power per year and 59 000 deaths annually. Leishmaniasis to influence the decisions of the government and a very is endemic in 88 countries on four continents. More limited capacity to assume the costs of the disease.” than 90% of cutaneous leishmaniasis (CL) cases occur in Iran, Afghanistan, Syria, Saudi Arabia, Brazil At international level, neglected diseases lack visi- and Peru. More than 90% of visceral leishmaniasis bility and the leishmaniases are no exception. Until (VL) cases occur in India, Bangladesh, Brazil, and

Figure 1. Number of kala azar cases 80 000 reported in India (1986–2000) by State Bihar and total 70 000 West Bengal

60 000 India

50 000

40 000

30 000

20 000

Source: compiled from annual reports 1992–2000 10 000 by Ministry of Health and Family Welfare, Govern- ment of India. Data available at http://mohfw.nic. 0 in/reports/index.htm (accessed 27 Jan 2004). 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 98 98 98 98 99 99 99 99 99 99 99 99 99 99 00 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

1 DALY: disability-adjusted life-year i.e. the number of healthy years of life lost to premature death and disability

82 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Sudan (Desjeux, 1996). The total number of reported of productive life are lost due to this severely debil- VL cases in the world was 82 000 in 1992 (Ashford itating disease. Thakur (2000) described the socio- et al. 1992), but Desjeux (1996) found a 1:5 ratio of economic conditions of a cohort of 938 patients of declared to undeclared VL cases in community sur- parasitologically confirmed VL from Bihar in India. veys in India. More recent figures by WHO therefore Seventy-five per cent of those patients were classi- estimate VL incidence at 500 000 new cases per year fied as poor (daily income < US$ 1) and 82% were (UNDP/World Bank/WHO Special Programme for engaged in agriculture and/or animal husbandry. Research and Training in Tropical Diseases, 1997). Seventy-seven per cent lived in mud or grass cov- However, this global figure has not been updated ered houses. Delays between onset of disease and since, and repeating the same incidence figure in all medical care seeking were considerable, as only 33% published documents does not capture the disease of patients attended within 1 month of onset. trends. This is illustrated by Figure 1, which is based on surveillance data from India over the period Adhikari & Maskay (2003) discuss in a letter to the 1986 to 2000. Especially when regions or countries editor an unpublished study of the direct and indi- announce efforts to “reduce the human reservoir rect costs related to kala azar for households in of VL by 2012” or even “to eliminate kala azar by two districts of Nepal. The analysis suggested that 2010”, adequate surveillance data become increas- the total cost (US$ 210) to a sampled household ingly important to guide control efforts. of a kala azar episode consumed almost two and a half times an average annual per capita house- As VL has a very focal distribution, the global fig- hold income (US$ 82). Fourteen out of 18 kala azar ures do not reflect the real impact VL has in affected affected households fell below the absolute pov- communities. VL is a deadly disease if left untreated, erty line (US$ 78/inhabitant/year) with the other and can have disastrous consequences when it 4 not far behind. The results of this household sur- strikes a non-immune population. In the Sudan, a vey indicate that the poor are especially susceptible VL epidemic caused major disruption of a famine- to the disease and that disease-related expenditure stricken society when an estimated 100 000 persons was catastrophic. died between 1984 and 1994 in Western Upper Nile Province (Seaman et al., 1996). Reported incidence An Indian health economist recently looked into the rates of kala azar in endemic communities vary household costs related to kala azar by surveying between 2/1000 person-years in Kenya (Schaefer et kala azar patients in villages in Bihar (n= 31) and al., 1995), to 14/1000 person-years in Ethiopia (Ali & West Bengal (n= 37) (Annigeri, unpublished report). Ashford, 1994), to, in a community in eastern Sudan, Mean annual income of patients was US$ 439.2 an incidence rate of 40/1000 person-years (Zijlstra and 191.2 respectively, while their mean expendi- et al., 1994). ture on the kala azar episode was US$ 401.6 and 59.8 respectively. The differences between the states could be related to observed differences in duration COST-OF-ILLNESS STUDIES of illness and in care-seeking behaviour of patients. Despite the considerable burden of disease caused Both might be explained by differences in accessibil- by the leishmaniases, there has been little attempt to ity of the health system. Annigeri relates that Bihar quantify the economic implications of the disease in patients had to borrow 47 per cent and West Bengal affected communities. Dedet et al. (1991) studied the patients 81 per cent of total treatment expenditure. socioeconomic cost of cutaneous leishmaniasis in French Guyana where it is considered a serious hin- COST-EFFECTIVENESS ANALYSIS OF drance for economic development of the forest area. The authors estimated the direct costs for health COMPETING CONTROL STRATEGIES services related to the care of 175 patients in 1979– A few studies have addressed the choice of most 1980 at FF 3216 per case (approximately US$ 480 per cost-effective strategy for VL control. The first pub- case). Interestingly, hospitalization costs accounted lished cost-effectiveness analysis (CEA) study tried for 82% of the total cost, more related to the need to answer the question of whether VL control was for supervised administration of the relatively toxic cost-effective in an emergency setting in compari- drugs (meglumine antimoniate and pentamidine) son to other relief measures (e.g. diarrhoea control). than to the condition itself. Wijeyaratne et al. (1994) Griekspoor et al. (1999) estimated the cost-effective- have drawn attention to the tremendous barrier ness ratio of VL treatment in a relief programme the visceral form of the disease (VL) poses to the in Sudan at US$ 18.40 per DALY averted (uncer- development efforts of affected communities, with- tainty range between US$ 13.53 and US$ 27.63), and out quantifying their observation. In every mem- US$ 595 per life saved. The average cost of this pro- ber of a family where a VL case occurs, many days gramme was US$ 394 per VL patient treated. As

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 83 any health intervention costing less than US$ 25 per the conclusion remained the same when prices of DALY averted is considered ’very good value for generic drugs were used instead. The overall cost of money’ by the World Bank, the authors concluded test-treatment strategies was much more dependent that in the local conditions, their programme was on the cost of treatment than on the cost of testing. cost-effective. Calculations were based on the use In a subsequent paper, the same authors performed of branded pentostam, at an average cost of US$ a further sensitivity analysis including a variety 100 per patient. Since that time, the nongovernmen- of scenarios for potential improvements in drug tal organization has switched to generic antimonials, therapy, which did not alter the above conclusion which reduce the drug cost by a factor 5. (Boelaert et al., 2002). Whenever splenic aspiration is not feasible, a serological test-treatment strategy In 1999, for an M.Sc thesis in economics, S. Pokhrel remains the optimal choice, even when better drugs of Chulalongkorn University in Thailand compared become available. patient and provider costs related to two alternative programmes: outreach case detection, and Recently a number of therapeutic breakthroughs health facility based detection. Patients’ costs were have occurred, such as the adoption of generic anti- assessed by interviewing a sample of 50 VL cases monials for VL control in East Africa (Veeken et al., detected by the two programmes in 1998–99. Median 2000), the clinical development of miltefosine as the costs to patients in the outreach programme were first oral drug for VL (Sundar et al., 2002), the eval- found to be US$ 25, but were six times higher in the uation of low-cost formulations of lipid formula- health facility based programme (US$ 146). Indirect tions of amphotericin B (Sundar et al., 2004), and the costs due to absence from work accounted for more evaluation of paromomycin in combination therapy than half the costs in both programmes. The study with short-course sodium stibogluconate (Thakur et showed that the outreach programme detected 40.9 al., 2000b). So far, only limited cost data are avail- cases per 100 000 population at risk while the health able to compare regimens; a formal CEA could facil- facility based programme detected 34.1 cases. The itate the decision on optimal therapeutic regimen. cost per case detected in the outreach programme See Table 1 for an overview by V. Vanlerberghe et was US$ 124 and in the health facility based pro- al. of published cost and efficacy estimates of avail- gramme it was US$ 191. The outreach and health able drug regimens, most of them obtained in clin- facility based programmes incurred US$ 131 and ical trials. The table shows that efficacy values are US$ 200 respectively for aversion of one death. quite comparable between the different regimens, hence the importance of careful costing of drugs and Boelaert et al. (1999) used CEA to address the ques- related care. The mode and duration of administration of most appropriate diagnostic test strategy tion play a crucial role, as the cost of care has a huge for VL if splenic aspirates are not feasible. The fol- impact on the overall cost-effectiveness ratios. In lowing strategies were compared: treatment of this respect, we should be very cautious, as the cost- all suspects on clinical grounds (strategy A); paring of patient care was carried out in hospital set- asitological investigation of bone marrow aspirate tings and this may not reflect the reality of a first-line (strategy B); two-step testing by means of the direct health service. Also, effectiveness in real-life condi- agglutination test (DAT) followed by treatment of tions might be quite different from efficacy obtained patients with high titres as well as those with par- in clinical trials, and moreover, compliance and thus asitologically confirmed borderline titres (strategy effectiveness is dependent on duration of regimen. C); and a single serological test, the DAT (strategy Last but not least, the data in the table apply only to D). The results for each strategy were expressed in HIV-negative patients, and the effectiveness of VL cost in US$ per life saved. The most effective strat- drugs in HIV co-infected patients is not at all com- egy in terms of number of lives saved was strategy A parable. (treating all suspects). The effectiveness of strategies C and D was close to that of strategy A and far better than that of strategy B. Diagnosis by parasitological investigation (strategy B) was the most cost-effective strategy at US$ 448 per life saved. As the cost- effectiveness ratio of serological diagnosis (US$ 465 per life saved) was not substantially higher than that of parasitological diagnosis (incremental cost-effectiveness ratio US$ 491 per life saved), the authors recommended serology because of its higher effectiveness. Though the baseline analysis was prepared using the cost of branded antimonials (Pentostam®),

84 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Seaman et al.,1993; Thakur et al., 2000 al., et Thakur al.,1993; et Seaman Meyerhoff, 1999 Meyerhoff, Sundar et al.,1997; Sundar et al., 2004; 2004; al., et Sundar al.,1997; et Sundar 2000 al., et Sundar 2000; Murray, Thakur et al., 2000; Murray, Murray, 2000; al., et Thakur 2000 Murray, 2001; Sundar et al., 2002(a); Sundar et al., al., et Sundar 2002(a); al., et Sundar 2004 al., et Bhattacharya 2003; References Sundar et al., 2002a; Sundar et al., 2004; 2004; al., et Sundar 2002a; al., et Sundar 2001; al., et Sundar 1999; al., et Thakur 2000 Murray, 1997; al., et Sundar Sundar et al., 2001; Sundar et al., 1998 al., et Sundar 2001; al., et Sundar al., et Sundar 2002(b); al., et Sundar et Sundar 1998; al., et Sundar 2001; al., et Sundar 2000; Murray, al.,1997; 2003 al., et Syriopoulou 2004; Seaman et al., 1993; Griekspoor et al., al., et Griekspoor 1993; al., et Seaman al., et Sundar 2000; al., et Veeken 1999; et Ritmeijer 2000; al., et Thakur 2000; 2001 Murray, 2000; Murray, 2001; al., (US$) 4 40 (range) (82–102) 82 (82–95) 82 82 (82–102) 82 75 (75–244) 75 35 (35–480) 35 112 (112–359) 112 240 ( 240–370) ( 240 Care Care cost

3 250 (US$) (110–283 ) (110–283 (range) 7 42 (42–72) 42 Drug cost (250–3760) 90 (90–220) 90 22 (22–200) 22 110 500 (500–1880) 500 1050 (1050–3948) 1050

2 92% 100% 90–94% (range) Efficacy (78–100%) 91% (70–91%) 91% 94% (88–94%) 94% 97% (96–99%) 97% 92% (90–96%) 92% 92% (36–96%) 92% 5 10 21 15 70 600 5–20 SSG 420 SSG PM 252+ 252+ PM (mg/kg) Total dose Total

6 8 5 2.5 MKD, per os, 28 days 28 os, per MKD, 2.5 5 mg/kg, single infusion single mg/kg, 5 Other regimens regimens Other PM 12 MKD + SSG 20 MKD, 21 days IM days 21 MKD, 20 SSG + MKD 12 PM Regimen 20 MKD for 30 days IM days 30 for MKD 20 2 MKD, 5 days 5 MKD, 2 Various regimens Various 7 on given mg/kg 21 FDA: period days 21 over days 1 MKD, 15 infusions on alternate days alternate on infusions 15 MKD, 1 Miltefosine SSG/paromomycin Drug Sodium stibogluconate Lipid formulations of Lipid formulations B amphotericin (Ambisome®, Abelcet®) Amphotericin B Amphotericin Efﬁcacy estimated as proportion of cases with negative parasitology /no clinical signs at end six months follow-up period Efﬁcacy estimated as proportion All cost values computed for a 50 kg patient is included : cost of a hospitalization day (4 US$), doctor’s In the cost of care fees, laboratory costs, infusion sets, syringes/ alternatively: intravenous (IV), 28 days alternatively: 1 MKD, 20 daily infusions (accessed 27 Jan 2004) http://stuenings-nt.stuenings.de/medeor/order/englisch/newsletter/leishmaniose alternatively: PM 18 MKD + SSG 20 MKD, 21 days or 15 17 Baseline value and range of efficacyavailable of currently range of and estimatesvalue Baseline 1. Table patients immunocompetent in leishmaniasis visceral chemotherapyagainst 2 3 4 Based on costing data of India/Nepal (S. Rijal, personal communication) needles, adjuvant treatment. 5 6 7 8 MKD = mg/kg/day; SSG = Sodium stibogluconate; PM = paromomycin; IM = intramuscular; FDA = Food and Drug Administration Drug and Food = FDA intramuscular; = IM paromomycin; = PM stibogluconate; Sodium = SSG mg/kg/day; = MKD

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 85 CONCLUSION: SOCIOECONOMIC to take place. Furthermore, if the efficiency of RESEARCH QUESTIONS IN THE alternative methods of vector control and con- AREA OF VISCERAL LEISHMANIASIS trol of the animal reservoir was better known, CONTROL this might provide leverage for intensification of leishmaniasis control efforts. To support control efforts and to argue the case for greater investment in this disease, there is a true If we want to translate the substantial gains in scien- need for undertaking socioeconomic studies. We tific knowledge and tools for leishmaniasis control suggest that the following issues should be tackled: achieved over the past decade into demonstrable • A more accurate estimate of the burden of changes in disease burden, it seems necessary to leishmaniasis and of disease trends is required invest in more accurate monitoring of disease trends, for reasons of advocacy as well as management in better advocacy for disease control within the of control efforts. The fact that the disease is so global aim of poverty reduction, and in more ratio- under-reported and under-recognized has hin- nal allocation of resources to the most cost-effective dered its profile at both national and interna- control strategies. tional level; it has also prevented health delivery systems in affected countries from allocat- ing the appropriate resources for disease con- References trol. Control efforts will not become coordinated Adhikari SR, Maskay NM. The economic bur- until proper surveillance data are fed into the den of kala-azar in households of the Danusha and management cycle; in this respect, a regional Mahottari districts of Nepal. Acta Tropica, 2003, 88:1–2. approach to surveillance might well be justified Ali A, Ashford RW. Visceral leishmaniasis in Ethiopia. in some endemic areas, as disease foci are often IV. Prevalence, incidence and relation of infection to situated across borders. disease in an endemic area. Annals of Tropical Medicine • Micro-economic studies (household costs/cost- and Parasitology of-illness studies) should demonstrate much , 1994, 88:289–293. more clearly that leishmaniasis is a poverty- Ashford DA, Desjeux P, de Raadt P. Estimation of pop- related disease, and how it impacts on develop- ulation at risk of infection and number of cases of ment. The millennium goals with their emphasis leishmaniasis. Parasitology Today, 1992, 8:104–105. on poverty reduction provide a unique win- Bhattacharya SK et al. Efficacy and tolerability of dow of opportunity to raise awareness on miltefosine for childhood visceral leishmaniasis in the neglected diseases at international level. India. Clinical Infectious Diseases, 2004, 38:217–221. Whereas in the past, ill-health was seen as the consequence of poverty, there is growing con- Boelaert M et al. Cost-effectiveness of competing diag- sensus amongst macro-economists that disease nostic-therapeutic strategies for visceral leishmani- in fact is a root cause of poverty (Canning, 2003). asis. Bulletin of the World Health Organization, 1999, Hence the strategic importance of research to 77:667–674. demonstrate (the obvious): the link between Boelaert M, Le Ray D, Van der Stuyft P. How better leishmaniasis and poverty. drugs could change kala-azar control. Lessons from • Cost-effectiveness analysis of competing a cost- effectiveness analysis. Tropical Medicine and choices in VL control strategies could provide International Health, 2002, 7:955–959. welcome advice in difficult choices on resource allocation within control programmes. The few Dedet JP, Pillot B, Gentilini M. [Evaluation of the soci- studies we retrieved discuss competing choices oeconomic costs of cutaneous leishmaniasis in French for test-treatment of VL, which is indeed the Guiana]. Revue d’epidemiologie et de sante publique, 1991, principal control strategy. One of the main lim- 39:129–133. itations for CEA in this respect is that reliable Desjeux P. Leishmaniasis. Public health aspects and cost data are lacking. The published clinical tri- control. Clinics in Dermatology, 1996, 14:417–423. als include some cost data, but patient care costs Griekspoor A, Sondorp E, Vos T. Cost-effectiveness in tertiary care centres cannot be extrapolated analysis of humanitarian relief interventions: viscer- to district health systems. As the optimal choice al leishmaniasis treatment in the Sudan. Health Policy of drug regimen is so sensitive to patient care and Planning, 1999, 14:70–76. costs, it is important to elucidate this question. These CEA analyses should not be limited to the Meyerhoff A. US Food and Drug Administration choice of a drug regimen or the choice of a diag- approval of AmBisome (liposomal amphotericin B) nostic test, but relate to the overall context of the for treatment of visceral leishmaniasis [see comments]. health system in which case management has Clinical Infectious Diseases, 1999, 28:42–48.

