Faculty of Medicine and Health Sciences

Onchocerciasis and lymphatic filariasis in Mali: preparedness for mass drug administration interruption and morbidity management

Dissertation submitted for the degree of Doctor in Medical Sciences to be defended by

Housseini Dolo

Promotor: Prof. Dr. Robert Colebunders, University of Antwerp,

Co -Promotors: Dr. Thomas B. Nutman, National Institute of Health, USA Prof. Maria Gloria Basáñez, Imperial College London, UK Dr. Martin Walker, Royal veterinary College London, UK

Antwerp, 2020

Cover image courtesy to Lymphatic Filariasis Unit, International Center of Excellence in Research, Faculty of Medicine and Odontostomatology

2

Table of contents

Dedication ...... 8 Acknowledgments ...... 9 Samenvatting ...... 10 Summary ...... 12 List of abbreviations ...... 14 Chapter 1 General introduction ...... 17 1.1. Background ...... 18 1.2. Onchocerciasis and lymphatic filariasis epidemiology ...... 19 1.2.1. Onchocerciasis life cycle and epidemiology ...... 19 1.2.2. Lymphatic filariasis life cycle and epidemiology ...... 21 1.2.3. Mansonella perstans epidemiology ...... 24 1.3. Pathogenesis of onchocerciasis and lymphatic filariasis ...... 25 1.3.1. Pathogenesis of onchocerciasis ...... 25 1.3.2. Pathogenesis of lymphatic filariasis ...... 26 1.3.3. Pathogenesis of Mansonella perstans ...... 27 1.4. Clinical manifestations of onchocerciasis and lymphatic filariasis ...... 28 1.4.1. Clinical manifestations of onchocerciasis ...... 28 1.4.2. Clinical manifestations of lymphatic filariasis ...... 29 1.4.3. Clinical manifestation of Mansonella perstans ...... 30 1.5. Current control and prevention strategies ...... 31 1.5.1. Drugs used for onchocerciasis and lymphatic filariasis treatment ...... 31 1.5.2. Treatment of onchocerciasis and lymphatic filariasis ...... 34 1.5.2.1. Onchocerciasis ...... 34 1.5.2.2. Lymphatic filariasis ...... 34 1.5.3.1. Prevention of onchocerciasis ...... 35 1.5.3.2. Prevention of lymphatic filariasis and Global Programme to Eliminate LF ...... 39 1.5. Onchocerciasis and lymphatic filariasis guidelines for Mass drug administration stopping and diagnostic tools ...... 41 1.5.1. Onchocerciasis guidelines for Ivermectin mass drug administration stopping and diagnostic tools ...... 41 1.5.1.1. Serodiagnostics for onchocerciasis ...... 41 1.5.1.2. Skin Snip test ...... 41 1.5.1.3. O-150 PCR in Simuliidae ...... 42 1.5.2. Lymphatic filariasis guidelines for Ivermectin and albendazole mass drug administration stopping and diagnostic tools ...... 42 1.5.2.1. Night thick smear ...... 43 1.5.2.2. Immunochromatographic test (ICT) ...... 43

3

1.5.2.3. Filariasis Test Strip (FTS) ...... 43 1.5.2.4. Wb123 antibodies test ...... 43 1.6. Mali country profile ...... 44 1.6.1. Geography and climate ...... 44 1.6.1. Demography ...... 46 1.6.2. Economic context ...... 46 1.6.3. Health system organization ...... 47 1.6.4. Lymphatic filariasis and onchocerciasis in Mali ...... 48 1.6.4.1. Epidemiology of onchocerciasis in Mali ...... 48 1.6.4.2. Epidemiology of lymphatic filariasis in Mali ...... 52 1.7. Rationale ...... 56 1.7. Scientific Objectives ...... 57 1.8. Organization of the thesis ...... 57 1.9. Studies team contribution ...... 59 1.10. References ...... 60 Chapter 2 Integrated seroprevalence-based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test ...... 71 Abstract ...... 72 2.1. Introduction ...... 73 2.2. Methods ...... 75 2.3. Results ...... 82 2.4. Discussion ...... 90 2.5. Conclusion ...... 92 2.6. References ...... 93 Chapter 3 Serological Evaluation of Onchocerciasis and Lymphatic Filariasis Elimination in the Bakoye and Falémé foci, Mali ...... 95 Abstract ...... 96 3.1. Background ...... 97 3.2. Material and methods ...... 99 3.3. Results ...... 107 3.4. Discussion ...... 120 3.5. Conclusion ...... 124 3.6. References ...... 125 Chapter 4 Lymphedema in three previously Wuchereria bancrofti-endemic health districts in Mali after cessation of mass drug administration ...... 129 Abstract ...... 130 4.1. Introduction ...... 131 4.2. Material and Methods ...... 132