86 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Murray HW. Treatment of visceral leishmaniasis (kala- Sundar S et al. Amphotericin B treatment for Indian azar): a decade of progress and future approach- visceral leishmaniasis: conventional versus lipid for- es. International Journal of Infectious Diseases, 2000, mulations. Clinical Infectious Diseases, 2004, 38:377–383. 4:158–177. Syriopoulou V et al. Two doses of a lipid formulation Murray HW. Clinical and experimental advances of amphotericin B for the treatment of Mediterranean in treatment of visceral leishmaniasis. Antimicrobial visceral leishmaniasis. Clinical Infectious Diseases, 2003, Agents and Chemotherapy, 2001, 45:2185–2197. 36:560–566. Ritmeijer K et al. Ethiopian visceral leishmania- Thakur CP et al. Amphotericin B deoxycholate treat- sis: generic and proprietary sodium stibogluconate ment of visceral leishmaniasis with newer modes of are equivalent; HIV co-infected patients have a poor administration and precautions: a study of 938 cases. outcome. Transactions of the Royal Society of Tropical Transactions of the Royal Society of Tropical Medicine and Medicine and Hygiene, 2001, 95:668–672. Hygiene, 1999, 93:319–323. Seaman J et al. Epidemic visceral leishmaniasis in Thakur CP. Socio-economics of visceral leishmania- Sudan: a randomized trial of aminosidine plus sodi- sis in Bihar (India). Transactions of the Royal Society of um stibogluconate versus sodium stibogluconate Tropical Medicine and Hygiene, 2000, 94:156–157. alone. Journal of Infectious Diseases, 1993, 168:715–720. Thakur CP et al. A prospective randomized, com- Seaman J, Mercer AJ, Sondorp E. The epidemic of vis- parative, open-label trial of the safety and efficacy of ceral leishmaniasis in western Upper Nile, south- paromomycin (aminosidine) plus sodium stiboglu- ern Sudan: course and impact from 1984 to 1994. conate versus sodium stibogluconate alone for the International Journal of Epidemiology, 1996, 25:862–871. treatment of visceral leishmaniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2000, Schaefer KU et al. A prospective sero-epidemiological 94:429–431. study of visceral leishmaniasis in Baringo District, Rift Valley Province, Kenya. Transactions of the Royal Society UNDP/World Bank/WHO Special Programme for of Tropical Medicine and Hygiene, 1995, 89(5):471–5. Research and Training in Tropical Diseases. Tropical Disease Research: progress 1995–1996. Thirteenth pro- Sundar S et al. Short-course, low-dose amphotericin B gramme report of the UNDP/World Bank/WHO Special lipid complex therapy for visceral leishmaniasis unre- Programme for Research and Training in Tropical Diseases. sponsive to antimony. Annals of Internal Medicine, 1997, Geneva, World Health Organization, 1997. 127:133–137. Veeken H et al. A randomized comparison of branded Sundar S et al. Treatment of antimony-unresponsive sodium stibogluconate and generic sodium stiboglu- Indian visceral leishmaniasis with ultra-short cours- conate for the treatment of visceral leishmaniasis es of amphotericin-B-lipid complex. Annals of Tropical under field conditions in Sudan. Tropical Medicine and Medicine and Parasitology, 1998, 92:755–764. International Health, 2000, 5:312–317. Sundar S et al. Short-course, cost-effective treatment Yamey G, Torreele E. The world’s most neglected dis- with amphotericin B-fat emulsion cures visceral leish- eases. British Medical Journal, 2002, 325:176–177. maniasis. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2000, 94:200–204. Zijlstra EE et al. Endemic kala-azar in eastern Sudan: a longitudinal study on the incidence of clinical and Sundar S et al. Treatment of Indian visceral leish- subclinical infection and post-kala-azar dermal leishmaniasis with single or daily infusions of low dose maniasis. American Journal of Tropical Medicine and liposomal amphotericin B: randomised trial. British Hygiene, 1994, 51:826–836. Medical Journal, 2001, 323:419–422. Wijeyaratne PM, Arsenault LK, Murphy CJ. Endemic Sundar S et al. Oral miltefosine for Indian visceral disease and development: the leishmaniases. Acta leishmaniasis. New England Journal of Medicine, 2002(a), Tropica, 1994, 56(4):349–64. 347:1739–1746. Sundar S et al. Low-dose liposomal amphotericin B in refractory Indian visceral leishmaniasis: a multicenter study. American Journal of Tropical Medicine and Hygiene, 2002(b), 66:143–146. Sundar S et al. Oral miltefosine treatment in children with mild to moderate Indian visceral leishmaniasis. The Pediatric Infectious Disease Journal, 2003, 22:434–438.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 87 5.2 CLIMATE VARIABILITY AND factors on disease risk, the possible impacts of future VISCERAL LEISHMANIASIS climate, and future research needs. IN EUROPE1 CURRENT DISTRIBUTION AND RISKS 1 2 3 Elisabeth Lindgren , Torsten Naucke , Bettina Menne In Europe, VL is considered to be a rare disease, 1 Department of Systems Ecology, Stockholm University, although its incidence increased significantly in Sweden the region during the 1990s. This was due in part 2 Institute for Medical Parasitology, Bonn University, to the large proportion of Leishmania/HIV co-infec- Germany tions (approximately 25%–70% of adult VL cases are 3 Global Change and Health, WHO European Centre co-infected with HIV) and to better reporting after for Environment and Health, Via Francesco Crispi 10, the establishment of a WHO surveillance network Rome 00187, Italy in 1994 (Desjeux et al., 2000). Non-HIV VL incidence rates have increased in Italy and France (including a four-fold increase in Alpes-Maritimes), and new endemic areas have been detected where no previous INTRODUCTION autochthonous cases had been reported, e.g. in northern Italy, North Croatia, Switzerland and Germany. A three-year pan-European project entitled Climate change and adaptation strategies for human health The distribution of leishmaniasis worldwide is lim- (cCASHh), funded under the European Union the- ited by the distribution of its vector sandfly and by matic programme on Energy, Environment and the latter’s susceptibility to cold climate, ability to Sustainable Development (EESD-1999), began in support internal development of specific species of 2000. The aim of this project was to provide a sci- Leishmania, and tendency to take blood from ani- entific basis for the development within the health mals or humans only (WHO, 2000[b]). Populations sector of response strategies to climate variability of Phlebotomus perniciosus, one of the known vectors and change. The project included academic partners of Mediterranean VL, have been found as far north in the United Kingdom, Sweden, the Netherlands, as Paris (Parrot, 1922), and recently also in Gerweiler Germany, Italy, and the Czech Republic. in Germany (Naucke & Schmitt, 2004). Table 1 lists the distribution of the different species that may act This paper reports the findings of a review of impact as vector for L. infantum, the pathogen that causes of climate variability and change on leishmaniasis, VL in Europe. and the results of discussions at a meeting on vector- borne diseases and climate change held in Prague, Since the mid-1990s, the worldwide geographical 5–7 May 2003. A literature search was conducted to distribution of endemic leishmaniasis has expanded answer questions about the current distribution of (WHO, 2000[a]). This spread is probably due to a visceral leishmaniasis (VL) in the WHO European combination of factors, among them increased mon- Region, the influence of environmental and climatic itoring, intensified research, demographic change,

Table 1. Distribution in Europe of sandfly Vector Countries species that may act as P. ariasi France, Italy, Portugal, Spain vector for L. infantum, P. neglectus Albania, Greece, Italya, Malta, Romania, former Socialist Republic of Yugoslaviab, southern Austriac the cause of European b visceral leishmaniasis P. perfiliewi Albania, Greece, Italy, former Socialist Republic of Yugoslavia P. perniciosus France (incl. Corsica), Germany, Italy, Malta, Portugal, Spain, southern Austriac P. tobbi Albania, Cyprus, Greece, Italy (Sicily), former Socialist Republic of Yugoslaviab P. mascittii d Belgium, Germany, France (incl. Corsica), Italy, Monaco, former Socialist Republic of Yugoslaviab, Spain, Switzerland

a Maroli et al., 2002. b Bosnia and Herzegovina, Croatia, Macedonia, Yugoslavia. c Suspected geographical distribution because of autochthonous VL cases (Dornbusch et al., 1999; Kollaritsch et al., 1989; Beyreder, 1965). d Suspected vector species because of autochthonous VL cases in humans and animals in Germany (Deplazes & Mettler, 2003; Naucke & Pesson, 2000; Naucke & Schmitt, 2004; Köhler et al., 2002; Bogdan et al., 2001; Gothe, 1991).

1 Extract from: Leishmaniasis: inﬂuences of climate and climate change epidemiology, ecology and adaptation measures. Copenhagen, WHO Regional Ofﬁce for Europe, 2004.

88 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 land-use/land cover changes that create new hab- mitted in the adjoining countries of Azerbaijan, itats and/or changes in microclimate, and changes Cyprus, Georgia, Kazakhstan, Tajikistan, Turkey, in seasonal climate. The overlap between geograph- Turkmenistan and Uzbekistan. Figure 1 shows the ical areas with high risk of both leishmaniasis and regional distribution of VL compared to cutaneous HIV is increasing with the spread of leishmania- leishmaniasis (CL). sis (typically a rural disease) into urban areas and the increased spread of HIV into rural areas (WHO, Most cases of co-infection in Europe are reported 2000[a]). from the most densely populated areas and prov- inces and, as shown in Figure 2, there is a pre- Within the European Region, cases of VL have been dominance of cases in coastal areas (75%). In reported from Albania, Bosnia & Herzegovina, south-western Europe, 80% of co-infection cases are Bulgaria, Croatia, France, Greece, Hungary, Italy, from urban areas, the main cities being Lisbon and Macedonia, Malta, Monaco, Portugal, Romania, Porto in Portugal; Barcelona, Granada, Madrid and Spain, and Yugoslavia. Leishmaniasis is also trans- Seville in Spain; Marseille and Nice in France; and

Figure 1. Approximate distribution in Europe of visceral leishmaniasis and its vectors compared to cutaneous leishmaniasis and its vectors

Note: Cases of VL reported from the Paris metropolitan area were probably the result of infection elsewhere.

= distribution of visceral leishmaniasis = distribution of cutaneous leishmaniasis = distribution of visceral leishmaniasis = distribution of cutaneous leishmaniasis vector vector sandflies sandflies

Figure 2. Cases of Leishmania/HIV

�

�� co-infection per locality and population �� density, 1990–1998. �

� � � � � � � � ��

� ��

�

� � � � � � �

� �� N � � 70 0 70 140 Kilometers � � � �� Global distribution of leishmaniasis Canary Islands Global distribution of Leishmania/ HIV co-infection � Modified from Desjeux et al., 2000

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 89 Genoa, Milan and Catania (Sicily) in Italy. This dis- Most of the co-infection cases have been reported tribution pattern, however, is probably partly related from France, Italy, Portugal and Spain, but co-infec- to the current location of the WHO surveillance cen- tion is prevalent in Albania, Croatia and Greece as tres (which specifically monitor Leishmania/HIV co- well. On average, the number of reported cases of infections) rather than to other factors. Most of the Leishmania/HIV co-infection increased during the centres are located near cities like Rome and Catania late 1990s relative to the number of reported HIV in Italy; Madrid, Barcelona, Seville, Bilbao, Granada cases in the same period (Desjeux et al., 2000). and Palma de Mallorca in Spain; Paris, Marseille, However, the number of cases of co-infection has Montpellier and Nice in France; and Lisbon in recently started to fall in southern Europe owing Portugal. to the use of new therapeutic methods, i.e. highly active antiretroviral therapies (HAARTs) (Table 2). Populations at risk include people living in rural and periurban areas where both sandflies and reservoir INFLUENCE OF ENVIRONMENTAL animals are prevalent. VL used to be found predom- inately in children but in recent years an increas- AND CLIMATIC FACTORS ON ing proportion of adult cases (non-HIV) has been DISEASE RISK reported. This change in age distribution is probably The distribution of VL in Europe is significantly less caused by several factors, such as changes in human than the distribution of the sandfly vectors. The exposure patterns, environmental changes, and occurrence of disease transmission within the range improvements in case diagnosis and notification. of the vectors depends on vector abundance, vector Improvements in nutritional and general health sta- survival, vector biting rate (i.e. gonotrophic cycle), tus in European children have probably played a the extrinsic incubation period, and the length of roll in reducing the characteristic high susceptibility the transmission season. Each of these parameters of children to this disease. is climate dependent, but the precise relationship with climate needs to be further studied and eval- The sandfly vector is mainly active during the night, uated. Caution is also required in the interpretation and the highest risk for contracting the disease from of laboratory experiments as sandflies are able to sandfly bites is therefore between dusk and dawn. evade extreme weather conditions in the field by their choice of resting site and time of activity. Urban areas have become high-risk locations of late. High-risk populations in urban environments Temperature and humidity are the two most impor- are people who are HIV-positive. In Europe, 77% tant climatic factors for sandfly survival, develop- of Leishmania/HIV co-infected patients are aged ment and activity. Phlebotomus sandflies can survive between 31 and 50 years, and 83% of them are men cold temperatures in diapause (overwintering), (WHO, 1997). Another risk group is intravenous which is initiated by a combination of low tempera- drug users who share syringes (Cruz et al., 2002; ture and reduced daylight and can last 4 to 8 months Pineda et al., 2002; Amela et al., 1996). The highest depending on location. In Europe, the biting activ- risk for VL is found among HIV-infected intrave- ity of sandflies is strongly seasonal, and restricted nous drug users, who account for 71% of co-infec- to the summer months in most areas. Adult activity tion cases (Desjeux et al., 2000). as well as larval development slows down considerably below 20°C. However, some species are active In addition, Leishmania can be transferred through even at much lower temperatures, e.g. P. neglectus in blood transfusion (Kubar et al., 1997); the trans-pla- Greece at 13°C and P. mascittii in Germany at 13.5°C cental route of infection is also possible (Meinecke (Naucke, 1998; Schmitt, 2002). et al., 1999).

Leishmania/HIV co-infections accelerate and aggra- vate both leishmaniasis and AIDS symptoms (Desjeux et al., 2000). Also, relapses of leishmania- Table 2. Number of reported cases of VL/HIV co-infection sis after treatment are common among persons with in southern Europe, 1990–2001 HIV co-infection. The mean survival of co-infected patients is only 13 months (WHO, 2000[b]). The Country 1990–1995 1996–1998 1999–2001 current number of HIV-infected persons in west- France 127 132 59 ern and eastern Europe (not including the Russian Italy 144 85 106 Federation) is approximately 580 000 (WHO, 2001). Portugal 29 88 42 In the Mediterranean basin, 1.5%–9% of AIDS Spain 473 412 214 patients develop VL (WHO, 2000[a]).

90 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Temperature also affects the parasite itself; evidence (2) the local vegetation (important as resting sites indicates that L. infantum is prevalent within the 5– and sugar sources); and (3) the patterns of human 10°C January and 20–30°C July isotherms, usually exposure to sandfly vectors. located below latitude 45 N and below 400–600 m above sea level (Kuhn, 1999). The worldwide distri- POSSIBLE IMPACT OF FUTURE bution of sandflies is considered to be confined to areas that have at least one month with a mean tem- CLIMATE RISKS perature of 20°C (WHO, 1984). However, it has been With climate change, the distribution range of both suggested that the limit of the European distribution the sandfly vector and the pathogen may extend of vectors and parasites better corresponds to the northwards and into higher altitudes (WHO, 1999). 10°C annual isotherm (Maier et al., 2003). This is in In currently endemic areas, higher seasonal temper- accordance with recent findings of vectors and iso- atures would lead to prolonged activity periods and lated autochthonous cases of leishmaniasis in south- shorter diapause periods. This could result in an ern Germany, where the July isotherm is 16°C to increased number of sandfly generations per year. 18°C. The highest focus of leishmaniasis in Europe In addition, higher temperatures are likely to accel- is the Guadix focus (Andalusia, Spain), at an alti- erate maturation of the protozoan parasite, thereby tude of 900 to 950 m above sea level. increasing the risk of infection (McCarthy et al., 2001; Rioux et al., 1985). However, if the climate becomes Sandflies are sensitive to sudden temperature too hot and dry for the vector to survive, the disease changes and usually prefer regions with small differ- may disappear from some localities even though the ences between the maximum and minimum temper- vector may adapt by resting in cool, humid places ature. Sandfly survival can be reduced if the climate during the daytime. gets too hot and dry, even though the flies may rest in cold, humid places during the daytime (McCarthy Imported infected dogs can contribute to the emer- et al., 2001). The resting sites of adults are known for gence of leishmaniasis in new locations. They are a few species of sylvatic sandfly; they include tree a potential source of the pathogen if the vector holes and trunks. Peridomestic species rest on walls expands its geographical distribution due to change and, at hot times of the day, retreat into cracks and in climate (WHO, 1999). Several imported cases of crevices (WHO, 1984). Poroton stone buildings, for canine leishmaniasis are, for example, reported from example, have the ability to store humidity during Germany (Gothe et al., 1997) and the Netherlands the night and evaporate it during the day, produc- (Slappendel, 1988) every year. ing favourable conditions for adult sandflies to survive the hot, dry summer days. In order to develop more sophisticated climate–vector–disease scenario models, increased knowledge In addition to the direct association between climate is needed on the short- and long-term effects of cli- and leishmaniasis transmission, climate has indirect mate variation on leishmaniasis risk. This can be impacts by influencing (1) the distribution of hosts; achieved in several ways: (1) experimental evidence

Table 3. Important Vector Minimum Optimum Maximum Preferred Preferred Causative Phlebotomus species temperature temperature temperature humidity altitude organism in Europe and their (larvae) (larvae) (larvae) (adults) climate thresholds P. perniciosus 0°C 25°C <900 m L. infantum a P. perfiliewi -4°C 25°C 33°C 60–80% <450 m L. infantum a P. ariasi 5 C b 30°C b 300–500 m c L. infantum

P. neglectus -4°C 25°C 30°C 60–80% 300–1000 m L. infantum a P. papatasi 5°C 28–34°C c 35°C 36–45% c 0–1000 m L. infantum a -4°C (Greece) P. sergenti 31–33°C c 0–45% c L. tropica b

P. mascittii d -4°C 19°C 28°C 60–80% 0–400 m

a Killick-Kendrick, 1999; b Rioux et al., 1985; c Singh, 1999; d Naucke & Schmitt, 2004.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 91 measuring the influence of temperature or humid- References ity (for example) on sandfly or Leishmania biology; Amela C et al. Injecting drug use as risk factor for vis- (2) field evidence that sandfly biological parame- ceral leishmaniasis in AIDS patients. European Journal ters vary with climate; (3) field evidence that geo- of Epidemiology, 1996, 12:91–92. graphic variation in sandfly abundance (including presence vs. absence) or Leishmania infection rate Beyreder J. Ein Fall von Leishmaniose in correlates with climatic variables; and (4) field evi- Niederösterreich. [A case of Leishmania in dence that temporal variation in sandfly abundance Niederösterreich] Wiener Medizinische Wochenschrift, or Leishmania infection rate (including seasonal 1965, 115:900–901. and long-term trends) correlates with climatic vari- Bogdan C et al. Visceral leishmaniasis in a German ables. As different sandfly species are responsible child who had never entered a known endemic area: for Leishmania transmission across Europe, climate Case report and review of the literature. Clinical change will have different impacts on VL risk in dif- Infectious Diseases, 2001, 32:302–306. ferent parts of the region. Cruz I et al. Leishmania in discarded syringes from Regional climate models for leishmaniasis should intravenous drug users. Lancet, 2002, 359:1124–1125. therefore be constructed according to the local vec- Deplazes P, Mettler M. Epidemiologische und kli- tor species. nische Aspekte der caninen Leishmaniose in Zentraleuropa. Tagung der DVG-Fachgruppe. FUTURE RESEARCH NEEDS [Epidemiology and clinical aspects of dog leishmania in Central Europe.] Parasitologie und Parasitäre There is a need for further research on visceral leish- Krankheiten, Leipzig, 20–21 March 2003. maniasis and its vectors in Europe. Better understanding of the current situation would allow more Desjeux P et al. Leishmania/HIV co-infection in south- specific risk evaluation and form the basis for pre- western Europe 1990–1998. Retrospective analysis of 965 dicting change in distribution and endemicity due cases. Geneva, World Health Organization, 2000 (doc- to environmental and climatic changes in different ument WHO/LEISH/2000.42). parts of Europe. Some main issues are to: Dornbusch HJ et al. Viszerale Leishmaniose bei einem 10 Monate alten österreichischen Mädchen: • Collate available data on sandfly and parasite XXXIII. Tagung der Österreichischen Gesellschaft für distribution, and sandfly seasonality, in relation Tropenmedizin und Parasitologie. [Visceral Leishmania to information and data on climate and environ- in a 10 months old girl in Austria. XXXIII. Meeting mental variables. of the Austrian Society of Tropical Medicine and • Collect new field data on sandfly biological Parasitology], Innsbruck 18–20 November 1999. parameters (including activity patterns, sea- Gothe R. Leishmaniasis in dogs in Germany: sonality, survival, infection rates) in relation to Aethiology, biology, epidemiology, clinic patho- defined climatic and environmental variables – genesis, diagnosis, therapy and disease prevention. latitude and altitude transects over full seasons. Develop a network of fieldworkers across Kleintierpraxis, 1991, 36:69–84. Europe able to use standard methodology for Gothe R, Nolte I, Kraft W. Leishmaniasis of dogs in measuring these parameters (especially infec- Germany: epidemiological case analysis and alterna- tion rate). tive to conventional causal therapy. Tierarztiche Praxis, • Collect laboratory data on vectorial competence, 1997, 25:68–73. including extrinsic incubation period and dia- Killick-Kendrick R. The biology and control of phle- pause determinants, of different European vec- botomine sandflies. Clinics in Dermatology, 1999, tor species. 17:279–289. • Collect new field data on dog prevalence, and set up a network of veterinarians across Europe Kollaritsch H et al. Suspected autochthonous kala- to use standardized methodology. azar in Austria. Lancet, 1989, 901–902. • Further analysis of HIV-leishmaniasis data. Kubar J et al. Transmission of L. infantum by blood donors. Nature Medicine, 1997, 3:368–368. Köhler K et al. Cutaneous leishmaniosis in a horse in southern Germany caused by Leishmania infantum. Veterinary Parasitology, 2002, 109:9–17.

92 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Kuhn KG. Global warming and leishmaniasis in Italy. WHO. Early human health effects of climate change and Bulletin of Tropical Medicine and International Health, stratospheric ozone depletion in Europe. Copenhagen, 1999, 7(2):1–2. WHO Regional Office for Europe, 1991 (document EUR/ICP/EHCO 02 02 05/15). Maier WA et al. Possible effects of climatic change on the distribution of arthropode (vector)-borne infec- WHO. Leishmaniasis and Leishmania/HIV co-infectious diseases and human parasites in Germany. tion. In: WHO report on global surveillance of epidem- Umweltbundesamt, 2003, 1–386. ic-prone infectious diseases. Geneva, World Health Organization, 2000[a] (document WHO/CDS/CSR/ Maroli M et al. Recent findings of Phlebotomus neglec- ISR/2000.1). tus Tonnoir, 1921 in Italy and its western limit of distribution. Parassitologia, 2002, 44:103–109. WHO. Leishmaniases and Leishmania/HIV co-infections. Geneva, World Health Organization, 2000[b] (WHO McCarthy JJ et al. Climate change 2001. Impacts, adap- Fact Sheet No. 116; http://www.who.int/inf-fs/en/ tation, and vulnerability. Cambridge, Cambridge fact116.html, accessed 26 January 2003). University Press, 2001. WHO. Report on the global HIV/AIDS epidemic 2002 Meinecke CK et al. Congenital transmission of viscer- (http://www.who.int/pub/epidemiology/hiv_aids_ al leishmaniasis (Kala Azar) from an asymptomatic 2001.xls, accessed 23 January 2003). mother to her child. Pediatrics, 19991, 104:1–5. WHO (1997). Leishmania/HIV co-infection. Naucke TJ. Investigation on vector control of phlebot- Epidemiological analysis of 692 retrospective cases. omine sandflies in northeastern Greece. Regensburg, S. Weekly Epidemiological Record, 72:49–54. Roderer Verlag, 1998. Naucke TJ, Pesson B. Presence of Phlebotomus (Transphlebotomus) mascittii Grassi, 1908 (Diptera: Psychodidae) in Germany. Parasitology Research, 2000, 86:335–336. Naucke TJ, Schmitt C. Is leishmaniasis becoming endemic in Germany? International Journal of Medical Microbiology, 2004, 293;37(Suppl.):179–181. Parrot LM. Présence de Phlebotomus perniciosus Newstead dans la région parisienne. Bulletin de la Société de Pathologie Exotique, 1922, 15:694–694. Pineda JA et al. Leishmania spp. infection in injecting drug users. Lancet, 2002, 360:950–951. Rioux JA et al. Ecology of leishmaniasis in the south of France. 21. Influence of temperature on the development of Leishmania infantum Nicolle, 1908 in Phlebotomus ariasi Tonnoir, 1921. Experimental study. Annales de Parasitologie Humaine et Compare, 1985, 60:221–229. Schmitt C. Untersuchungen zu Biologie und Verbreitung von Phlebotomus (Transphlebotomus) mascittii, Grassi 1908 (Diptera: Psychodidae) in Deutschland [thesis]. Bonn, Institut for Medical Parasitologie, 2002. Singh KV. Studies on the role of climatological factors in the distribution of Phlebotomine sandflies (Diptera: Psycodidae) in semi-arid areas of Rajasthan, India. Journal of Arid Environments,1999, 42:43–48. Slappendel RJ. Canine leishmaniasis: A review based on 95 cases in the Netherlands. Veterinary Quarterly, 1988, 10:1–16. WHO. The leishmaniases: report of a WHO Expert Committee. Geneva, World Health Organization, 1984 (WHO Technical Report Series, No. 701).

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 93 5.3 TOOLS AND STRATEGIES to mesh treated with 1 g a.i./m2; biting rates were FOR USE OF INSECTICIDE also reduced in both species. IMPREGNATED FABRICS IN Most studies have utilized polyester or cotton net- THE CONTROL OF SANDFLIES ting material similar to that used for malaria con- AND LEISHMANIASIS trol. Because of their small size however, it is often argued that sandflies can pass through wide-mesh Dia-Eldin A. Elnaiem bednets; on the other hand, there is some concern Department of Entomology that fine-mesh bednets are not acceptable to poten- UC-Davis, CA 95616, tial users in hot climates. Therefore some workers USA have attempted to test the efficacy of impregnated wide-mesh ITNs. Maroli & Lane (1989) found that permethrin-impregnated nets of 1 cm sq. mesh placed over windows significantly reduced the num- The use of insecticide impregnated fabrics (ITNs) bers of P. perfiliewi entering houses in Italy. Similarly, is one of the most effective methods of reducing Alexander et al. (1995), Elnaiem et al. (1994), and man-vector contact and intra- and peri-domiciliary Elnaiem, Elnahas & Aboud (1999) showed that the transmission of vector-borne diseases. During the sandfly biting rate on human volunteers under past two decades, encouraging results on the use impregnated wide-mesh bednets was significantly of ITNs against phlebotomine sandflies and leish- lower than that under untreated bednets. In contrast maniasis have been obtained from several coun- to these findings, Feliciangeli et al. (1995) reported tries, including Italy (Maroli & Lane, 1989), Burkina that sandflies (mainly Lutzomyia ovallesi) were able Faso (Majori et al., 1989), Syria (Tayeh et al., 1997; to pass through 6 mm mesh curtains treated with 2 Desjeux, 2000), Sudan (Elnaiem, 1994, 1997, 1998; deltamethrin at 15 mg/m and bite volunteers. Elnaiem et al., 1999; Elnaiem, Elnahas & Aboud, However, when the mesh size was reduced to 4 mm 1999), Kenya (Mutinga et al., 1992, 1993; Basimike and the concentration of permethrin raised to 60 2 & Mutinga, 1995), Colombia (Alexander et al., 1995) mg/m , all sandflies were killed within 30 minutes and Venezuela (Feliciangeli et al., 1995; Kroeger et after 10 minutes of exposure. al., 2002). This paper reviews the literature on use of ITNs against sandflies and leishmaniasis and EFFECTS OF INSECTICIDE TREATED attempts to highlight the remaining gaps in knowl- FABRICS ON ABUNDANCE, INDOOR edge and the further research needed to be under- RESTING, AND BITING RATES OF taken. SANDFLIES

WHAT INSECTICIDE, WHAT DOSE, Maroli & Lane (1989) provided the first evidence that impregnated window curtains reduce the num- WHAT FABRIC? ber of sandflies entering houses in Italy. Similarly in In most studies, the insecticides used have been Burkina Faso, Majori et al (1989) showed that use synthetic pyrethroids, which combine the proper- of permethrin-impregnated cotton curtains almost ties of low to moderate mammalian toxicity (Wells, completely eliminated the occurrence of endophilic Grayson & Langer, 1986), low volatility, and high Sergentomyia sandflies. These results were substanti- insecticidal activity. Due to their low vapour pres- ated by Elnaiem et al. (1999), who showed that the sure, the pyrethroid insecticides used for impregnat- biting activity indoors and the resting density of P. ing nets do not repel insects, and therefore the ITNs papatasi were significantly reduced in rooms fitted act as “baited traps”. with permethrin impregnated curtains as compared to control rooms left without curtains or fitted with The dose of insecticide used to impregnate nets non-impregnated curtains; no significant difference against sandflies depends on the insecticide used, was found between the number of nocturnally active and has varied in the different trials (Alexander & sandflies captured on sticky paper traps in the inter- Maroli, 2003). In field experiments, Elnaiem, Elnahas vention and non-intervention rooms. Interestingly, & Aboud (1999) found that only 30 seconds of expo- when nocturnal activity was assessed by hourly sure to bednets impregnated with 10 mg a.i./m2 of night trap collection, it was noticed that most sand- lambda-cyhalothrin were sufficient to kill 100% of P. flies entering the intervention rooms died immedi- orientalis within 1 hour of contact. Maroli & Majori ately after capture. It was therefore concluded that (1991) recorded over 90% mortality within 24 h in P. sandflies entering the rooms were not repelled by papatasi and P. perniciosus exposed in the laboratory the permethrin-impregnated curtains but, on entry,

94 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 they picked up a lethal dose of the insecticide that efficacy against anthroponotic cutaneous leishman- prevented their blood-feeding and resulted in sub- iasis (ACL), whereas lambda-cyhalothrin residual sequent death. This particular study raised concerns house spraying resulted in 40% protective efficacy about using sticky paper traps to assess the number (Reyburn et al., 2000). Similarly, in a pilot study in of sandflies entering rooms fitted with insecticide- eastern Sudan, Elnaiem (1997, 1998) found that the impregnated curtains, since this method cannot dis- inhabitants of two villages using bednets impreg- tinguish between healthy and dying individuals. In nated with lambda-cyhalothrin insecticides had sig- contradiction to these findings, Feliciangeli (1995) nificantly less incidence of kala azar as compared to reported that curtains impregnated with deltame- a non-intervention village (1.6% in the intervention thrin at 60 mg/m2 produced no significant reduc- vs. 12.4% in the non-intervention village). The study tion in the number of Lu. ovallesi sandflies entering provided the intriguing result that the ratio of clini- houses in El Ingenio, Mirnada State, Venzuela; the cal to subclinical infection with L. donovani changed authors attributed this failure to low compliance from 7:1 in the non-intervention village to 1:3 in the with the control measure, as discomfort due to high intervention villages, probably due to reduction in night temperatures frequently prompted local res- the transmission of L. donovani and/or malaria co- idents to remove the nets from their windows and infection. The impact of the ITNs was maintained doors. However, more recently, in Trujillo, Venezuela, throughout the three years of the study. However, Kroeger et al. (2002) reported a significant reduction the trial was limited by lack of additional villages in the number of Lu. ovallesi and Lu. youngi entering because of their inclusion in a visceral leishmaniasis houses fitted with lambda-cyhalothrin impregnated (VL) vaccine trial. No other studies on the impact of curtains, as compared to non-intervention houses ITNs on VL are reported. However, in a recent case fitted with un-impregnated curtains. control study in Nepal, people using conventional, untreated, nets were shown to be 70% less likely to Studies on the impact of impregnated bednets have develop visceral leishmaniasis (Bern et al., 2000). focused mainly on the sandfly biting rate, with little assessment of the impact of nets on sandfly density. The impact of insecticide impregnated curtains on In Colombia, Alexander et al. (1995) showed that the incidence of cutaneous leishmaniasis was stud- the sandfly biting rate on volunteers sleeping under ied thoroughly in a cluster randomized trial in impregnated bednets was significantly lower than Trujillo, Venezuela, where most transmission takes on those sleeping under untreated bednets. Similar place within the domestic setting (Kroeger et al., results were obtained by Elnaiem (1994), who found 2002). The authors found that insecticide impreg- that permethrin impregnated bednets reduced the nated curtains provided a high degree of protection biting rate of P. papatasi from 9.4 to 0.2 bites per against indoor transmission of cutaneous leishman- night. In a second study, the authors recorded that iasis. After 12 months of intervention using curtains the number of P. orientalis (vector of L. donovani) impregnated with lambda-cyhalothrin in 13 sectors females landing on human collectors without bed- of the city, the incidence of leishmaniasis dropped nets or using untreated nets averaged 32 and 7 per from 4% to 0%. In comparison, the incidence in 13 man-night respectively, whereas volunteers using control sectors, using untreated curtains, rose from bednets impregnated with lambdacyhalothrin insec- 4.5% to 8%. ticide received no sandfly bites (Elnaiem, Elnahas & Aboud, 1999). Recently some studies have been carried out on the protective efficacy of insecticide impregnated cloth- ing, mainly military uniforms. Soto et al. (1995) EFFECTS OF INSECTICIDE TREATED showed that permethrin-impregnated uniforms FABRICS ON THE INCIDENCE OF reduced the incidence of cutaneous leishmaniasis LEISHMANIASIS (CL) in Colombian soldiers on patrol by 75%. These From the few trials and retrospective studies con- findings, however, were challenged by Asilian et al ducted in different parts of the world, there is strong (2003), who observed that permethrin impregnation evidence that impregnated bednets and curtains of military uniforms at a dose of 850mg/m2 did not can significantly reduce the incidence of leishman- significantly protect soldiers in Iran from CL. iasis (Davies et al., 2003). In a controlled study in Syria, Tayeh et al. (1997) demonstrated a sharp and ACCEPTABILITY OF INSECTICIDE consistent reduction in the incidence of cutane- TREATED FABRICS, AND ous leishmaniasis caused by L. tropica. A random- COMPLIANCE BY LOCAL ized controlled trial in Kabul, Afghanistan, also showed that permethrin treated nets and chadors POPULATIONS (Islamic cloth wraps) each provided 65% protective Little information has been provided on the accept-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 95 ability and other human behavioural factors affect- References ing ITN use against leishmaniasis. However, some Alexander B, Maroli M. Control of phlebotmine sand- studies have provided valuable information on the flies. Medical and Veterinary Entomology, 2003, 17:1–17. efficacy of implementation. In eastern Sudan, for example, a major question was whether bednets are Alexander B et al. Evaluation of deltamethrin-impreg- protective against VL since a great deal of man-vec- nated bednets and curtains against phlebotomine tor contact takes place outdoors, before people go to sandflies in Valle del Cauca, Colombia. Medical and bed. However, a socio-behavioural study indicated Veterinary Entomology, 1995, 9:279–283. that the duration of exposure was significantly asso- Asilian A. Efficacy of permethrin-impregnated uni- ciated with gender as well as age group (Elnaiem, forms in the prevention of cutaneous leishmania- Elnahas & Aboud, 1999). Impregnated bednets were sis in Iranian soldiers. Journal of Clinical Pharmacy and potentially more protective against P. orientalis for Therapeutics, 2003, 28:175–178. women and children than for adult men. Overall, the proportion of people and their duration of expo- Basimike M, Mutinga MJ. Effects of permethrin-treat- sure to risk of sandlfy bites (i.e. after sunset until ed screens on phlebotmine sandflies, with reference to they went to bed) was 40% unprotected for <1h, 50% Phlebotmus martini (Diptera: Psychodidae). Journal of unprotected for 1–2h, and 10% unprotected for >2h. Medical Entomology, 1995, 32:428–432. Most children in the age groups of less than 5 years Bern C et al. Factors associated with visceral leish- and 6–15 years were potentially exposed for <1 h maniasis in Nepal: bed-net use is strongly protective. and 1–2 h, respectively. Because VL in Sudan occurs American Journal of Tropical Medicine and Hygiene, 2000, mainly in children, it was concluded that the use of 63:184–188. impregnated bednets (outdoors as well as indoors) is highly protective. Davies CR et al. Leishmaniasis: new approaches to disease control. Clinical review. British Medical Journal, 2003, 326:377–382. REMAINING GAPS IN KNOWLEDGE AND FUTURE RESEARCH PRIORITIES Desjeux P. Pyrethroid impregnated bed nets: an alternative vector control approach for leishmaniasis. In: The studies reviewed above clearly show that ITNs Caglar S et al, eds. Proceedings of the 13th European are efficacious against sandflies and leishmaniasis. SOVE Meeting 2000, Belek, Antalia, Turkey. USA, However, further evaluations of impact of ITNs on Society for Vector Ecology, 2000. VL should be encouraged. Furthermore, research on Elnaiem DA. A pilot study to control cutaneous leishma- ITNs should move from efficacy studies into evalu- niasis in Khartoum using insecticide impregnated curtains ation of effectiveness; to do this, more operational and bednets. Report on a WHO-funded project (ID research is needed to address the socioeconomic L3/181/147), World Health Organization, 1994. dimensions of ITN use against leishmaniasis and to make knowledge available on practical aspects of Elnaiem DA. Use of pyrethroid impregnated bednets for bednet intervention. Needed is/are: control of visceral leishmaniasis in eastern Sudan. Report on WHO-funded project (ID L3/181/147), World • Definition of indicators, for decision-makers, on Health Organization, 1997. when to implement ITNs. This can be achieved Elnaiem DA. Use of pyrethroid-impregnated bednets for by strengthening knowledge of sandfly ecol- control of visceral leishmaniasis in eastern Sudan: fol- ogy and leishmaniasis epidemiology at regional low-up study. Report on WHO-funded project (ID levels. L3/181/147), World Health Organization, 1998. • Assessment of financial, managerial, human and material elements needed for large-scale inter- Elnaiem DA et al. Impact of permethrin-impregnated vention programmes. This can be done through curtains on Phlebotomus papatasi sandflies indoors at regional-level cost-effectiveness studies. Khartoum, Sudan. Medical and Veterinary Entomology, • Socioeconomic studies addressing behavioural 1999, 13:191–197. aspects of ITN use, and how to motivate people Elnaiem DA, Elnahas AM, Aboud MA. Protective to use ITNs during leishmaniasis epidemics. efficacy of lambdacyhaothrin-impregnated bed- • New research initiatives to encourage the inven- nets against Phlebotmus orientalis, the vector of vis- tion of new impregnated fabric tools that can ceral leishmaniasis in Sudan. Medical and Veterinary be used outdoors during the early hours of the Entomology, 1999, 13:310–314. evening. Lessons from the use of insecticide- impregnated dog collars should lead us to new ideas for controlling human leishmaniasis.

96 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Feliciangeli MD et al. Sandfly control trial with deltamethrin-impregnated curtains in ElIngenio, Miranda State, Venezuela. Boletin de la Direccion de Malaraiologia y Saneamiento Ambiental, 1995, 35:127–132. Kroeger A, Avila EV, Morison L. Insecticide impregnated curtains to control domestic transmission of cutaneous leishmaniasis: cluster randomized trial. British Medical Journal, 2002, 325:810–813. Majori G et al. Efficacy of permethrin-impregnated curtains against endophilic phlebotomine sandflies in Burkina Faso. Medical and Veterinary Entomology, 1989, 3:441–444. Maroli M, Lane RP. The effect of permethrin impregnated nets on Phlebotomus (Diptera: Psychodidae) in central Italy. In: Leishmaniasis - the current status and new strategies for control, pp. 217–223. New York, Plenum Press, 1989. Maroli M, Majori G. Permethrin-impregnated curtains against phlebotmine sandflies (Diptera: Psychodidae): laboratory and field studies. Parassitologia, 1991, 33:399–404. Mutinga M et al. The use of permethrin-impregnated wall cloth (MBU cloth) for control of vectors of malaria and leishmanisis in Kenya – II. Effect on Phlebotmine sandfly populations. Insect Science Applications, 1992, 13:163–172. Mutinga MJ et al. A bioassay to evaluate the efficacy of permethrin-impregnated screens used against phlebotmine sand flies (Diptera: Psychodidae) in Baringo district of Kenya. East African Medical Journal, 1993, 70:168–170. Reyburn H et al. A randomized controlled trial of insecticide-treated bednets or top sheets and residual spraying of interior rooms for the prevention of cutaneous leishmaniasis in Kabul, Afghanistan. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2000, 94:361–366. Soto J et al. Efficacy of permethrin-impregnated uni- form in the prevention of malaria and leishmaniasis in Colombian soldiers. Clinical Infectious Diseases, 1995, 21:599–602. Tayeh A, Jlouk L, Al-Kahaimi A. A cutaneous leishmaniasis control trial using pyrethroid-impregnated bednets in villages near Aleppo, Syria. Geneva, World Health Organization, 1997 (WHO/LEISH/97.41). Wells D, Grayson BT, Langer E. Vapour pressure of permethrin. Pesticide Science, 1986, 17:473–477.

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 97 98 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 6 PARTNERSHIP AND CAPACITY BUILDING

6.1 The importance of risk factors in the control of leishmaniasis ...... 100

6.2 Challenges and opportunities in research and control ...... 103 Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 99 6.1 THE IMPORTANCE OF RISK the 1980s, the majority of studies were observational FACTORS IN THE CONTROL in nature, but since the 1980s, analytic research has increased, looking more at causality, pathogenic OF LEISHMANIASIS mechanisms, transmission patterns and the interrelationship between vectors, reservoirs, humans, Alejandro Llanos-Cuentas climate and ecology. Research is necessary to pro- Facultad de Salud Pública y Administración vide solid information for improving intervention Universidad Peruana Cayetano Heredia measures and prevention programmes; however, Lima despite the important questions raised, only a few Peru scientific results have been incorporated into control programmes, and these have been without significant impact for the people living in endemic areas.