4

4.3. Results ...... 136 4.4. Discussion ...... 143 4.5. Conclusion ...... 145 4.6. References ...... 146 Chapter 5 Factors associated with Wuchereria bancrofti microfilaremia in an endemic area of Mali 149 Abstract ...... 150 5.1. Background ...... 151 5.2. Material and methods ...... 152 5.3. Results ...... 156 5.4. Discussion ...... 164 5.5. Conclusion ...... 166 5.6. References ...... 167 Chapter 6 General discussion ...... 169 6.1. General discussion ...... 170 6.2. Conclusion ...... 175 6.3. Research priorities ...... 175 6.4. References ...... 177 Curriculum vitae ...... 180

5

List of tables

Table 2-1 Lymphatic filariasis and onchocerciasis pre-control endemicity and current status of mass drug distribution per district ...... 76 Table 2-2 Study area description per evaluation unit and details on number of villages and mean number of expected children ...... 79 Table 2-3 Study sites and population tested ...... 83 Table 2-4 Circulating filarial antigen and Wuchereria bancrofti seroprevalence per district and evaluation unit ...... 85 Table 2-5 Onchocerca volvulus seroprevalence per district and evaluation unit according to pre- control endemicity ...... 87 Table 3-1 Prevalence and Intensity of Onchocerca volvulus Microfilariae in the Pre-Ivermectin MDA Period (1985–1988) for 13 Villages in Bakoye, Mali ...... 102 Table 3-2 Prevalence and Intensity of Onchocerca volvulus Microfilariae in the Pre-Ivermectin MDA Period (1986–1990) for 14 Villages in Falémé, Mali ...... 103 Table 3-3 Description of the Study Population Tested with Onchocerciasis/LF IgG4 Rapid Diagnostic Test in Bakoye and Falémé, Mali, 2017–2018 ...... 108 Table 3-4 . Ov16 and Wb123 IgG4 Seroprevalence by Village in 3–10-year Old Children in Bakoye, Mali, 2017–2018 ...... 113 Table 3-5. Ov16 and Wb123 IgG4 Seroprevalence by Village in 3–10-year Old Children in Falémé, Mali, 2017–2018 ...... 114 Table 3-6 Ov16 and Wb123 IgG4 Seroprevalence by Age Group in Three Bakoye Villages Hyperendemic for Onchocerciasis at Baseline, Mali, 2017–2018 ...... 117 Table 4-1 Distribution of the lymphedema cases according to gender, age and body localization in three health districts of Mali ...... 137 Table 4-2 Number and percentage of lymphedema cases recorded per health district and per gender according to the method of identification ...... 139 Table 4-3 Estimation of lymphedema prevalence per health district in three health districts of Mali 140 Table 5-1 Characteristics of the study population ...... 157 Table 5-2 Risk factors associated with Wuchereria bancrofti microfilaremia in Tieneguebougou and Bougoudiana ...... 160 Table 5-3 Risk factors distribution by village ...... 163

6

List of figures

Figure 1-1 Life cycle of Onchocerca volvulus ...... 21 Figure 1-2 Life Cycle of Wuchereria bancrofti ...... 23 Figure 1-3 Image of filaria worm with Wolbachia before and after treatment with doxycycline [40] . 26 Figure 1-4 Onchocerciasis elimination framework [105] ...... 36 Figure 1-5 lymphatic filariasis framework for MDA implementation from mapping to MDA interruption and surveillance period [124] ...... 40 Figure 1-6 Lymphatic filariasis elimination strategies [122] ...... 40 Figure 1-7 Map of Mali showing the main rivers and cities ...... 45 Figure 1-8 Description of Malian health system organization ...... 48 Figure 1-9 Pre-control mapping of onchocerciasis between 1974- 1987 in Mali ...... 50 Figure 1-10 Onchocerciasis endemicity in the different districts between 2008 and 2015 evaluation in Mali ...... 51 Figure 1-11 Planning of onchocerciasis activities in elimination context ...... 52 Figure 1-12 Pre-control mapping of Lymphatic filariasis in 2004 ...... 53 Figure 1-13 Mapping neglected tropical diseases targeted for preventive chemotherapy in Mali in 2012 [8]...... 54 Figure 1-14 Lymphatic filariasis elimination status in Mali as of 2017 ...... 55 Figure 2-1 Distribution of circulating filarial antigen (orange dots) IgG4 antibodies to Wb123 (red dots) and to Ov16 (blue dots) in the evaluation units of Kadiolo –Kolondieba and Bafoulabe -Kita- Oussoubidiagna-Yelimane ...... 89 Figure 3-1 Map of the study area and the location of the study villages in the Kayes region of Mali 101 Figure 3-2 Trends in Onchocerca volvulus microfilarial infection in Bakoye from 1985 to 2010 ..... 110 Figure 3-3 Trends in Onchocerca volvulus microfilarial infection in Falémé from 1986 to 2010 ..... 111 Figure 3-4 Age-specific Ov16 and Wb123 seroprevalence profiles in the villages of Kantila, Nioumala and Galé combined ...... 116 Figure 3-5 Age-specific Ov16 Seroprevalence in Three Historically Hyperendemic Villages of the Bakoye Focus, Mali, and Three River Basins in the Kédougou Region, Senegal...... 119 Figure 4-1 Map of Mali showing the three study districts (Kolondieba, Bougouni and Kolokani in Read) ...... 133 Figure 4-2 Variability of clinical presentation in patients with lymphedema in Mali ...... 142 Figure 5-1 Map of Koulikoro district with the two study villages indicated by black dots ...... 153 Figure 5-2 Wuchereria bancrofti load among Mansonella perstans negative and positive subjects .. 159 Figure 5-3 Correlation between M. perstans and W. bancrofti microfilaremia loads within study subjects ...... 159 Figure 5-4 Geographic distribution of Wuchereria bancrofti antigen and Mansonella perstans ...... 162