The leishmaniases are vector-borne diseases, and are Table 1 summarizes the intervention targets rec- highly complex: they are usually zoonotic; a large ommended for leishmaniasis by the WHO Expert number of Leishmania species infect humans; the Committee [WHO, 1990]. These actions have been biological cycles and epidemiological interrelation- applied frequently, partially and independently of ships between vector, reservoir, humans, climate each other. The most common intervention is early and ecology are highly variable; they have multi- diagnosis and treatment (action against the para- faceted clinical manifestations (due to the genetic site), but this does not reduce transmission. The vec- heterogenicity of both parasite and humans) and tor is usually controlled (inside houses and in the outcomes (which vary from spontaneously cured to peridomiciliary and sylvatic environments) only not curable) as a consequence of disease. in outbreaks, with limited benefit in the long term, while control of the animal reservoirs (domestic and The magnitude of leishmaniasis has changed little wild) is restricted to special situations. Personal pro- in the last 50 years in some endemic areas of the tection measures (which reduce man-fly contact) are New World. For instance, in some areas of Peru used only on a small scale and in time-limited situa- endemic for cutaneous leishmaniasis (CL), the prev- tions, usually by tourists or personnel employed by alence during the 1990s was similar to the preva- extraction companies working in the jungle. A vac- lence before the 1950s (Davies et al., 1994); although cine is an important alternative, but is still in the the DDT house spraying campaign between the experimental stages. 1950s and 1970s reduced the annual incidence of leishmaniasis, when spraying was stopped around These actions have not so far been successful in con- 1972, CL rose again until it had reached its tradi- trolling leishmaniasis in the New World. The weak- tional level. The global incidence and prevalence of ness of the strategy is the biomedical approach, the leishmaniasis increased generally in the Americas application of interventions through vertical pro- between the 1980s and 1990s, despite the interven- grammes, the lack of sustainability in the long term, tion measures and efforts of the health authorities. and the low or absent participation of people from Other vector-borne diseases such us malaria, dengue, yellow fever, and bartonellosis have increased too. The growth of the population, poor levels of hygiene, and increasing periurban poverty are creating favourable habitats for the proliferation of vectors and reservoirs. As a consequence, vector-borne Table 1. Vector control disease such us malaria, dengue, and lymphatic fil- Intervention targets Insecticide spraying in leishmaniasis ariasis are becoming major public health problems Genetic control (Knudsen & Slooft, 1992); the same is now happen- Biological control ing with leishmaniasis, which is rapidly becom- Chemical control ing urbanized, as reported from Manaus and Belo Control of animal reservoirs Horizonte (Profeta da Luz et al., 2001). (domestic and wild) Drugs The contribution that scientific knowledge is making Vaccine to New World leishmaniasis is indisputable. A large Personal protection number of papers have been published since the Vaccine early 60s, including detailed reviews of parasitolog- Repellents ical, ecological, entomological, immunopathological, clinical, therapeutic and public health aspects. Before Early diagnosis and treatment

100 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 endemic areas. The approach using risk factors is an reservoir behaviours, together influence the trans- alternative to this classical approach. mission pattern, vector species, abundance of vectors, rate of infection of vectors and reservoirs, and In the Latin American literature, several factors incidence and prevalence of human infection. The have been associated with a high risk of transmis- frequency of the disease, its severity and rate of cure sion. These include occupational activities mainly are more dependant on the host–parasite relation- related to deforestation, extraction of natural prod- ship and the antiparasitic action of the drugs used. ucts, hunting and exploring (Pessoa & Barretto, 1948; Castro, 1986). Age (young adults), sex (male), and Despite the fact that risk factors have been deter- low socioeconomic status are factors closely related mined in several countries, they have been used very with these occupations in the jungle areas of Brazil, little. Only in one study (Llanos-Cuentas, 1993) in Peru and Mexico (Gomes et al 1992, Bartolini, 1988; Peru was estimation of the population attributable Andrade-Narvaez et al., 1992). The location of homes risk (PAR) for an individual subset of factors simul- close to the forest has also been related to increased taneously adjusted for the risk attributable to the risk of disease (Castro, 1986; Netto et al., 1986). All of remaining factors in the model. A group of risk fac- these risks have been detected on the basis of statisti- tors in central West Peru (region 1) was significantly cal association, but are not representative (in terms of associated with transmission inside houses because sample size) or adjusted for multifactorial determi- the suggested intervention was to use a single mea- nants; however, the results have provided a hypoth- sure (spraying insecticide indoors). The combined esis for analytic studies. As a disease very closely PAR for this group was 0.792, which implies that associated with poverty, leishmaniasis usually affects removal of this group of factors with a specific inter- the poorest people of the poorest countries; 72 of 88 vention would lead to a 79.2% reduction in CL inci- countries affected are developing, and 13 of them are dence in the region. In order to evaluate the impact among the least developed. It is here, where more of the same intervention measure in North-West than 80% of the population earns less than US$ 2 per Peru (region 2), a group of risk factors related to day (Davies et al., 2003), that the majority of cases are indoors transmission was selected and the best PAR reported every year (Desjeux, 2001). was found to be only 5.1. For five years, both regions were systematically studied (spraying lambda-cyha- Most properly designed assessments of risk fac- lothrin in the home and peridomestic area every six tors were published after 1990. Risk factors associ- months for two years, and measuring the incidence ated with the transmission of Leishmania infections rate for an additional three years); reduction in the have been demonstrated in Peru (Llanos-Cuentas, incidence of CL in region 1 was 81%, but in region 2 1993; Davies et al., 1997), Colombia (Weigle et al., was only 4.6%. Thus, the risk assessment approach 1993), Costa Rica (Rojas et al., 1988), and Argentina allows the results of studies to be drawn upon for (Yadon et al., 2003; Sosa-Estani et al., 2001), among prioritizing the use of limited resources and con- other countries; they vary in importance and nature centrating efforts against those hazards with greater according to region, epidemiological and ecological impact. characteristics, and pattern of transmission. The risk factors are quite different if the transmission is in The studies on risk factors have taught us several dwellings (indoors or peridomestic) or outside the lessons: home (in forests or rural places). In addition, there • Leishmaniasis has a dynamic epidemiology; fac- is often more than one pattern of transmission in the tors can change under the influence of climate, same region. For instance, in the Andean regions ecology and behaviour of humans, vectors and where indoor transmission has been classically rec- reservoirs. ognized, peridomestic and rural transmission occur • We cannot apply the same intervention strategy at the same time. In some endemic areas, transmis- in all places, even when the infection is caused sion around the home causes about 80% of cases; in by a unique species. other Andean places, where transmission occurs in • There are limitations of the control strategy used rural places (Llanos-Cuentas & Davies C., 1992), a in the majority of countries. The classical end- single Leishmania species (L. peruviana) may cause points of control programmes are control of the more than 90% of the CL cases. A similar situation disease (early diagnosis and treatment) and con- occurs when the main transmission pattern is in the trol of the vector(s) or reservoir(s), but in fact forest; however domestic transmission is always the primary endpoints are climate, ecology and more frequent (Weigle et al., 1993). human behaviour.

The studies about risk factors suggest that ecologic In future, leishmaniasis control programmes in and climatic characteristics, and human, vector and developing countries should be focused on reduc-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 101 tion of poverty and development of the health rights Knudsen AB, Slooft R. Vector-borne disease prob- of the people in order to achieve sustainable suc- lems in rapid urbanization: new approaches to vector cess through control activities. So health and eco- control. Bulletin of the World Health Organization, 1992, nomic improvement must be critical issues in the 70(1):1–6. national policy. The community should be ade- Llanos-Cuentas EA, Davies C. Epidemiological quately educated, in a long-term programme, about studies on Andean cutaneous leishmaniasis and the strategies. Thus, a combined risk assessment and their significance for designing a control strate- ecosystem approach is a new alternative based on gy. In: Wijeyaratne P, Goodman T, Espinzal C, eds. the interrelationship between scientific knowledge, Leishmaniasis control strategies: A critical evaluation participation of the communities living in endemic of IDRC-supported research. Proceedings of a workshop areas, and economic support by the government. held in Mérida, Mexico, November 25–29, 1991. Ottawa, Canada, International Development Research Centre References (IDRC), MR 322e, 1992. Andrade-Narvaez FJ et al. Risk factors associat- Llanos-Cuentas EA. Risk factors associated with the ed with cutaneous leishmaniasis infection and dis- transmission of Andean cutaneous leishmaniasis [PhD ease in the State of Campeche, Peninsula de Yucatán, thesis]. London, University of London, 1993. México. In: Wijeyaratne P, Goodman T, Espinzal C, Netto EM et al. Origin of patients with mucosal leish- eds. Leishmaniasis control strategies: A critical evaluation maniasis in an endemic area of Bahia, Brasil. Revista of IDRC-supported research. Proceedings of a workshop da Sociedade Brasileira de Medicina Tropical, 1986, 19:121. held in Mérida, Mexico, November 25–29, 1991. Ottawa, Canada, International Development Research Centre Pessoa SB, Barreto MP. Leishmaniose Tegumentar (IDRC), MR 322e, 1992. Americana. Rio de Janeiro, Brasil: Ministerio da Educacao e Saude, 1948. Bartolini R et al. Social epidemiology of leishmaniasis in the central jungle of Peru. In: Walton BC, Profeta da Luz ZM et al. Leishmaniasis urbaniza- Wijeyaratne PM, Moddaber F, eds. Research on control tion and low diagnosis capacity in the Metropolitan strategies for the leishmaniases. Proceedings of an interna- Region of Belo Horizonte. Revista da Sociedade tional workshop held in Ottawa, Canada, 1–4 June, 1987. Brasileira de Medicina Tropical, 2001, 34(3):249–54. Ottawa, Canada, International Development Research Rojas JC et al. Identification of risk factors associ- Centre (IDRC), 1988. ated with cutaneous leishmaniasis in Costa Rica. Castro GA. American leishmaniasis epidemiology in In: Walton BC, Wijeyaratne PM, Moddaber F, eds. Brazil. Insect Science and its Application, 1986, 7:161–169. Research on control strategies for the leishmaniases. Proceedings of an international workshop held in Ottawa, Davies CR et al. Cutaneous leishmaniasis in the Canada, 1–4 June, 1987. Ottawa, Canada, International Peruvian Andes: risk factors identified from a village Development Research Centre (IDRC), 1988. cohort study. American Journal of Tropical Medicine and Hygiene, 1997, 56:85–95. Sosa-Estani S et al. Cutaneous leishmaniasis in Northern Argentina: identification of risk factors in Davies CR et al. The fall and rise of Andean cutane- a case-cohort study of three municipalities in Salta. ous leishmaniasis: transient impact of the DDT cam- Revista da Sociedade Brasileira de Medicina Tropical, 2001, paign in Peru. Transactions of the Royal Society Medicine 34 (6):511–517. and Hygiene, 1994, 88:389–393. Weigle KA et al. Epidemiology of cutaneous leishma- Davies CR et al. Leishmaniasis: new approaches to dis- niasis in Colombia: environmental and behavioral risk ease control. British Medical Journal, 2003, 326:377–382. factors for infection, clinical manifestations, and patho- Desjeux P. The increase in risk factors for leishma- genicity. Journal of Infectious Diseases, 1993, 168:709–714. niasis worldwide. Transactions of the Royal Society of World Health Organization. Control of the leishmanias- Tropical Medicine and Hygiene, 2001, 95:239–243. es. Report of a WHO Expert Committee. Geneva, World Gomes AC et al. Aspectos ecológicos da leishmani- Health Organization, 1990 (WHO Technical Report ose tegumentar americana: prevalencia/incidencia da Series, No. 793). infeccao humana nos municipios de Pedro de Toledo Yadón ZE et al. Indoor and peridomestic transmission e Miracatu, Sao Paulo, Brasil. Revista do Instituto de of American cutaneous leishmaniasis in the north- Medicina Tropical de Sao Paulo, 1992, 34:149–158. western Argentina: a retrospective case-control study. American Journal of Tropical Medicine and Hygiene, 2003, 68 (5):519–26.

102 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 6.2 CHALLENGES AND nomic characters. For any automated interpretation OPPORTUNITIES IN of these, there should be at least 30 characters, and the state of each should be described for each stock. RESEARCH AND CONTROL With this information, operational taxonomic units can be nominated, and a classification can be com- Richard Ashford (retired) piled. Only then can limits between strains, subspe- Liverpool School of Tropical Medicine cies and species be proposed, on largely subjective Liverpool L3 5QA grounds. UK Only when a reliable classification has been compiled does identification become meaningful.

These notes reflect personal opinion. They are delib- Isoenzymes provided the first, and for a long time erately controversial, and are intended rather to apparently ideal, set of valid, comparable taxo- stimulate discussion than to determine policy. In nomic characters, and allowed the classification that my opinion, there has been a tendency over many largely stands today. However, several shortcom- years for TDR to follow what is fashionable and ings have become apparent in the isoenzyme system, technologically advanced. I believe this has not only and additional methods are required, especially for diverted TDR funds away from the goals of disease certain difficult groups of strains. control, but has influenced other funders to do the same. TDR has a particularly important responsibil- When a classification has become established, marker ity in that it serves as a trendsetter every bit as much strains of species, and marker stocks of strains, can as a sponsor of research. What follows are a few per- be established. Only then may it be acceptable to sonal opinions highlighting areas where I believe reduce the number of taxonomic characters to be we have tended to lose sight of our real objectives. used for identification. Such simplification is per- fectly acceptable for quantitative studies, where the In the words of Burl Ives, let us try to ‘watch the range of strains has been well established. However, doughnut, not the hole’. for stocks from new areas or from unfamiliar hosts, a full standardized description is still required. TAXONOMY: DETECTION, DESCRIPTION, CLASSIFICATION Whether or not it is still necessary to refine the exist- AND IDENTIFICATION OF THE ing classification for the purposes of TDR is debat- PARASITES able. I suggest that there have been several instances, notably in the reports of visceralizing L. tropica, and Taxonomy is at the very heart of almost any aspect in the apparent existence of three ‘species’ in a sin- of biology. In recent years, several happenings have gle Sudanese epidemic area, where the current sys- affected the problem of taxonomy of Leishmania. tem has been shown to be inadequate. Further, there are several instances where vectors and reservoir The advent of polymerase chain reaction (PCR) hosts still need to be identified. Better methods are methods for amplifying DNA has greatly facilitated needed in these areas. the detection of parasite DNA, thereby allowing detection of infection in candidate reservoir hosts. Monoclonal antibodies, whether singly or in batteries, and so-called specific DNA probes may provide Several studies, particularly in Sudan, have shown excellent tools for identification of stocks for quan- the standard battery of isoenzymes to be inadequate titative purposes, but can be very misleading if they for the classification of L. donovani strains. are over-interpreted. They are far from adequate for the ‘identification’ of new, aberrant or surprising Microsatellite batteries promise new and more eas- results. Their use should be limited to the identifi- ily standardized protocols for describing series of cation of stocks belonging to strains that contributed strains. to the classification being used.

In any taxonomic study, collection of specimens is the first objective. Long series of specimens (stocks EPIDEMIOLOGY in our instance) are required from as many popu- Methods using remotely sensed information, ana- lations as possible, in order to assess the variation lysed using global positioning systems (GPS) soft- within each population. Each stock must then be ware, now allow much more detailed description of described by a range of descriptors, known as taxo-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 103 the habitat requirements of each ‘nosodemiological DIAGNOSTIC TESTS unit’, and the development of detailed risk maps. Many biologists justify their studies on genomics, or on the immunology of the domestic mouse, under Risk maps are particularly needed in control of the umbrella of ‘developing a diagnostic test’ (or diseases like the leishmaniases: outbreaks are fre- more commonly but even less realistically, ‘devel- quently mobile, and the prepatent periods are often oping a vaccine’). Usually this involves a relatively so long that, by the time the infection is recognized banal, stamp-collecting analysis of some antigen, in a place, very many people may have become justified because ‘it might lead to a diagnostic test’ infected. Ideally control intervention must include (in fact, the most it will usually do is teach a student not only areas where the infection is known, but some fashionable techniques). also peripheral areas which are suitable for transmission, where the infection may have recently been Almost always, once a ‘test’ has been ‘developed’ introduced but has not yet appeared. (usually in a PhD-with-no-Ph project), it is promptly set aside in favour of the next fashionable study. VACCINE DEVELOPMENT Such studies should be justified in terms of their potential contribution to training, or to basic sci- While the idea of a vaccine is all very well, we should ence (not much, usually), and should not be funded consider whether or not it would be desirable to under the banner of tropical diseases research. actually use any vaccine, and whether it might even be counterproductive to do so. As I see it, any diagnostic test may serve one or more of at least four main functions; each function In Nepal we have estimated that a vaccine pro- requires a test with particular specifications. Table 1 gramme would need to cover some 6 million people below compares some of the specifications desirable initially, and 240 000 annually thereafter for several of tests in the main diagnostic contexts. years at least. The number of people requiring treatment is unlikely ever to exceed 6000 annually. We Only rarely is the purpose of diagnosis precisely estimated that, in Nepal, the unit cost of vaccination described, and only exceptionally is a project spe- with a perfect vaccine would need to be at the very cifically aimed at a clearly defined goal. Diagnostics least 90 times less than that of treatment in order to research to be funded by TDR should be clearly be economically competitive. In the absence of any identified within one or more categories of test, and reliable model, there is no way of knowing whether the potential advantages over existing tests should a vaccine programme or an enhanced treatment be clearly identified. programme would be more effective in controlling the disease, but an enhanced treatment programme using active case detection should have numerous CONCLUSIONS beneficial side effects. After all the years of TDR, now is a good time to take stock of achievements, not in terms of scientific, per- On balance, it may well be that an enhanced treat- sonal or institutional interest, but in terms of relief ment programme would be both more cost effec- of suffering. tive and more beneficial than any vaccination programme. The greatly improved classification of the parasites has allowed meaningful identification and, thereby, I suggest that we should consider abandoning sup- enormous improvement of our understanding of port for vaccine development, and concentrate the structure of both zoonotic and anthroponotic research on: foci. Serious gaps still exist in this vital area. • how to detect early cases • how to distinguish cases who should be treated First the direct agglutination test (DAT), then the from those who will self-cure, and dipstick rK39 and, dare we hope, the urine antigen • how to deliver effective treatment to every per- test, materially advance diagnosis at village clinic son who needs it. and treatment centre level. Rapid, reliable diagnosis must be the basis for intervention relying on active This consideration is particularly appropriate for case detection and treatment. kala azar, which is relatively uncommon even in epidemics, and can be cured without lasting disfig- Developments in treatment are limited: excel- urement, and for which the idea of an economically lent new drug formulations or combinations have viable vaccine is probably ‘pie in the sky’. not radically reduced the cost, nor the duration of

104 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 treatment. Oral miltefosine promises to be advanta- geous, but has serious limitations.

Vaccination remains pie in the sky.

We know little more of sandfly ecology than we did at the last expert committee, and that was not much. Sandfly ecology remains one of the most important areas where research is required.

Our work in Tunisia developed guidelines for prediction of zoonotic cutaneous leishmaniasis (ZCL) outbreaks: these need to be refined and could then easily be implemented. As ever, it is the link between the ministry and the laboratory that needs to be reinforced.

As far as kala azar in the Indian subcontinent is con- cerned, current technology should be quite adequate for the disease to be eliminated to the extent that there are very few cases and no deaths. It is the implementation of such technology that is wanting. The same is probably true of infantile visceral leishmaniasis. For both of these, the emphasis of research should be on the socio-politico-economic aspects, advocacy, and delivery of education to the politi- cians, medical profession and villagers.

Table 1. Some specifications for clinical and epidemiological diagnostic methods

Clinical (passive case Clinical (active case Epidemiological (for Epidemiological (for detection – PCD) detection – ACD) numbers of cases) distribution of agent) Aim Indicates: To treat Indicates: To refer for Indicates: Density and Indicates: Presence of agent, or not to treat confirmation or not dynamics of agent in host by geography, host and vector or vector population Objective Must identify active infection Must detect all active Should distinguish infecteds, Must identify active infection infections susceptibles and immunes Sensitivity Screening test (optional) Screening test must Single test desirable; Sensitivity not important must be highly sensitive be highly sensitive sensitivity must be known Specificity Specificity important (Refer positives to Specificity must be known Specificity essential, at species level PCD methods) to strain level Expense Expense not the major issue Must be very cheap Must be very cheap Complex, expensive procedures acceptable Reference material Positive and negative Reference material of Reference material of Reference collection controls required lesser importance – refer lesser importance – test material vital positives to specific test must be well calibrated Direct/indirect Indirect/direct tests OK Indirect/direct tests OK Indirect/direct tests OK Direct test essential (parasite or DNA) Shelf life Important, especially in Unimportant – if used Unimportant – usually Unimportant – usually acute or rare diseases in special projects limited to special projects limited to special projects Technical level of Clinician/clinical technologist, Technologist, may Technologist, may Research scientist: operator should be basic level require high level require high level may involve hi-tech for identification of agent

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 105 106 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 Annex 7 COMMISSIONED PAPERS

7.1 Drug resistance in leishmaniasis ...... 108

7.2 From genome to discovery in Leishmania: recommendations for the development of drugs, vaccines and diagnostics ...... 120

7.3 Can leishmaniasis still be considered as a category 1 disease? If moved, what is required? ...... 130 Leishmaniasis

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 107 7.1 DRUG RESISTANCE major clinical forms of the disease, visceral leish- IN LEISHMANIASIS maniasis (VL) and cutaneous leishmaniasis (CL), there are other cutaneous manifestations including mucocutaneous leishmaniasis (MCL), diffuse cuta- Simon L. Croft neous leishmaniasis (DCL), recidivans leishmani- Department of Infectious and Tropical Diseases asis (LR) and post kala azar dermal leishmaniasis London School of Hygiene and Tropical Medicine (PKDL). The current situation for the chemother- London WC1E 7HT, UK apy of leishmaniasis is more promising than it has been for several years with both new drugs and new formulations of old drugs either recently approved or on clinical trial (Croft and Coombs, 2003; Croft SUMMARY and Yardley, 2002; Murray, 2001). In the past decade, four new potential therapies for VL have been intro- The efficacy of drugs used in the treatment of vis- duced: a parenteral formulation of aminosidine ceral and cutaneous leishmaniasis is influenced by (paromomycin) (Thakur et al., 2000), amphotericin both host and parasite factors. The latter includes B liposomes (Berman et al.,1998; Meyerhoff, 1999), both an intrinsic variation in the sensitivity of and the orally active drugs miltefosine (Sundar et Leishmania species as well as acquired drug resis- al., 2002) and sitamaquine (WR6026) (Sherwood et tance due to selection. Intrinsic variation in sensitiv- al., 1994; Dietze et al., 2001). Treatment of CL has ity has been described to pentavalent antimonials, been improved by various topical formulations of paromomycin, miltefosine, azoles and other drugs paromomycin (El-On et al., 1992; Asilian et al., 2003; that have reached clinical trials. Acquired resistance Soto et al., 2002; reviewed Garnier and Croft, 2002) to anti-leishmanial drugs has been reported from and could also benefit from oral miltefosine (Soto et laboratory studies over several decades but it is only al., 2001). Several other drugs, in particular the anti- recently that this has been shown in Leishmania don- fungal azoles itraconazole, ketoconazole and flucon- ovani visceral leishmaniasis clinical resistance to azole, have been on limited clinical trials, often with antimonials. The development of resistance to anti- equivocal results. monials and the potential for resistance to other drugs has several implications. It makes the search Drug treatment is complicated by the variation in for new drugs more urgent, it focuses attention on sensitivity of Leishmania species, the different dis- our lack of knowledge about the molecular and ease manifestations, the variation in host response, biochemical mechanisms of action of antimonial and the absence of baseline data from controlled drugs on Leishmania, it places emphasis on strate- clinical trials of many old drugs for CL. The aims of gies and policies for use of anti-leishmanial drugs this review are to dissect the problems of variation in endemic areas, and it brings into focus the need in drug sensitivity and acquired drug resistance, to to consider drug combinations in anti-leishmanial evaluate their importance in anti-leishmanial che- therapy. There is a need to develop systems for the motherapy and, finally, to consider implications for monitoring and surveillance of resistance. However, drug use, research, surveillance, and public health. for antimonials and most drugs this is problematic due to the absence of molecular markers and a reliance on the amastigote-macrophage culture assay to CONFOUNDING ISSUES – IMMUNE adequately correlate clinical response with parasite STATUS AND PHARMACOKINETICS phenotype. Firstly, it is important to briefly acknowledge that host factors affect drug efficacy and must be con- INTRODUCTION sidered in interpretation of data. The immune sta- Leishmaniasis is a disease complex caused by spe- tus of leishmaniasis patients has long been known cies of haemoflagellate protozoa parasites belong- to effect drug activity. This has proved to be of paring to the genus Leishmania. There are an estimated ticular importance in relation to pentavalent anti- 12 million humans infected, with an incidence of 0.5 monial treatment of DCL (Ercoli, 1966) and HIV/VL million cases of the visceral form of the disease and co-infections (Alvar et al., 1997; Berhe et al., 1999) 1.5 to 2.0 million cases of the cutaneous form of the where there is an absence of specific T cell-mediated disease. The numbers are probably underestimated immune response and mutual exacerbation of infec- as leishmaniasis is a reportable disease in only 40 tion. The basis of this lack of activity of pentavalent of the 88 countries worldwide (http://www.who. antimonials has been explored in immunodeficient int/tdr/diseases/leish/diseaseinfo.htm) where mouse models where the effects are probably due it is known to be present. In addition to the two to deficiencies of both Th1 cell-mediated and macrophage responses (Murray et al., 1989; Murray

108 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 and Delph-Etienne, 2000). Experimental models sitive to sodium stibogluconate than L. major, L. trop- have also shown that the anti-leishmanial activities ica and L. mexicana (Allen and Neal, 1989; Neal et al., of amphotericin B and miltefosine are T cell inde- 1995). Other studies by Berman et al. (1982), using pendent, whereas pentamidine is T cell dependent another amastigote-macrophage model, and Grogl (Murray et al., 1993; Murray & Delph-Etienne, 2000; et al (1992), using promastigote cultures, demon- Escobar et al., 2001). strated wide variation in the sensitivity of isolates from cutaneous leishmaniasis cases to pentavalent As the sites of infection in leishmaniasis are nor- antimonials, and suggested that there was a correla- mally in either the visceral organs or the skin, phar- tion between sub-curative treatment and decreased macokinetic properties can also affect drug efficacy. clinical drug sensitivity. This has been confirmed These properties could influence the activity of in one of the few controlled clinical trials that com- sitamaquine, an 8-aminoquinoline, a class of drugs pared the cure rate to antimonials of CL caused by that is well distributed to the liver (Carson, 1984), different species. Pentostam produced a signifi- or itraconazole (a triazole), that is well distributed cantly higher cure rate for L. braziliensis lesions than to the skin (Leyden, 1998). Other pharmacokinetic for L. mexicana lesions (Navin et al., 1992). properties, metabolism and excretion, must also be considered. In a study on the treatment of CL in Paromomycin (aminosidine) Saudi Arabia, patients showed marked variation This aminoglycoside antibiotic has been used for the in response to Pentostam, but no differences were treatment of both VL, in a parenteral formulation, observed in the sensitivity of Leishmania major iso- and CL, in both topical and parenteral formulations. lates to this drug from patients in the amastigote In experimental models and in clinical treatment, – macrophage model (Al-Jaser, 1995). However, sig- lesions caused by L. major that were treated with nificant differences were observed between patients paromomycin ointment resolved faster and more in the elimination rate of antimonials, and area completely than lesions caused by L. amazonen- under the curve (AUC) analysis suggested that dif- sis and L. panamensis (El-On & Hamburger, 1987). ferences in the length of exposure to antimony could These observations have been supported by an in influence response in CL treatment (Al Jaser et al., vitro study of the sensitivity of amastigotes, where 1995). L. major and L. tropica (ED50 values in the range of 1– 5 µM) were more sensitive than L. braziliensis (ED50 DRUG RESISTANCE – THE PROBLEM < 12µM) and L. mexicana (ED50 39µM), while L. donovani showed intermediate sensitivity (ED 6–18 Drug sensitivity and Leishmania species 50 µM except DD8 strain which had an ED50 > 150µM) There are over seventeen species of Leishmania (Neal et al., 1995). known to be infective to humans and they have been characterized by biochemical and molecular differ- Amphotericin B ences that provide the basis for phylogenetic anal- This polyene antibiotic has selective activity against ysis and several methods of diagnosis (Cupolillo fungi, as well as Leishmania and Trypanosoma cruzi, et al., 2000). These biochemical and molecular dif- through a higher affinity for the predominant ferences between species are reflected in variation microbial sterol ergosterol than for host cell cho- of the intrinsic sensitivity of Leishmania species to lesterol. Differences in species sensitivity might be a range of drugs from different chemical classes. expected due to variation in the type and quantity Examples of intrinsic variation have been defined in of sterols in membranes of different species (Beach a number of laboratory studies, but interpretation of et al., 1988), and have been characterized in rela- these studies is important as different assay condition to ergosterol biosynthesis in fungi (Young et tions lead to several-fold differences in activity val- al., 2003). These observations were extended in a ues (Croft and Brun, 2003). recent in vitro study on amastigotes of six species in macrophages, where L. mexicana was less sensitive Antimonials to this antibiotic (Escobar et al., 2002). The higher The variation in clinical response to the pentavalent efficacy of liposomal amphotericin B (AmBisome) antimonials sodium stibogluconate (Pentostam) and against L. donovani than L. infantum/L. chagasi infec- meglumine antimoniate (Glucantime) has been a tions (Berman et al., 1998) is probably unrelated to persistent problem in the treatment of leishmania- species sensitivity, and more affected by parasite sis over the past 50 years. There is an intrinsic differ- load, pharmacology and pathology. ence in the sensitivity of species to these antimonials. In studies using the amastigote–macrophage model, L. donovani and L. braziliensis were 3-5 fold more sen-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 109 Azoles Nucleoside analogues The biosynthetic pathway of ergosterol, the major Wide variations in the sensitivity of promastigotes sterol in fungi as well as Leishmania spp. and T. cruzi, of different species to the pyrazolopyrimidines allo- is a target for the most important antifungal drugs. purinol and allopurinol riboside have been reported, There has been an interest in two classes of these suggesting differences in the affinity of enzymes of drugs as anti-leishmanial agents, the allylamines the purine salvage pathway (Avila and Casanova, (for example, terbinafine) that inhibit squalene epox- 1982; Nelson et al., 1979). idase, and the azoles (for example, ketoconazole and itraconazole) that inhibit C14α-demethylase. A num- Miltefosine (hexadecylphosphocholine) and ber of clinical studies have suggested that these ste- edelfosine (ET-18-OCH3) rol biosynthesis inhibitors are more effective against Variation in the sensitivities of promastigote and L. major and L. mexicana infections than against L. amastigote stages of L. donovani, L. major, L. tropica, L. donovani or L. braziliensis infections. One placebo aethiopica, L. mexicana and L. panamensis were shown controlled trial on the treatment of CL showed that in vitro to these phospholipid drugs (Escobar et al., L. mexicana infections were more responsive than 2002). In all assays, L. donovani was the most sensi- L. braziliensis infections to ketoconazole (Navin et tive species, with ED values in the range of 0.12 al., 1992). The results from in vitro studies that have 50 to 1.32 µM against promastigotes and 1.2 to 4.6 µM investigated the intrinsic differences in sensitivity against amastigotes. L. major was the least sensitive of Leishmania species to sterol biosynthesis inhibi- species in the majority of assays, with ED values tors support this clinical observation. In a compar- 50 for miltefosine in the range of 4.8 to 13.1 µM against ative study on the sensitivity of promastigotes to promastigotes and for HPC and ET-18-OCH in the ketoconazole, L. donovani, L. braziliensis and L. ama- 3 range of 7.5 to 37.1 µM against amastigotes. zonensis were found to be more sensitive than L. aethiopica, L. major, L. tropica and L. mexicana (Beach Sitamaquine et al., 1988). However, these results contrast with those of Rangel et al. (1996), who found that L. bra- There have been no direct comparative studies. ziliensis was relatively insensitive to ketoconazole Earlier studies reported similar ED50 values for two and the bis-triazole D0870 whereas L. mexicana was species, 2.6 µM against L. tropica (Berman and Lee, sensitive to ketoconazole. Both sets of results con- 1983) and 1.5 µM against L. donovani (Neal and Croft, trast with an earlier study using an amastigote-mac- 1984). rophage model, which showed that L. donovani was more sensitive to ketoconazole than L. mexicana or L. Acquired drug resistance major (Berman, 1982); in this case the ability of amastigotes to salvage sterols from host cell macrophages The selection of drug resistant pathogens is a major might play a part (Roberts et al., 2003). and well known threat to the treatment of bacterial

Figure 1. Changes in clinical visceral leishmaniasis response in India: Decline in efficacy of pentavalent antimony regimens in Bihar, India, in the 1980s and 1990s

Figure courtesy of Prof Shyam Sundar and Dr Philippe Desjeux d = days; MKD = milligram per kilogram body weight per day

110 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 infections as well as malaria. However, although the enon. Other biological data do support the idea selection of resistant Leishmania has long been a part of acquired resistance. The potential for selection of laboratory studies, it is only in the past decade of antimonial resistance in L. infantum has been that acquired resistance has become a clinical threat, reported from immunodeficient and immunocom- and this is mainly restricted to pentavalent antimo- petent VL patients in France (Faraut-Gambarelli et nial drugs in India. al., 1997). Isolates from 14 patients were made prior to and after Glucantime treatment, and their sensi- Antimonials tivity was measured in an amastigote–macrophage assay. Thirteen of the 14 isolates were less sensitive Studies over the past decade have demonstrated an to the antimonial after treatment. Similar decreased ever decreasing response rate of VL cases to antimo- sensitivity was observed in L. infantum isolates nial treatment in Bihar, India (Figure 1) and that, in taken from dogs before and after Glucantime treat- this major focus of VL, over 60% of cases are treatment (Gramiccia et al., 1992). In the laboratory, L. ment failures to pentavalent antimonials (Sundar, donovani resistance to antimonials is easily gener- 2001). This has had a major impact on the treatment ated, for example in promastigotes (Grogl et al., of visceral leishmaniasis. It is important to note that 1989), a rodent model (see Berman et al., 1982), and the focus of resistance is in districts of Bihar north of more recently in axenic amastigote cultures of L. the Ganges, in comparison to Bihar districts south of donovani and L. infantum (Ephros et al., 1997; Sereno the Ganges and neighbouring Uttar Pradesh where and Lemesre,1997). patients remain responsive to antimonial treatment (see map in Sundar, 2001). So, if there is acquired resistance, do we have a han- dle on the mechanisms? After 60 years of use, the Sundar (2001) has outlined reasons for the possi- anti-leishmanial mechanism of action of pentava- ble increase in failure of response: use of unquali- lent antimonials is still not clearly defined and this fied practitioners, failure to follow WHO guidelines, makes a full description of mechanism of resistance and use of poor quality drug resulting in inadequate impossible. However, recent studies have pointed to treatment. particular biochemical events that are involved in antimonial sensitivity (Figure 2): So is this failure of response due to acquired resistance? It must be assumed that the problems out- • The lack of sensitivity of promastigotes and the lined by Sundar are also to be found in districts sensitivity of amastigotes to pentavalent anti- where antimonials are still relatively effective. In monials depends upon transformation of pen- a study to determine whether acquired resistance tavalent compound to trivalent compound in was present in Bihar, L. donovani isolates were taken the latter but not the former stage (Shaked- from responders and non-responders (Lira et al., Mishan et al., 2001; Santos Ferriera et al., 2002). 1999). In vitro, amastigote–macrophage isolates The question is whether “resistant” amastigotes from patients who did respond to Pentostam were are also deficient in their ability to reduce SbV three fold more sensitive (ED values around 2.5 50 to SbIII ? A recent report started to make some µg Sb/ml) compared to isolates from patients who connections: in vivo L. panamensis amastigotes did not respond to Pentostam (ED values around 50 derived from an SbIII resistant promastigote line 7.5 µg Sb/ml). There was no difference in the sen- were more resistant to SbV treatment than wild sitivity of isolates when the promastigote assay type (Travi et al., 2003). The accumulation of SbV was used (Lira et al., 1999). The significant differ- and SbIII into promastigotes and amastigotes has ence in amastigote sensitivity and the correlation been shown to be by different transport systems supports acquired resistance. However, the sample (Brochu et al., 2003), and though Sb accumula- size was small (15 non-responders, 9 responders), tion was lower in resistant than sensitive forms, and a three-fold difference in sensitivity can be seen levels of accumulation could not be correlated between experiments in this model (Croft and Brun, to sensitivity in wild type cells. 2003). As already mentioned, there are strains in cir- • Whatever, the role of the parasite is unlikely to culation that are insensitive to antimonials. An ear- be an absolute factor as thiols of host cell mac- lier report on VL isolates from Sudan also showed rophages also reduce SbV to SbIII (Frezard et al., that clinical response to Pentostam was reflected in 2001; Santos Ferriera et al., 2002). the amastigote-macrophage model (but not in pro- • Parasite thiols (trypanothione, glutathione) are mastigotes) in an area where there was still a good considered by many to have a critical role in the response to antimonials (Ibrahim et al., 1994). More process of detoxication of heavy metals in try- biological evidence is required to support the tem- panosomatids. Studies on trivalent antimony poral and spatial parameters of the Bihar phenom- (and arsenic) resistant Leishmania parasites have

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 111 shown increased levels of thiols (trypanothi- Amphotericin B one, glucathione, ovothiol). In these lines, this Although this antibiotic has been widely used in the has been related to amplification of enzymes treatment of mycoses for over 30 years, resistance involved in the synthesis of glutathione (γ-glu- in fungal isolates has been reported only rarely tamylcysteine synthetase, gsh1) and polyamines (Georgopapadakou, 1998). However, resistance is (ornithine decarboxylase), the two precur- possible, and a resistant L. donovani clone of promas- sor molecules of trypanothione, in some stud- tigotes was selected through a step-wise increase in ies (Haimeur et al., 2000; Mukhopadhyay et al., amphotericin B concentration in culture. Resistant 1996) but not others (Arana et al., 1998). promastigotes showed a significant change in the • The next part of detoxification is the conjugation plasmamembrane sterol profile by gas chromatog- of antimony with a thiol. Glutathione-S-transfer- raphy/mass spectrometry, ergosterol being replaced ase (GST) could mediate this activity. Increased by a precursor cholesta-5, 7, 24-trien-3β-ol (Mbongo GST expression and levels have recently been et al., 1998). This is probably due to an effect on C - reported in antimony resistant isolates with con- 24 transmethylation due to changes in S-adenosyl-L- comitant increased levels of spermidine and methionine-C -∆-sterol-methyltransferase (SCMT) glutathione (Chatterjee et al., 2003). A trypano- 24 (Loiseau et al., 2002), the same enzyme that con- thione-S-transferase, if present, could play a role fers resistance to some Candida (Young et al., 2003). here as well. There have been two small inconclusive studies on • The efflux of Sb is the final step. Sb-thiol conju- the emergence of amphotericin B resistance in L. gates are amplified in some cases (Arana et al., infantum/HIV infected cases in France. One study 1998; Haimeur et al., 2000; Ouellette et al., 1998), failed to find a change in sensitivity in isolates taken effluxed from Leishmania by PGPA-like ABC before and after treatment of one patient (Durand et transporters (Roberts et al., 1995), or sequestered al., 1998). In contrast, a decrease in sensitivity was into a vacuole (Legare et al., 2001). observed in isolates taken over several relapses from another patient (Di Giorgio et al., 1999). There has Two notes of caution: many of the studies on this been increased use of amphotericin B for visceral complex process have been carried out in vitro on leishmaniasis, both in the deoxycholate (Thakur et the promastigote stage which, as already described, al., 1999; Sundar et al., 2002) and lipid formulations has many differences to the relevant amastigote (Berman et al., 1998; Sundar et al., 2003) following stage (ibid); and some studies were performed on L. failure of antimonial treatment. Although ampho- tarentolae, a distant relative of L. donovani which has, tericin B resistant L. donovani promastigotes have for example, no transporter for miltefosine (Perez- been selected by increasing drug pressure (Mbongo Victoria et al., 2003b). In field isolates, no ampli- et al.,1998), this has yet to be demonstrated in amas- fication of genes seen in laboratory studies was tigotes or under field conditions. observed; rather an amplification on chromosome 9 was related to protein phosphorylation (Singh, Paromomycin Singh and Sundar, 2003). This aminoglycoside is in phase III trials as a parenteral formulation for VL and in topical formulations for CL (two commercial products). The mechanisms Figure 2. Proposed Sb metabolism in Leishmania of resistance in bacteria to this class of antibiotic are well characterized; most commonly the drug is metabolized to an inactive form by acetylases and phosphorylases. In studies on selected populations of promastigotes, resistance was related to decreased SbV SbIII drug uptake in L. donovani (Maarouf et al.,1998), but not to enzymatic modification or to any mutation of GST the small sub-unit ribosomal gene in L. tropica (Fong et al., 1994). The mechanisms of resistance to par- ODC SbIII – thiol conjugate omomycin in Leishmania have not been sufficiently GSH defined.

There is potential for the development of clinical vesicle resistance. Following a 60-day parenteral course for PGPA transporter L. aethiopica cases, and using an amastigote–macrophage assay, isolates taken from relapse patients were 3-5 fold less sensitive to the drug after treatment

112 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 than isolates taken before treatment (Teklemariam perhaps we have to address the measures and policy et al., 1994). Monitoring of resistance could be of that should be put into place. importance if the parenteral formulation is introduced as a first line treatment for L. donovani in an Two important considerations in analysing the seri- anthroponotic focus. ousness of the drug resistance problem are (a) the ease with which resistant individual microbes can Miltefosine be selected for by a particular drug, and (b) potential spread of resistance in a population and there- Promastigote lines of L. donovani resistant to hexa- fore the public health importance. Firstly, the spread decylphosphocholine were generated in the labora- of drug resistant genotypes through a population tory. Resistance was stable after withdrawal of drug of microorganisms is primarily governed by certain pressure, but the lines showed little cross-resis- measurable parameters: (i) the volume of drug used, tance to standard anti-leishmanial drugs (Seifert (ii) the probability that a drug-sensitive infection et al., 2003). The mechanism of resistance in the 40 becomes resistant upon infection, (iii) the duration µM miltefosine resistant line has been determined of infection in individuals, (iv) the fitness costs (divi- as due to defective uptake of miltefosine (Perez- sion rate, transmissibility) for the pathogen incurred Victoria et al., 2003a) through point mutations on a by being resistant in the absence of drugs, and (v) plasmamembrane aminophospholipid translocase the degree to which compensatory mechanisms (Perez-Victoria et al., 2003b). The potential relevance develop that offset these fitness costs (Bjorkman et of these observations needs to be extended to milt- al., 2000; Davison, Low & Woolhouse, 2000; Levin, efosine-resistant amastigotes before clinical implica- Perrot & Walker, 2000). In zoonotic diseases, like tions can be properly considered. In addition, it has most cutaneous leishmaniasis and most L. infantum/ been shown that multidrug resistant L. tropica lines L. chagasi visceral leishmaniasis, the parasite is pri- that overexpress a P-glycoprotein are less sensitive marily an infection of a feral or domestic mamma- to miltefosine (Perez-Victoria et al., 2001). lian host and only occasionally humans. The volume of drug used in relation to the time that a parasite Sitamaquine population is exposed to a drug is insignificant for L. donovani promastigote lines resistant to sitama- most forms of leishmaniasis unless the mammalian quine have been selected in the laboratory (Philippe host is also treated. This is a consideration when Loiseau, pers. comm.), but no further information is control methods for canine leishmaniasis are imple- available. mented, where extensive treatment of the domestic canine host has been shown to lead to changes in Pentamidine sensitivity of isolates (Gramiccia, Gradoni & Orsini, Pentamidine has been used as a second line treat- 1992; Gradoni, Gramiccia & Scalone, 2003). Effects ment for VL, CL and DCL, but its anti-leishman- of drug resistance on the fitness (and virulence), for ial mechanism of action, after 40 years of use, is still example on the abilities to transform, to establish not defined (see Bray et al., 2003). It was shown that an infection in the sandfly vector, to outgrow a non- pentamidine resistant clones of L. donovani and L. resistant wildtype, and R0 of Leishmania, have not amazonensis promastigotes have reduced uptake and been estimated. Current knowledge of the epidemi- increased efflux of the drug due to specific trans- ology and transmission of leishmaniasis suggests porters (Bray et al., 2003; Coelho, Beverley & Cotrim, that the spread of acquired drug resistance is not 2003). A recent review of resistance to diamidines in a factor to be considered in cutaneous leishmania- protozoa also noted the importance of drug accu- sis, except maybe in anthroponotic foci of L. tropica, mulation into the Leishmania mitochondrion (Bray et but that it is a factor that requires consideration in L. al., 2003). The limited use of pentamidine, often in a infantum leishmaniasis, where there is needle trans- zoonotic setting, suggests that resistance should not mission (Alvar et al., 1997). However, it is clearly a be a problem. factor of major importance in anthroponotic disease foci such as L. donovani in Bihar State, India (Sundar, 2001). This does not preclude a number of studies WHAT TO DO ABOUT THE PROBLEM? that describe the development of resistance in par- In a 2001 review, Bryceson stated “At the moment, asites normally associated with zoonotic infections there seems to be no policy at an international or in animals (Gramiccia Gradoni & Orsini, 1992) or national level to prevent the emergence of para- in humans during long courses of treatment, espe- site resistance to anti-leishmanial drugs”. Given the cially in immunocompromised patients (Faraut- problems discussed above, and the tools available, Gambarelli et al.,1997); rather, these studies must be seen in the context of the wider population. Also, it is not ignoring observations that indicate that

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 113 within zoonotic populations of leishmaniasis there DOTS are populations that are highly drug insensitive (e.g. The introduction of oral drugs for leishmaniasis Grogl et al., 1992). These populations probably have offers advantages of improved compliance, self- a highly stable “resistance” phenotype (and geno- administration and reduced costs. In the phase IV type) and are transmitted from host to host. trial for miltefosine, a seven-day supply is issued to patients who have to return to the clinic each week So what strategies are available to combat the prob- for examination and re-supply. For drugs like milt- lems of drug resistance? efosine which have a long half-life and a propensity for selection of resistant forms, the monitoring of Monitoring drug resistance daily dosing and the completion of a course of treatment is essential. The directly observed treatment In all cases we require better methods to monitor (DOTS) strategy for TB chemotherapy was success- drug resistance through either (i) phenotypic sensi- fully introduced in India by the Revised National tivity of parasite isolates, or (ii) molecular changes TB Control Programme (RNTCP) in 1997 (see WHO that indicate alterations in either the drug target Report, 2003. Global Tuberculosis Control – www. or mechanisms that alter the intra-parasite level of who.int/gtb/publications/globerep/index.html). active drug. The potential for use of a parallel system for the control of leishmaniasis, both for miltefosine now There are problems with both approaches: and maybe sitamaquine in the future, should be (i) The determination of drug sensitivity of clini- considered. cal isolates is open to the criticism that pathogen adaptation from host to culture medium imme- Diagnostic methods diately selects for a sub-population of pathogens best suited for growth in that medium. The The development of non-invasive serological diag- drug sensitivity of parasites must therefore be nostic methods with high sensitivity and specificity, tested as soon as possible after isolation from for example DAT, K39, and Katex (urine dipstick), the patient using carefully defined protocols. is a major advance in the control of leishmaniasis Although promastigote assays are easiest and (see Guerin et al., 2002). In this context, it is impor- quickest, this assay is not predictive for pen- tant to evaluate these kits for the monitoring of drug tavalent antimonials, and possibly not for other response in patients. The antigen detecting katex anti-leishmanials, for example paromomycin, (Sakari, Chance & Hommel, 2002) might be of par- pentamidine and miltefosine. The amastigote- ticular interest in this case. macrophage assay is currently the only model able to correlate clinical response to the sensi- The variation in species sensitivity has greatest clini- tivity of the isolate, as demonstrated in relation cal significance in Central and South America where to pentavalent antimonials (Ibrahim et al., 1994; the distribution of L. mexicana, L. amazonensis, L. pan- Lira et al., 1999). Axenic amastigotes are sensi- amensis, L. braziliensis and other members of these tive to antimonials but adaptation of isolates is groups overlap. Diagnosis by microscopy is possi- both too selective and too lengthy a process to ble for trained staff, otherwise molecular tools have be used in this type of assay (Ephros, Waldman been developed that could be implemented. & Zilberstein, 1997; Sereno and Lemesre, 1997). (ii) The ability to develop molecular probes or poly- Combinations merase chain reaction (PCR) based diagnos- Drug combinations have proved to be an essential tics to monitor the development and spread feature of antimicrobial treatment through design of drug resistance is severely limited by a lack or use to: (i) increase activity through use of com- of knowledge of the molecular and biochemi- pounds with synergistic or additive activity, (ii) pre- cal mechanisms of action and resistance of most vent the emergence of drug resistance, (iii) lower anti-leishmanial drugs, especially in clinical iso- required doses, reducing chances of toxic side effects lates (see above). Molecular techniques using and cost, and/or (iv) increase the spectrum of activ- cosmid vectors have been successfully used to ity, for example anti-leishmanial and anti-inflamma- identify drug resistant loci in Leishmania, in par- tory or immunodulator in cutaneous leishmaniasis. ticular those to antifolates, pentamidine and ste- Previous studies on drug combinations for VL, for rol biosynthesis inhibitors (Cotrim, Garrity & example allopurinol plus sodium stibogluconate Beverly, 1999; Coelho, Beverley & Cotrim, 2003). (Chunge et al., 1985) and paromomycin plus sodium stibogluconate (Chunge et al., 1990; Neal et al., 1995; Thakur et al., 2000), have aimed to improve efficacy.

114 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 The use of combinations to prevent resistance has iasis however, especially India, acquired resistance been well rehearsed for antimalarials. Resistance due to pentavalent antimonials has occurred and effec- to point mutations has been estimated in symptom- tive monitoring of drug resistance is needed. No atic individuals that harbour up to about 1012 para- molecular markers of resistance are available for sites. If a target enzyme has a mutation rate of 10-7, currently used anti-leishmanial drugs. The only reli- the chance of resistance developing to a single agent able method to monitor resistance of isolates is the is high, but the likelihood of resistance to two com- technically demanding in vitro amastigote-macro- pounds with different targets is very low (White and phage model. New treatments for visceral leishman- Pongtavornpinyo, 2002). Studies to identify such iasis have been introduced and others are on clinical combinations are new for leishmaniasis; limited trial. Care needs to be taken that resistance does not studies are under way to examine the interactions develop to these drugs, and regimens of simultane- between miltefosine and other anti-leishmanials to ous or sequential combinations need to be consid- identify suitable combinations (Seifert and Croft, ered as well as systems to monitor drug use, drug unpublished). Bryceson (2001) has made the case for response and spread of resistance. examining combinations of strong anti-leishmanials with “weak” drugs (for example, azoles); this is an Acknowledgements approach also used in malaria treatment, for example the inclusion of clindamycin or azithromycin in Thanks to all the team of the EC INCO-DEV proj- combinations. Combinations should also consider ect “Molecular tools for monitoring emergence and both simultaneous and sequential administration. spread of drug resistance among natural populations of Leishmania” [ICA4-CT-2001-10076], led by Reversal agents Jean-Claude Dujardin, for many stimulating discussions. The strategy to reverse resistance has long been discussed in relation to chloroquine resistance in Plasmodium falciparum, and has looked interesting References experimentally but not clinically. In laboratory stud- Al-Jaser MH. Drug Sensitivity of cutaneous leishmani- ies, a series of sesquiterpenes have been shown to asis in Al-Kharj, Saudi Arabia [PhD thesis]. London, reverse drug resistance due to P-glycoproteins in a University of London, 1995. L. tropica clone (Perez-Victoria et al., 2002). Another study suggested a strategy of inhibition of thiol lev- Al-Jaser M, El-Yazigi A, Croft SL. Pharmacokinetics els by co-administration of antimony with an inhibi- of antimony in patients treated with sodium stiboglu- tor of glutathione synthesis (Carter et al., 2003). It is, conate for cutaneous leishmaniasis. Pharmaceutical however, unlikely that these approaches will have Research, 1995, 12:113–116. any clinical relevance. Allen S, Neal RA. The in vitro susceptibility of macrophages infected with amastigotes of Leishmania spp. to New targets, new drugs pentavalent antimonial drugs and other compounds There are few better ways to avoid drug resistance with special relevance to cutaneous isolates. In: Hart than to have an adequate armoury of drugs with DT, ed. Leishmaniasis. New York, Plenum Press, 1989. different targets and no cross-resistance. Although Alvar J et al. Leishmania and human immunodefi- miltefosine, paromomycin and sitamaquine are in ciency virus coinfection: the first 10 years. Clinical various stages of development for leishmaniasis Microbiological Reviews, 1997, 10:298–319. treatment (Croft and Coombs, 2003), not all have Arana FE et al. Involvement of thiol metabolism completed the final steps for registration and all in resistance to Glucantime in Leishmania tropica. have clear limitations. There are still insufficient Biochemical Pharmacology, 1998, 56:1201–1208. data as to whether the compounds fulfil all necessary criteria and a need for new drugs. Asilian A et al. Treatment of cutaneous leishmaniasis with aminosidine (paromomycin) ointment: double- CONCLUSIONS blind, randomized trial in the Islamic Republic of Iran. Bulletin of the World Health Organization, 2003, 81:353– Variation in the efficacy of drugs in the treatment 359. of leishmaniasis is frequently due to differences in Avila JL, Casanova MA. Comparative effects of 4- drug sensitivity of Leishmania species, the immune aminopyrazolopyrimidine, its 2’-deoxyriboside status of the patient, or the pharmacokinetic proper- derivative, and allopurinol on in vitro growth of ties of the drug. Most leishmaniasis is zoonotic, and American Leishmania species. Antimicrobial Agents and acquired drug resistance is not an important consid- Chemotherapy, 1982, 22:380–385. eration. In areas of anthroponotic visceral leishman-

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Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 119 7.2 FROM GENOME TO mal inoculation. Direct examination works best for DISCOVERY IN LEISHMANIA: visceral leishmaniasis (VL) due to parasite density in the smears, with spleen aspirates being more sen- RECOMMENDATIONS FOR THE sitive than bone marrow or lymph node aspirates. DEVELOPMENT OF DRUGS, Parasitological diagnosis of cutaneous leishmaniasis VACCINES AND DIAGNOSTICS (CL) is made by demonstrating amastigotes in skin lesions using skin biopsy and culture of these spec- Dan Zilberstein,1 Kenneth Stuart2 and imens. Culture-based diagnosis of mucocutaneous Alan Fairlamb3 leishmaniasis has very low sensitivity as the organ- 1 Department of biology, Technion-Israel Institute of isms are often scant. Technology, Haifa, Israel 2 Seattle Biomedical Research Institute, Seattle, USA Serological diagnosis is based on the presence of a 3 Division of Biological Chemistry and Molecular specific humoral response, as in visceral leishman- Microbiology, Wellcome Trust Biocentre, University of iasis, or on the cell-mediated immune response, as Dundee, Scotland, UK in cutaneous and mucocutaneous leishmaniasis [1]. A wide range of serological methods, which vary in sensitivity and specificity, are available for the diagnosis of visceral leishmaniasis; these tests can INTRODUCTION be grouped into non-specific and specific. Non-specific tests have been used in the past but are rarely The completion of the Leishmania major genome used today; they include: indirect haemagglutina- sequence is a landmark that, for the first time, identi- tion, counter-current immunoelectrophoresis, and fies all genes that encode proteins and processes that immunodiffusion. These are cumbersome tests and may be targets for drug development and vaccine tend to have lower sensitivity and specificity; an candidates, and useful for diagnostics. However, it example is the Montenegro skin test, a delayed-type is not known how these pathogens utilize the infor- hypersensitivity test. To date, no specific antigen has mation in their genome to establish infection, pre- been identified as a standard for this test, although vent elimination by the host, and cause disease. a few candidates are currently being studied in TDR- financed projects. Recent technological advances in experimental and in silico analyses create a promising environment Specific serological diagnostic methods include the for generating the knowledge needed to combat direct agglutination test (DAT), the immunofluo- the spectrum of leishmanial diseases. These can be rescence antibody test (IFAT), the enzyme-linked utilized, for example, for comparative genomics at immunosorbent assay (ELISA), immunoblotting, both the gene sequence and RNA expression lev- and antigen detection [4]. els, for parasite and host cell expression profiling at the RNA (DNA microarrays) and protein (mass The DAT is highly specific, sensitive, simple, and spectrometry) levels, and for comprehensive analy- inexpensive; it uses whole, stained promastigotes ses of host immune responses to infection or exper- either in suspension or in freeze-dried form. The imental immunization. This review summarizes the freeze-dried form is heat stable and enables applica- current status and recommends future directions for tion of the DAT in the field. However, theDAT has improving the diagnosis, prevention and treatment a major disadvantage: the relatively long incuba- of leishmaniasis using genome-wide technologies. tion time of 18 hours and the requirement for serial dilution of serum [5, 6]. Also, the DAT has no prognostic value, since the test may remain positive for DIAGNOSIS OF LEISHMANIASIS several years. Recently, a modified version of DAT, Current methods in molecular diagnosis of the fast agglutination screen test (FAST), has been leishmaniasis developed for use in endemic regions and for large- scale population screening. Several methods are used for diagnosis of leishmaniasis; these can be classified into three groups The IFAT is one of the most sensitive tests available. – parasitological, serological, molecular. Reliable It is based on detecting antibodies that appear in diagnosis of leishmaniasis is made by demonstrat- the very early stages of infection and are undetect- ing the parasite in biopsy samples or tissue aspi- able six to nine months after cure. If the antibodies rates of the spleen or bone marrow. Identification persist in low titres, it is good indication of relapse by microscopy can be either by direct examination [2, 3]. of tissue smears or following tissue culture or ani-

120 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 The ELISA is the most commonly used test for immu- The best targets for PCR, as for DNA probes, are nodiagnosis of leishmaniasis. The antigens used are still the repetitive sequences. They are either based traditionally derived from axenic promastigotes and on kDNA minicircle conserved regions or complete consist of a repertoire of at least 30 somatic antigens minicircle amplification [14, 15], miniexon-derived and several surface components. The major prob- RNA genes [16], small subunit ribosomal genes [17] lems are specificity and cross-reactivity of species or gp63 [18]. within a family as well as with phylogenetically distant microorganisms [4]. Cross-reactivity also occurs A recently developed combined PCR-ELISA tech- in regions endemic for both leishmaniasis and try- nique has been reported to be sufficiently sensitive panosomiasis. The use of recombinant antigens has and specific for use as a diagnostic test in muco- improved sensitivity and specificity [7]. cutaneous leishmaniasis. PCR is perhaps the most reliable diagnostic method. However, it has not yet Most work concerning the use of immunoblotting been developed for practical use in the field and for diagnosis of leishmaniasis has been carried out awaits further improvement. on VL. Although anti-leishmania antibodies are detectable in CL, they are present only at low titres; Real time PCR is also being applied as a molecular hence immunoblotting has not been widely adopted tool for diagnosis. However, this method is complex, for diagnosis of CL [3]. requires many internal controls, and may also be too complicated and expensive for use in the field. Antigen detection is, in theory, an ideal method for diagnosing an infection, particularly since antigen Both molecular and serological diagnostic methods levels are expected to correlate with the parasite rely on using single antigens or sequences that are load. However, although a few reports have been specific enough to enable identification of the infec- published, no satisfactory antigen detection system tious agent. To date, methods for improving speci- is currently available [8]. Despite the large number ficity and sensitivity are still being developed. of serological tests available, there is no gold standard diagnostic test for VL, mainly because none of New approaches in molecular diagnosis: the tests are 100% sensitive and specific [3]. application of genome-wide technologies High-throughput technologies introduce a new Molecular diagnostic methods include monoclo- approach for diagnosis, namely the dimension of nal antibodies (MAbs), DNA probes, and the poly- simultaneous analysis of many genes and/or pro- merase chain reaction (PCR). MAbs and isoenzyme teins at the same time. The number of diagnostically electrophoresis are undoubtedly the most precise relevant targets identified is growing. Paraphrasing methods for Leishmania identification, but these Clare Fraser of The Institute for Genomic Research techniques are not applicable to the routine diag- (Rockville, MD, USA) in her presentation at the nosis of leishmaniasis. MAbs have the advantage of Association for Molecular Pathology Meeting allowing the characterization of the parasite species (Philadelphia, PA, 17 November 2001), “genomics using ELISA, IFAT or immunohistochemistry. has provided us with a parts list without the instruc- tion manual”. In this context, genomics paves the The successful use of a DNA probe depends on the way for the upstream search for diagnostics, vac- target sequence. Examples are kinetoplast minicir- cines and therapeutics, so that the results can be cle DNA (due to its high copy number, kDNA pro- realized sooner [19]. vides multiple targets for DNA probes and contains a conserved region of at least 120 base pairs), ribo- The world of diagnostics and therapeutics is chang- somal RNA genes, miniexon-derived RNA genes or ing towards greater complexity, and the era of the genomic repeats [3, 9]. PCR provides the most sen- ‘blockbuster drug’ may soon be over, making way sitive diagnostic technique for species specific genes. for the ‘tailor made’ treatment regime. This ‘phar- The PCR assay can detect parasite DNA or RNA a macogenomic strategy’ requires screening and cata- few weeks ahead of the appearance of any clinical loguing of patients’ gene and protein profiles prior to, signs or symptoms. Different DNA sequences in during and following treatment, using efficient high- the genome of Leishmania, e.g. the ITS region, gp63 throughput technology [20]. Pharmacogenomics is locus, telomeric sequences, and targets in rRNA unlikely to be practically useful or affordable for genes such as 18s rRNA and SSU-rRNA, and both orphan diseases for the foreseeable future. conserved and variable regions in kinetoplast DNA (kDNA) minicircles, are being used by numerous groups [10–13]. DNA microarray DNA microarrays make it possible to analyse the

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 121 mRNA expression of thousands of genes simultane- evance of candidate genes. TMAs are microscope ously. The resulting comprehensive gene expression slides containing samples from hundreds of individ- surveys lead to the identification of new genes and ual tumour specimens. They can be used for large- pathways with importance in cancer development scale, massively parallel, in situ analysis of genetic and progression, or as targets for new therapies. alterations on a DNA, RNA and protein level using Furthermore, some reference laboratories are carry- in situ hybridization or immunohistochemistry on ing out 1800 molecularly-based cystic fibrosis tests hundreds of tumour specimens at a time [22]. When per week [19]. A high-density microarray has been using tissues, the accuracy of quantitative gene described that identifies 54 different Mycobacterium profiling is critically dependent on microdissec- species, using 82 unique 16S rRNA sequences and tion, especially on the laser capture microdissection all known mutations associated with rifampicin and (LCM) of individual cells or cell populations [26]. isoniazid resistance in M. tuberculosis [21]. The reasons for this rapid increase are two-fold. First, the Protein microarray era of genomics has provided us with the sequences DNA array work is predicated on the assumption of important organisms including our own (Homo that quantification of cellular levels of mRNA tran- sapiens) as well as many bacterial and viral patho- scripts can function as a surrogate for measuring the gens. The validation and prioritization of genes expression or activity of the protein which is the final emerging from genome screening analyses in large functional product of gene expression. However, series of clinical tumours has become a new bottle- the correlation between the number of mRNA and neck in research [22]. protein molecules is generally not strong enough to predict one value from the measurement of the The current fashion for functional genomics has other [27], as there is at least a ten-fold range of pro- put the spotlight on microarray technologies that tein abundance for mRNAs of a given abundance are capable of comprehensive, quantitative analy- [28]. Certainly, gene expression levels per se tell us sis of RNA expression, with the promise of a global very little about the biological function of a given approach to the quantification of gene expression abundance. [23]. However, there are two conflicting opinions on the value of microarrays: they are either promoted Microarrays are already having a major impact on as an exploratory-driven replacement for hypoth- cancer biology, pharmacology and drug develop- esis-driven biology [24]; or they are criticized as ment and, with their rapid development, new tech- expensive fads that cannot substitute for the old- nologies, methods of analysis and applications are fashioned, low-throughput approach to experimen- emerging continuously. However, the major limiting tal biology. Unquestionably, microarrays can reveal factor in their further application is the current lack associations between gene-expression signatures of data comparability, which is essential for appro- and the biology and outcome of disease – for exam- priate comparisons between different array data. ple, by identifying clinically significant subtypes of cancers. This has raised expectations that the expres- Furthermore, a full understanding of pathogene- sion profile of a particular cancer will help in clinical sis of an infectious agent could ultimately require management of this disease [25]. knowledge of levels of proteins and their variants. Therefore, there is an increasing need to use and Fundamental issues of protocol and platform com- develop proteomics to explain complex disease phe- parability remain to be addressed. There is no stan- notypes. Identification of all protein variants, how dardization of protocols or reagents for sample they relate to each other and the functional conse- acquisition, or RNA or cDNA target preparation. All quences of changes in their levels must be consid- procedural errors are compounded by continuing ered in order to understand the clinical presentation uncertainties in the normalization step. The expres- of complex diseases. Hence, protein arrays appear sion of housekeeping genes, which are frequently to be promising tools for drug screening and used as internal standards, is variable. Therefore, diagnostics. validation of changes in expression in an unrelated, secondary assay remains a critical requirement, and Proteins can be arrayed either on flat solid phases is best accomplished using the real-time reverse or in capillary systems (microfluidic arrays [29]). transcriptase polymerase chain reaction (RT-PCR) Preferred solid phases are modified glass or fil- with its high dynamic range, bearing in mind the ter membranes, because of their low-fluorescence limitations of the technique. background. Nitrocellulose and polyvinylidene difluoride membranes as well as polyamine and Recently, a tissue microarray (TMA) technology has been developed that efficiently tests the clinical rel-

122 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 polyaldehyde derivatized slides have also been So far, a microarray-based immunoassay involving used [20]. 18 different autoantigens and high-throughput arrays featuring 196 distinct biomolecules and containing The concept of antibody arrays is derived from two the major autoantigens of eight distinct human auto- sources: classical immuno-capture assays and cDNA immune diseases, including systemic lupus erythe- arrays. First, high-throughput probing of hundreds matosis and rheumatoid arthritis, have been reported. of compounds in complex mixtures (e.g. body flu- These are the first, but extremely promising, steps ids or tissues) can be miniaturized. Second, robust, towards high-throughput protein arrays as diagnos- hybridization-like antibody–antigen binding can tic tools, eventually arriving at the doctor’s office and be used in a standard way to identify all or certain as over-the-counter applications [20]. marker proteins expressed from a genome. Their common molecular features make antibodies ideal Functional proteomics provides a powerful approach candidates for standard assay formats and the cre- to screen for alterations in protein expression and ation of protein-expression profiles, complementing posttranslational modifications under conditions of and extending diagnostic options offered by DNA human disease [35]. microarrays [30]. Multidimensional chromatography of cell and tis- Analyses sue lysates or cellular subfractions could become an important alternative to recombinant protein expres- As indicated above, the use of high-throughput sion. By consecutive chromatography and isoelectric technologies has lead to a new era in diagnosis. It focusing, cellular protein extracts have been directly enables screening and cataloguing of patients’ gene fractionated, arrayed and detected with antibod- and protein profiles prior to, during and follow- ies [36]. For example, protein screening to examine ing treatment, using efficient high-throughput tech- markers of melanoma progression, by profiling mela- nology. Based on the concept of the arrayed library, nocyte versus melanoma cell lines using two-dimen- cDNA clone arrays can be used for high-through- sional electrophoresis and mass spectrometry [35]. put expression and analysis of unknown proteins [31]. As a variant of this principle, antibodies can be screened and selected from phage display librar- Recommendations ies using bacterial clone arrays [32]. Building upon As indicated above, current methods of diagnosis of established DNA microarray technology, arrays of leishmaniasis include the parasitological and sero- purified proteins represent the next level diagnos- logical, using patient tissue smears from biopsy or tic tools, allowing direct analysis of gene functions. sera against specific parasite antigens. The emer- Protein arrays enable miniaturized and highly par- gence of genetic engineering allows molecular allel ligand binding assays for protein identification, diagnosis, which includes amplification of species- quantification and affinity studies [33]. specific parasite sequences using the PCR. We recommend TDR support the application of DNA and Protein affinity assays are used to analyse interac- protein microarray technologies for the diagnosis tions between proteins such as antibodies, receptors of leishmaniasis as well as for the study of parasite or enzymes with other proteins, peptides, low-molec- virulence, development and host-pathogen interac- ular-weight compounds, oligosaccharides or DNA. tions as follows: Protein profiles and interactions can already be correlated to states of cellular activity and disease [20]. • The use of DNA microarrays for the identification of better and more species-specific markers. Using new bioinformatics software (genetic algo- The following directions should be encouraged: rithms and cluster analysis), proteomic patterns in – Comparative genomics of the various patho- serum were shown to completely discriminate can- genic Leishmania strains/species. These cer from non-cancer within the ovary [34]. These studies should elucidate all possible species- findings indicate that proteomic pattern analysis specific genes. In addition, their expression might ultimately be applied as a screening tool for profiles during infection should be assessed. cancer in high-risk and general populations. This – Gene expression of host genes during infec- also applies to autoimmune diseases, by screening tion. This can be done using either DNA sera of patients or high-risk individuals for the pres- microarrays of infected vs. non infected mac- ence of specific autoantibodies, using arrays of large rophage cell lines or with patient tissues. The numbers of recombinant proteins of known identity results of these studies should provide impor- [20]. tant information on host-parasite interactions as well as the host reaction to parasite vir-

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 123 ulence. These results will also be useful for pare mRNA with protein abundance in order improved diagnosis. to assess whether it is necessary to use both • Proteomics is an additional tool to study gene for diagnosis as well as studying development. expression. Since, regulation of gene expres- – Development of protein microarrays of sion in Leishmania is post transcriptional, the Leishmania proteins. This can be done by pre- appearance of specific proteins is critical for the paring a Leishmania expression library of the study of pathogenesis, drug development and whole genome and expressing all in bacteria diagnosis. The following directions should be followed by printing the proteins/peptides encouraged: on a solid phase, as has been recently done – Identification and characterization of with yeast. This will be useful for the study Leishmania proteins in all developmental of phenotypic expression in Leishmania and stages, using state of the art methodology. more importantly for diagnosis. This should also include post-translational • Development of tools for genome wide-based modifications such as phosphorylation and diagnosis in the field for genomic and proteomic glycosylation microarray analyses. – Comparative proteomics of pathogenic species. Ideally, the diagnostics that are developed should – Comparative analyses of gene expression, e.g. be sensitive and detect infection and distinguish mRNA and protein abundance throughout between prior exposure and current infection. They the life stages of Leishmania. This includes the should be specific and distinguish Leishmania from development of bioinformatic tools to com- other pathogens and non-pathogens and distin-

Table 1. Drugs used to treat visceral leishmaniasis and their principal limitations (references 42–45)

Drug Structure Main limitations or issues Pentavalent antimonials Drug resistance (up to 65% in Bihar, India) Sodium stibogluconate (Pentostam) Parenteral administration (i.m. or i.v.) Meglumine antimonate (Glucantime) Prolonged treatment (up to 4 weeks) Toxicity (liver and pancreas, especially in HIV patients) Poor compliance Cost (US$ 120–150) Diamidines Parenteral administration (i.m.) 4,4’-Diamidino-α,ω-diphenoxypentane: Prolonged treatment (up to 4 weeks) Di-isethionate salt (Pentamidine) Toxicity (myalgia, nausea, Dimesylate salt (Lomidine) hypoglycaemia, diabetes) Poor response rates (around 75%) Cost (US$ 60–150) Polyene antibiotics Parenteral administration (slow i.v. infusion) Amphotericin B Prolonged hospitalization Desoxycholate formulation (Fungizone) Severe toxicity (hypokalaemia, renal failure, Lipid liposomal formulation (AmBisome) metabolic acidosis, cardiotoxicity) Cost (Fungizone US$ 60–150; AmBisome > US$ 1000)

Aminoglycoside antibiotics Parenteral administration (i.m.) Paromomycin sulphate (Aminosidine) Prolonged treatment (up to 3 weeks) Toxicity (nephrotoxicity, nerve deafness) Cost (US$ 50) Availability

Ether lipid analogues Toxicity (teratogenicity: contraindicated Hexadecylphosphorylcholine (Miltefosine) in pregnant women) Ease of resistance (long half life of drug) Patient compliance Cost (US$ 50)

124 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 guish parasites that are likely to cause serious or The genome sequence provides a substantial oppor- lethal disease from those that are likely to result in tunity for identifying novel potential drug tar- mild disease or be asymptomatic. They should be gets through bioinformatics and database mining. able to recognize drug resistant strains and assess Comparative genomic studies could identify bio- the consequences of response to therapy, especially chemical targets that are common to Leishmania and adverse responses such as post kala azar dermal trypanosomes, thereby reducing drug discovery and leishmaniasis. drug development costs of a broad spectrum anti- trypanosomatid drug. Many genes that are unique to Leishmania (or better still, are common with try- DRUG DISCOVERY panosomes but absent from humans) have unknown Current anti-leishmanial drugs functions that may be essential for parasite growth or survival. The essentiality/redundancy of such Current drug treatment of leishmaniasis is unsatis- genes needs to be investigated by gene disruption factory (see Table 1 for a list of principal drugs and or gene knockout, and the biological or biochemical their main limitations). Of particular concern is the function of their products established. Target vali- fact that the clinical value of the front-line pentava- dation is a major bottleneck in drug discovery, and lent antimonial drugs (Pentostam and Glucantime) WHO/TDR could play a coordinating role in this is seriously threatened by the emergence of wide- post genomic activity. As summarized in Table 2, a spread resistance in parts of India and Bangladesh powerful range of genetic tools are now available to [37]. In addition, treatment efficacy with many of address this question [46]. However, unlike African the available drugs is reduced by immunosuppres- trypanosomes [40], Leishmania do not appear to be sion, particularly in patients co-infected with HIV susceptible to RNA interference (RNAi), the current [38, 39]. Thus, new drugs are required primarily for method of choice, possibly due to lack of some or the treatment of the life-threatening visceral form all components of the RNAi machinery [41]. Thus, of leishmaniasis and secondarily for the disfiguring improved genetic methods for target validation mucocutaneous and cutaneous forms of the disease. need to be developed. WHO/TDR supports a num-

Table 2. Genetic tools in trypanosomatids (adapted from reference 46)

T. brucei T. cruzi L. major In vitro culture models ++ ++ +++ In vivo disease models +++ ++ +++ Transient transfection +++ + ++ Stable transfection +++ ++ +++ Expression vectors – Episomal + ++ +++ – Integrating +++ + +++ +++ - + – Regulatable Selectable markers – Positive 6 3 8 – Negative 1 1 2 Gene knockouts +++ ++ +++ Sexual crossing + ? - Positional cloning Possible ND - RNA interference (RNAi) +++ - - Functional rescue + ND +++ Transposon mutagenesis + - ++ Genome size (Mb) 35 40 35 Number of chromosomes 11 plus mini- >30 ~36 chromosomes

ND = not determined +, ++, +++ = the given method has been developed; the number of + symbols reflects an approximate assessment of how well the method works and/or the extent of its use

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 125 ber of centres carrying out small molecule screening VACCINE DEVELOPMENT against Leishmania and other parasites. Improved reporter methods to increase throughput in cellular Current anti-leishmanial vaccines assays are required to identify new classes of lead The immunity that follows recovery from Leishmania compounds with novel modes of action (chemical infection illustrates the feasibility of developing genomics). an effective vaccine; however, no such anti-leishmanial vaccine is available for practical use at this The Leishmania genome also provides new opportu- time. The protective immunity against Leishmania nities to further define the mode of action of existing entails strong Th1 T-cell responses with associated drugs, with a view to developing novel classes of IFN-γ and IL-2 production, while disease is associ- drugs against existing targets or providing a scien- ated with a Th2 response and production of associ- tific rationale for combination chemotherapy. Drug ated cytokines. The outcome of infection depends resistance is a serious threat to current therapy and on the balance between Th1 and Th2 responses, a more detailed understanding of resistance mecha- with the former being associated with disease reso- nisms in clinical isolates may reveal novel strategies lution as well as protective immunity. Experimental to circumvent drug resistance. The development of mouse model systems have been useful in gener- improved diagnostic methods could be useful in ating these insights, and generally parallel the monitoring clinical responses to drug therapy and as immune responses and disease outcome in human a predictor of drug sensitivity in clinical isolates. At infections. Mouse model systems have also been the present time, few compounds are in the late dis- used to explore the development of anti-leishman- covery or preclinical phases of development. WHO/ ial vaccines. Characteristically these models employ TDR should be proactive in identifying suitable val- strains of mice and parasites that differ in suscep- idated “druggable” targets and funding the devel- tibility and virulence, respectively. The host and opment of robust, cheap and reproducible assay pathogen strains are selected to mimic natural dis- methods for high-throughput screening. ease, which illustrates the importance of both host and pathogen factors in disease. As with any model Recommendations system, care must be taken when extrapolating to the human host as well as to parasite strains that occur The following activities are recommended in order in the field. Studies to develop leishmanial vaccine to translate genome information into drug discovery have utilized antigens ranging from live parasites to and development: subunit vaccines [47, 48]. These studies have illus- • Target identification by database mining and trated that development of an effective vaccine also metabolic pathway reconstruction (identify requires an appropriate immunization protocol and novel or unique/different pathways or enzymes adjuvant to elicit the needed Th1 response. common to trypanosomatids). • Target validation using reverse genetics (identify Live vaccines utilizing species of Leishmania that genes essential for amastigote growth/survival). cause cutaneous disease result in strong immune • Development of inducible systems for condi- responses after healing of the lesions in humans. tional gene knockout and RNAi, possibly by Attempts have been made to standardize the inocu- knock-in of the dicer system. lum for reasons of reliability, safety, and efficacy [49]. • Target characterization (establish biochemical Similarly, attenuated Leishmania that have been gen- function, molecular mechanism and structure). erated by genetic modification through elimination • Development of transgenic parasites with of gp63 protease [50] or DHFR-TS [51] genes have reporter systems for use in WHO/TDR screen- been examined in mouse model systems. Protection ing centres (in vitro and in vivo). is achieved with these vaccines, although it is not • Assay development and high-throughput fully analysed with respect to cross-species pro- screening (identify lead compounds). tection. However, the use of live or live attenuated • Screening of lead compounds against whole parasites as vaccines is unlikely to be practical for cells (chemical validation of target; demonstra- reasons of safety and economics. Non-live complex tion of selectivity for parasite versus host). vaccines consisting of killed cells or cellular fractions • Comparative studies at the DNA, RNA and pro- have also been explored as potential vaccines. These tein levels in drug sensitive and drug resistant include immunization with killed L. amazonenesis, L. lines (modes of drug action and drug resistance). major, L. mexicana, and L. braziliensis alone or in com- • Method development for predicting inhibitor bination; these were administered variously with effectiveness and drug toxicity. BCG or cytokines as adjuvants for both prophylactic and therapeutic vaccines, and were tested in mice and dogs and in human trials. They appear safe and

126 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 elicit significant protection from natural infection in bility and economy of such vaccines, make DNA humans and animal model systems, although the vaccines attractive. In addition, gene (and hence anti- proportion of protected humans and the utility of gen) variation among strains and species is likely to delayed-type hypersensitivity as a surrogate marker be an important consideration for the development of protection is uncertain [47, 52, 53]. of vaccines that broadly cross-protect among strains and species. Analysis of such variation at the DNA Several recombinant proteins have been assessed level, as well as accommodating it in a DNA vaccine, as vaccine antigens. Such antigens offer the obvious is reasonably straightforward. advantages of control of composition and reproducibility; however, they elicit a narrow immune response Recommendations and hence their efficacy is less certain. The antigens Substantial evidence exists for the ability to tested include proteins encoded in multi-gene fami- develop a vaccine, although the specific correlates lies such as the gp63 protease and the membrane anti- of acquired and induced immunity are yet to be gen known as gp46/M2 or PSA2. Analyses in animal defined. Therefore, we recommend support for vac- model systems have indicated species-specific dif- cine development with an emphasis on genome- ferences in protection and potential importance of wide approach. We suggest: conformational epitopes. Other antigens tested as • Proteome-wide analyses (protein microarray vaccine candidates include LACK, the homologue studies) to identify protein antigens that are pre- of receptor activated C kinase; Leishmania eukaryotic dicted to protect against infection or disease. ribosome protein (LeIF), the homologue of an initi- • Identification of amastigote-specific antigens in ation factor 4A; TSA (trypanothione peroxidase), a the entire proteome. homologue of a eukaryotic thiol-specific antioxidant • Characterization of immune responses specific protein; M15, a homologue of a yeast stress induc- for natural or induced protection. ible protein; and LCR1, which has homology with • Development of live attenuated (recombinant) a T. cruzi flagellar antigen. LeIF, TSA, and M15 have parasite vaccines. been tested alone and in combination. More recently • Combined vaccine development and immuno- identified vaccine candidates include amastigote pathogenesis studies. specific antigens A2, P4 and P8. Synthetic peptides • Studies of epitopes that may cross-react between derived from the above antigens, as well as the non- host and pathogen. protein antigen Leishmania lipophosphoglycan (LPG), • Assessment of protection in mouse model sys- induced protective immunity, but further study is tems with parallel human analyses. needed. In general, these antigens vary in ability to • Identification of T-cell receptor associated pep- induce protection, and this ability is often influenced tide antigens (mass spectrometry), and reactiv- by the adjuvant and immunization scheme. The ity to specific antigens by ELISPOT and protein protein antigens induce variable degrees of partial arrays. immunity with varying cross-species protection. The • Use of DNA vaccines as a tool for identifying peptide antigens result in limited protection, while vaccine candidates. protection with LPG is more robust. Detailed assess- • Exploration of immunization strategies – prime/ ment of these vaccine candidates must be considered boost and adjuvant combinations. as work in progress [54]. Mouse model systems have been very fruitful and Identification of vaccine candidates entails vari- should continue to be used in combination with ous approaches, ranging from studies unrelated to genetic manipulation of the pathogens and in con- vaccine development to various forms of immune cert with examination of human parallels to the interrogation of cellular or recombinant leishman- experimental mouse model systems. Such stud- ial proteins. DNA encoding these antigens has been ies will advance our understanding of factors that used not only as an immunogen to stimulate T-cell are critical for the development of disease and can responses but also as a method to screen numerous lead to insights that could be used to develop pre- potential protein antigens [55]. Complexities aris- ventative or therapeutic measures or for patient ing from the importance of post-translational mod- management. ification in inducing immunity and the possibility of inhibitory interactions among gene products potentially limit this approach. However, with the CONCLUDING REMARKS completion of the Leishmania genome sequence, the The near completion of genome sequencing for L. potential for a comprehensive survey of all possi- major and other kinetoplastids has provided us with ble Leishmania protein antigens is now feasible. The an unparalleled wealth of information relating to availability of appropriate assays, as well as the sta-

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Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 129 7.3 CAN LEISHMANIASIS sis in different settings is crucial to prevention and STILL BE CONSIDERED A TDR control and to mobilizing resources. For example, if case detection and treatment are important, then CATEGORY 1 DISEASE? IF educational strategies for affected populations that MOVED, WHAT IS REQUIRED? promote understanding of the disease, the urgency of early diagnosis, and adherence to treatment are Nancy Gore Saravia required, as are development and evaluation of Centro Internacionale de Entrenamiento e point-of-care diagnostic tools, and the capacity to Investigaciones Medicas (CIDEIM) evaluate alternative less toxic oral or topical thera- Cali pies. If vector control is a key issue, then the capac- COLOMBIA ity to conduct entomological assessment of areas of transmission in order to target control measures and evaluate the cost-effectiveness and sustainability of the intervention are needed. I would like to begin the assessment of research and capacity needs for the control of leishmaniasis with Worldwide, the epidemiological circumstances an authoritative view on science and problem solv- of Leishmania transmission vary widely (Desjeux, ing from a giant of innovation: 2001). Moreover, the circumstances of transmission are continually evolving in relation to environmen- “If we all did the things we were capable of doing, we tal, demographic and human behavioural fac- would literally astound ourselves.” —Thomas Edison tors (Lainson, 1989). Urbanization of transmission, spread of HIV co-infection, impact of land use on transmission, and internal and international conflict have changed the landscape of leishmaniasis. Data THE CHALLENGES OF PREVENTION on, and knowledge about, these changes and their AND CONTROL OF LEISHMANIASIS significance for control are scarce, almost anecdotal. Endemic leishmaniasis is perceived as a problem of Communication of the knowledge gained in investi- low priority by governments, society and patients gating specific foci of transmission and interventions themselves. Even though case detection and treat- is often delayed for years, or not undertaken due ment are the primary control measure in most to the arduous and unfamiliar demands of publica- endemic settings, and leishmaniasis is a disease of tion in national and international journals, language obligatory notification in 33 of 88 endemic coun- constraints and limited experience of endemic countries, passive detection and under-reporting prevail try scientists and control personnel in communicat- (Desjeux, 2004). Factors that contribute to the per- ing through this medium. Alternative mechanisms ception of low priority include: and media for systematically communicating epi- • other or urgent health problems of greater demiological trends, associated factors and results magnitude or impact (medical, public health, of interventions at the national and regional level political) are needed in order to make information oppor- • limited resources (human, organizational, tunely available. National epidemiological bulle- financial) tins along the lines of the United States Center for • lack of current information about the magnitude Disease Prevention and Control (CDC) Morbidity or nature of the problem and Mortality Weekly Report (MMWR) might begin • absence of knowledge about the most effec- to address this information gap. Capacity building tive points for intervention in the problem (as to implement such a medium of communication opposed to reacting to the ultimate, often epi- in developing countries could have a high payoff demic, manifestation of the problem) simply by making information available to public • ignorance in affected communities about the health authorities, investigators, and policy-makers. importance and availability of diagnosis and Likewise, development of capacity in epidemiolog- treatment. ical assessment, surveillance and reporting for disease control authorities would empower those on In fact, leishmaniasis is not a health priority in most the front line to convert the results of their work endemic countries, but it is important in all of them. into accessible information, which is fundamental to What is important about leishmaniasis in individ- resolving the problem. ual countries can be very different, and if what is important can be identified, then this may become Accessible epidemiological information is necessary a priority even if the disease is not. The capacity for integration of control measures. Information to determine what is important about leishmania- technologies such as geographic information sys-

130 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 tems have begun to be exploited in the surveillance the adage “No pain no gain”. But is it pathogenic- of risk for outbreaks of leishmaniasis (King et al, ity, or capacity to complete the life cycle in the host, 2004). Integration of leishmaniasis control with the including persistence, that determines immunoge- control of other vector borne diseases could allow nicity? Asymptomatic infections with Leishmania limited resources to be used more effectively, as has occur, and perhaps accumulate, in a variable pro- been proposed and widely recommended. However, portion of the populations in endemic settings this is not automatic, nor are data substantiating (Saravia, 1990; Weigle et al, 1993; Davies et al,1995); effectiveness and cost savings readily available. most of these remain asymptomatic indefinitely Investigating and planning how interventions for unless immune status changes. Hence pathogenic- different vector borne diseases can be integrated, ity does not appear to be essential to protection. Can evaluating the effectiveness of integrated measures, targeted gene deletion extricate pathogenicity and and determining the savings generated, constitute leave protective immunogenicity intact? Deletion requirements for successful and sustainable integra- of genes encoding “virulence factors” while retain- tion of leishmaniasis control with control of other ing infectivity and persistence in vivo, provides evi- vector borne diseases. Emerging problems such as dence that this may be achievable (Spath et al., 2003; yellow fever, dengue and West Nile virus can divert Alexander et al.,1998). The feasibility of enhancing resources from other vector borne diseases such as the immunogenicity of an attenuated live vaccine by leishmaniasis, but this is less likely to impair con- recombinant genetics or exploitation of non-specific trol if integrated programmes anticipate such cir- immune activation through CpG sequences (Rhee cumstances. et al., 2002; Mendez et al., 2003) or other Toll receptor ligands to achieve healing is also supported by experimental and clinical evidence (Buates and VACCINATION Matlashewski, 1999; Arevalo et al., 2001). For quite a long time, it has been known that, in most cases, symptomatic natural infection of immu- What are the technical specifications of a live atten- nocompetent individuals with Leishmania of any uated Leishmania vaccine? How will we know when species and in any epidemiological context leads we have a “viable” candidate? What is the appropri- to resistance (perhaps lifelong) against new disease. ate “gauntlet” of evaluation for this or any vaccine? The practice of leishmanization derived from this This information is lacking, yet would serve to focus empirical knowledge, and accessible data from some research and development efforts at the preclinical of the vast experience with leishmanization, substan- and clinical stages of evaluation. tiate the feasibility of inducing protective immunity by artificial infection (Gurges, 1971; Greenblatt,1980; Safety, production including quality control, stabil- Kellina, 1981; Nadim, Javadian & Mohebali, 1997). ity, and delivery are major concerns for all vaccines. So the feasibility of vaccination against leishmani- Live vaccines, especially, provoke concerns of vio- asis seems self-evident. How to accomplish this is lating the Hippocratic oath (“first do no harm”) and another issue. During the past two decades, thou- of legal and financial liability, though other types sands of research reports on leishmaniasis have been of vaccine are not without risks. On the other hand, published, much has been learned, and the feasibil- live vaccines contain the antigenic repertoire needed ity of immunoprevention has been experimentally to induce protection in genetically heterogeneous demonstrated with a range of antigens, adjuvants populations,and engage the local, regional and sys- and strategies of administration. Nevertheless, no temic immune system. Furthermore, attenuated vaccine candidate has approximated the protection Leishmania may persist indefinitely (Alexander,1998; afforded to humans by leishmanization. Streit, 2001), thereby maintaining a state of concomitant immunity. How do the potential benefits weigh Experience with leishmanization and with atten- in against the risks? While a molecularly defined uated Leishmania in experimental models has sug- subunit vaccine is being developed, are we missing gested that pathogenicity (“virulence” as defined by the opportunity to prevent the serious consequences the production of a lesion) is associated with protec- of leishmaniasis by failing to apply the lessons of tion. Avirulent strains either fail to induce protec- leishmanization, the immunobiology of experi- tion or do so only partially. Immunotherapy with mental leishmaniasis, the Leishmania genome and killed Leishmania has provided proof of principle recombinant genetics, in designing a live, but eradi- that intervention of the immune response can reduce cable, attenuated, non-revertible Leishmania vaccine? morbidity (Convit et al., 1987, 1989, 2004). Yet even Alternatively, could DNA vaccines in a replicating successful immunotherapy using killed Leishmania vector approximate a live vaccine (Gonzalo et al., has required BCG or multiple series of ten daily 2002)? inoculations of antigen, apparently vindicating

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 131 FIELD TRIALS AND CHALLENGE tutions in middle and advanced developing STUDIES countries or, alternatively, industrialized disease endemic countries Protection of humans from disease is the ultimate • international guidelines and mechanisms for criterion in evaluating a Leishmania vaccine. The quality control assurance in the design and con- availability of field sites with sufficiently high, pre- duct of challenge studies. dictable, incidence of new disease in the exposed population, and the accessibility of such high risk What about challenge studies via needle inoculation populations to follow-up, constitute significant of volunteers in unexposed populations? Are these constraints on the development of Leishmania vac- ethically and technically feasible in either devel- cines. In addition, efficacy trials in endemic pop- oped or developing countries? Would the develop- ulations encounter the confounding potential of ment of genetically attenuated Leishmania with drug prior exposure to infection, even if skin test positive susceptibility to a non-toxic, restricted-use antibi- individuals are excluded, and are long, (requiring otic, or other genetic marker that permitted eradi- two or more years after safety and immunogenicity cation, improve the ethical and technical feasibility testing),involve large numbers of participants, and of challenge studies? Does the molecular genetic are very labour intensive. While challenge studies technology required to create a minimally patho- are highly demanding in terms of legal and ethical genic, eradicable, protectively immunizing live vac- hurdles, knowledge of and strict adherence to good cine exist? In-depth, authoritative analysis of these clinical practices (Hope and McMillan, 2004), and questions in the light of technological advances is technical infrastructure, they can be conducted in needed to rally the scientific community for the small numbers of volunteers and provide definitive daunting task of challenge studies. results in a short period of time (WHO/TDR Expert Review, 1997; Levine, 1998). Challenge studies could therefore, expedite the identification of can- LESSONS FROM OLD DRUGS, didate vaccines that induce protection in humans RESISTANCE, AND NEW DRUGS but would not replace field trials of candidates that The toxicity and high cost of the most effective prove effective in challenge studies. They would drugs impose constraints on the treatment of leish- also allow correlates of protection to be identified maniasis. In consequence, many patients in disease by the evaluation of potential surrogate immune endemic countries do not receive adequate treat- parameters prior to challenge and subsequent corre- ment. Avoidance of over-treatment is a major con- lation with outcome (Levine, 1998). cern, This results in reluctance to treat based on clinical diagnsosis and referral to centres equipped The conduct of challenge studies in concert with to conduct parasitological diagnosis in order to leishmanization in endemic settings where leish- receive treatment. The availability of the oral drug manization is still practiced requires political com- miltefosine for visceral leishmaniasis (VL) could mitment, substantial investment in infrastructure, change the management of treatment so that serol- and technical and logistical assistance in the design, ogy, which is highly correlated with clinical VL, or planning, execution, data analysis and evalua- the detection of specific antigen in urine (Attar et al., tion inherent in this type of research. Ethical issues 2001), and standardized clinical criteria would be including informed consent in volunteer popu- sufficient to administer treatment, thereby allowing lations in developing countries, particularly the treatment to be administered closer to home. groups at high risk of leishmaniasis, are formida- ble. The experience gained in attempting to conduct The identification and licensing of the oral drug Leishmania vaccine trials in conjunction with leish- miltefosine for the treatment of visceral leishmania- manization in Uzbekistan and Iran offers invaluable sis caused by L. donovani in India is a major achieve- lessons for building challenge study capability. ment. Implementation of the use of miltefosine, and other drugs in the future, requires regulation and Capacity building to establish challenge study capa- education to prevent misuse and loss of effective- bility would require: ness. The efficacy of this drug for other species of • apprenticeship training at postdoctoral level, of Leishmania causing visceral disease, L. infantum/ highly qualified scientists from disease endemic chagasi, L. amazonensis, and dermatotrophic spe- countries, in vaccine development centres where cies in different geographic regions, is still uncer- challenge studies are safely and rigorously contain. Nevertheless, if this drug proves to be widely ducted effective against viscerotropic and dermatotrophic • scientific and technological infrastructure com- leishmaniasis, disease control through active case mensurate with the task, i.e. established insti- detection and treatment would become possible.

132 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 The teratogenicity of miltefosine, however, presents latently infected asymptomatic residents in emerg- a challenge in the design, execution and manage- ing or expanding foci, could be considered for ment of ambulatory treatment in order to avoid treatment. Furthermore, non-toxic oral or topically inappropriate use. Widespread use of any mono- administered drugs theoretically reduce the need therapy in circumstances of anthroponotic transmis- for etiologic diagnosis and medical supervision of sion creates vulnerability to loss of susceptibility to treatment, since over-treatment would then be less the drug. Adherence to therapy is crucial. Therefore of a concern than failure to treat or inability to make health education strategies to promote this and an etiologic diagnosis at the point of care. If “mass” other personal and community protection methods treatment was found to be a technically feasible con- constitute a priority need. trol strategy, as in the case of onchocercaisis and HIV, such an intervention would require consideration of In addition to proper administration of available cost vs. benefits and the possibility of increased risk drugs, surveillance for the emergence of drug resis- of generating resistant organisms. tance and the use of combined regimens have proven protective of drug utility in other microbial infec- Treatment of HIV co-infection with Leishmania pres- tions. Combinatorial therapy is now recommended ents special challenges. Although in experimental for treatment of P. falciparum malaria (Nosten and murine models amphotericin does not require an Brasseurs, 2002); similar strategies may be useful in intact immune response, as do pentavalent antimo- conserving the life of miltefosine and other new che- nial drugs, patients with HIV co-infection eventu- motherapeutic options. Knowledge of the efficacy, ally relapse following treatment with amphotericin pharmacokinetics, and desirable and undesirable (Alvar et al., 1997; and personal communication). interactions of drugs amenable to combination will Resistance to multiple drugs can develop and could be needed in order to rationally identify combined be transmissible among intravenous drug users or therapies. Since lifelong persistence of infection by sandflies. More effective drugs and therapeu- occurs, even after prolonged treatment with antimo- tic strategies are needed to address this expanding nial drugs, knowing whether miltefosine eradicates problem. Antiretroviral therapy has been shown to Leishmania or reduces the transmissibility of persis- reduce the incidence of VL in HIV-positive popu- tent parasites will be an asset in judiciously manag- lations (De La Rosa et al., 2002), and hence is a key ing the use of this drug in the treatment, prevention consideration in the prevention and control of HIV and control of leishmaniasis. A growing range of co-infection. reporter systems (green fluorescent protein, lucif- erase luc gene) and molecular amplification meth- All available anti-leishmanial drugs are costly in ods are now applicable to the experimental and relation to the financial capability of affected coun- clinical assessment of persistence of parasites post tries and populations. Drug production in devel- treatment, and researchers in endemic countries are oping endemic countries has already lowered the able to address these questions. Concerted proto- cost of antimonial drugs; other drugs such as lipo- col development and conduct of studies can pro- somal amphotericin formulations could be more mote the development of scientific community and accessible to the budgets of high burden countries bridge the gulf that frequently separates disease con- if production could be undertaken in a developing trol authorities and research communities. Problem country. Although few developing countries are in solving capacities, rather than promotion of compe- a position to undertake drug research and develop- tition for limited public resources, can be developed ment or have production capacity for drugs, tech- within the framework of such cooperative goal ori- nological transfer and capital investment could ented endeavours. empower some to generate long-term solutions (Trouiller et al., 2002). Moreover, production of bio- Epidemic anthroponotic leishmaniasis and treat- logicals and drugs could generate sustainable tech- ment failure linked to drug resistance have under- nological and research capacity since the lower cost scored the crucial importance of knowing whether of production and unsatisfied volume of demand parasites are eradicated or reduced to levels below would yield assets that could be reinvested in tech- the threshold of transmissibility by any individual nological capacity and otherwise support economic or combined treatment including immunotherapy. development. If treatment with miltefosine, or any other safe and easily administrable drug, definitively and dura- IMMUNOTHERAPY bly interrupts transmission, this knowledge could open the way for “mass” or community-based treat- Jacinto Convit in Venezuela pioneered immuno- ment. Then, individuals with scars from previ- therapeutic approaches to dermal leishmaniasis, ously untreated leishmaniasis or, conceivably, even unequivocally demonstrating the plausibility of

Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 133 inducing clinical cure by activating and presum- the incentive for early diagnosis. Since antibody ably modifying the ongoing host immune response titres and antigen excretion are generally lower in (Convit et al., 1987, 1989, 2004). Although repetitive the early stages of VL, serological tests and antigen injection of leishmanin and BCG is effective in pro- detection methods that are so useful at later stages moting healing of lesions, the adverse effects of BCG may be less sensitive for early diagnosis. In conse- in a variable but concerning proportion of patients, quence, each advance redefines the nature of the and the need for multiple parenteral inoculations problem as well as its management. over an extended period of time, have discour- aged wide general use of this approach to treatment. NUTRITIONAL INTERVENTION Multiple intralesional injections of killed Leishmania vaccine have also shown evidence of therapeutic Malnutrition has been linked to the development of benefit when administered with antimonial drug VL. Nevertheless, the role of nutrition, young age, (Machado-Pinto, 2002), but this strategy is impracti- and immunological maturity are not clear (Dye and cal for widespread use. Advances in understanding Williams, 1993). Since VL usually occurs in highly of the innate immune response have yielded inno- vulnerable individuals in the context of multifac- vations in topically applicable immunomodulatory tor distress, prevention and control strategies might drugs that can shift a Th2 to a Th1 type response. include “vulnerability reduction” by amelioration of Imiquimod and related compounds act through risk factors such as malnutrition. Rather than exam- Toll-like receptor 7. These compounds have shown ining malnutrition as a risk factor, might we instead promise as coadjuvants in the treatment of dermal examine nutritional supplementation and health leishmaniasis with antimonial drugs (Arevalo et education as protective factors? al., 2002; Buates et al., 1999), whose anti-leishmanial efficacy is dependent on immunocompetence. SOME MACRO CONSIDERATIONS OF Therapeutic gain is currently under investigation PREVENTION AND CONTROL in controlled clinical trials. Nevertheless, topical agents offer practical advantages and would surely Awareness of emerging and expanding foci of leish- be a more acceptable and feasible therapeutic strat- maniasis at an early stage is crucial to prevention egy than injectable formulations. and control. Likewise, understanding of the situation and the intervenable circumstances precludes Immunotherapeutic application of vaccines devel- effective action based on available diagnostic, treat- oped for immunoprophylactic use provides a com- ment and vector/reservoir control strategies. Timely, paratively rapid indicator of disease-modifying accurate information is generally not available for immunogenicity while potentially offering coadju- the control of leishmaniasis; we seem to have a sub- vant benefit. Specifications for immunotherapeutic stantial knowledge base about Leishmania and leish- interventions and consensus guidelines for evalu- maniasis but have not yet brought the collective ation are needed, and would provide a basis upon knowledge to effectively bear on the diverse prob- which to compare different products and regimens. lems that each outbreak, or novel expression of the mutability of the host/parasite interaction, presents. DIAGNOSTICS Bednets, insecticides, personal protection, dog collars, and environmental management have all dem- Despite noteworthy progress represented by the onstrated some utility in controlling transmission in direct agglutination test (DAT) and K39 dipstick different circumstances, yet leishmaniasis is a grow- serology for visceral leishmaniasis, simple, inexpen- ing problem. Hence we could probably say that, in sive, parasitological, diagnostic tools usable by non- fact, “existing tools have not been used effectively”. professional health workers in endemic areas are still However, perfect tools for every situation do not needed. Together with less toxic therapeutics, such exist, so we can also say that, in the case of leish- diagnostic tools would allow treatment and moni- maniasis, while “existing tools are inadequate”, oth- toring of treatment to occur close to the home and to ers, such as a vaccine, are non-existent. We must also be supervised by non-physician medical personnel. recognize that, even if we have learned quite a lot, This in turn would increase access to appropriate we do not have sufficient knowledge of the multiple medical care and lower its cost. Antigen detection in diseases that constitute the leishmaniases to design urine may address this need; however, field testing an effective vaccine, develop a portfolio of drugs, or in different settings of zoonotic and anthroponotic intervene effectively in every circumstance. VL are required to determine the sensitivity, specificity and robustness of this method. Knowledge brokering is a new theme and career opportunity in the global knowledge-based econ- The availability of an effective oral drug increases omy. Screening, organizing and reconfiguring infor-

134 Report of the Scientific Working Group on Leishmaniasis, 2004 • TDR/SWG/04 mation is fundamental to mediating communication Buates S, Matlashewski G. Treatment of experimen- with groups having different objectives, i.e. public tal leishmaniasis with immunomodulators imiquimod health authorities and investigators, communities and S28463: efficacy and mode of action. Journal of and policy-makers. The ability to identify aspects Infectious Diseases, 1999, 179:1485–94. of health policy and management that research can Campos-Neto A et al. Vaccination with plasmid DNA resolve is another essential feature of knowledge encoding TSA/LmSTI1 leishmanial fusion proteins brokering. Knowing what people need to know, and confers protection against Leishmania major infection being able to find that information through research, in susceptible BALB/c mice. Infection and Immunity, boils down to being responsive to demand or the 2002, 70:2828–2836. “market” for research. The ability to find and recognize evidence produced by research builds the Convit J et al. Immunotherapy versus chemotherapy credibility needed to convene different groups to in localized cutaneous leishmaniasis. The Lancet, 1987, interact and thereby articulate demand for knowl- 1(8530):401–405. edge and the supply chain to fulfill that demand Convit J et al. Immunotherapy of localized, intermedi- (Oldham and McLean, 1997). The skills of knowl- ate and diffuse forms of American cutaneous leishma- edge brokering are especially needed by health niasis. Journal of Infectious Diseases, 1989, 160:104–115. researchers in disease endemic countries in order to assure the effective use of available tools, to design Convit J et al. Therapy of Venezuelan patients with better, more acceptable, cost-effective strategies, and, severe mucocutaneous or early lesions of diffuse ultimately, to foster political will. cutaneous leishmaniasis with a vaccine containing pasteurized Leishmania promastigotes and bacillus In conclusion, leishmaniasis is still a Category 1 TDR Calmette-Guerin – preliminary report. Memorias do disease, but we have a huge inventory of assets. The Instituto Oswaldo Cruz, 2004, 99:57–62. challenge is to insightfully and strategically apply Copeland HW, Arana BA, Navin TR. Comparison of what we know, as well as continue to build the active and passive case detection of cutaneous leish- knowledge base to more efficiently and effectively maniasis in Guatemala. American Journal of Tropical detect, treat, and prevent this disease. Medicine and Hygiene, 1990, 43:257–259. Davies CR et al. Cutaneous leishmaniasis in the Acknowledgements: Peruvian Andes: An epidemiological study of infection and immunity. Epidemiology and Infection, 1995, I would like to thank Dr. MR Yaghoobi-Ershadi of 114:297–318. the School of Public Health and Institute of Public Health Research, Tehran University of Medical Davies CR et al. Cutaneous leishmaniasis in the Sciences, for kindly providing a perspective on Peruvian Andes: factors associated with clinical symp- research and capacity building priorities in Iran. toms, response to treatment and parasite isolation rate. Clinical Infectious Diseases, 1997, 25:302–310. References Davies CR et al. Leishmaniasis: New approaches to disease control. British Medical Journal, 2003, Alexander J, Coombs GH, Mottram JC. Leishmania 326:377–382. mexicana cysteine proteinase-deficient mutants have attenuated virulence for mice and potenti- Davies CR et al. The epidemiology and control of ate a Th1 response. Journal of Immunology, 1998, leishmaniasis in the Andean countries. Cadernos de 161(12):6794–801. Saude Publica, 2000, 16:925–950. Alvar et al. Leishmania and human immunodeficiency De La Rosa R et al. Incidence and risk factors for virus coinfection: the first 10 years. symptomatic visceral leishmaniasis among human Clinical Microbiology Reviews, 1997, 10(2):298–319. immunodificiency virus type 1 infected patients from Spain in the era of highly active antiretroviral therapy. Arevalo I et al. Successful treatment of drug resistant Journal of Clinical Microbiology, 2002, 40:762–7. cutaneous leishmaniasis in humans by use of imiquimod, an immunomodulator. Clinical Infectious Diseases, Desjeux P. Leishmaniasis: current situation and new 2001, 33:1847–51. perspectives. Comparative Immunology, Microbiology and Infectious Diseases, 2004, 27(5):305–18. Attar ZJ et al. Latex agglutination test for detection of urinary antigens in visceral leishmaniasis. Acta Tropica, Desjeux P. The increases in risk factors for leishma- 2001, 78:11 16. niasis worldwide. Transactions of the Royal Society of Tropical Medicine and Hygiene, 2001, 95:239–243.

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