7

Dedication

I thank Allah; for giving me a healthy life to achieve this thesis.

This thesis is entirely dedicated to my well-regarded father and mother, who have been my source of encouragement, who continuously provide their moral, spiritual, emotional, and financial support. To my loved wife Bintou and my cherished daughters Fatoumata Yatanou and Mariam Yaley who constructed in my happiness and accepted my leave for training and studies in the field in Mali and overseas during my PhD training period.

8

Acknowledgments

I would like to express my sincere gratitude to my promoters Prof. Dr Robert Colebunders and Dr

Thomas B. Nutman for their support, supervision and extensive help for conducting research and writing of this thesis.

I would like also to thank my co promotors Prof Maria Gloria Basáñez and Dr Martin Walker for their valuable contribution to this thesis, for their training during my fellowship and their social support.

I am grateful to Islamic Development Bank and the European Research Council funded NSETHIO project for their financial support.

I thank all my colleague PhD students of the Global Health Institute for sharing their social and scientific knowledge and experiences with me in Belgium and in the field.

I would like also to thank the NSETHIO project team lead by Prof Robert Colebunders and my supporting team for field work in Mali namely the Filariasis Research Unit in the International

Center of Excellence in Research of the Faculty of Medicine and Odontostomatology lead by Dr

Yaya Ibrahim Coulibaly.

My special thank goes to the Dean of the Faculty Medicine and Odontostomatology, Prof. Seydou

Doumbia for his assistance and advice for starting a PhD and for supporting my teaching at the

Faculty.

My thanks go also to both the lymphatic filariasis elimination programme in Mali as well as to onchocerciasis control programme for their respective support and willingness to allow me access to their data and reports.

My thanks go to all the participants and villages leaders who accepted and made our field studies easy.

My special thanks to my brothers and sisters for their support during the thesis period.

My special thanks to Handy and Prof Maria Gloria Basáñez and their daughter Hiwot for welcoming me in their beautiful house in Oxford many times for work and good times.

9

Samenvatting

Onchocercose en lymfatische filariasis (LF) zijn verwaarloosde tropische ziekten (NTD) die miljoenen mensen treffen in de tropen, vooral in afgelegen en ontoegankelijke gebieden.

Onchocercose is verantwoordelijk voor jeuk, huidletsels, blindheid en epilepsie, terwijl lymfatische filariasis lymfoedeem en hydrocele veroorzaakt. Deze twee ziekten hebben een belangrijke sociaal- economische impact oa door het veroorzaken van elephantiasis en blindheid die gemeenschappen naar extreme armoede drijft. Na vele jaren succesvolle massale toediening van geneesmiddelen

(MDA) is Mali, net als vele andere landen, geïnteresseerd om het onderbreken van de transmissie van onchocercose en LF te kunnen vaststellen, om de MDA te kunnen stoppen en om over te kunnen gaan in een uitroeiing en opvolging fase.

De bedoeling van dit proefschrift is om te evalueren of Mali klaar is om de MDA voor onchocercose en LF te onderbreken en om de opvang van mensen met lymphoedeem te evalueren. Eerst evalueerden we een nieuwe snelle diagnostische test (de SD Bioline Onchocercose / LF IgG4

Rapid-test) voor detectie van Onchocerca volvulus en Wuchereria. bancrofti-infectie bij kinderen van 6 - 7 jaar in twee evaluatie-eenheden volgens de LF Transmission Assessment Survey (TAS) richtlijnen. De test bleek betrouwbaar te zijn voor de opvolging van onchocercose en LF- blootstelling in co-endemische gebieden. Alle Ov16-antilichaam positieve kinderen werden geïdentificeerd in de twee voormalige meso- en hyperendemische onchocercose districten. De O. volvulus antilichaam prevalentie in de twee districten waren 0,19% en 0,18%. Hoewel deze prevalenties hoger zijn dan de 0,1% -drempel voorgesteld door de Wereldgezondheidsorganisatie voor het stoppen van MDA, worden deze waarden, wegens de specificiteit van de Ov 16 Elisa test, toch beschouwd als een indicatie dat in vele onchocercose hypo-endemische gebieden in Mali

MDA kan gestopt worden.

In een tweede veldstudie werd een onchocercose en LF serologisch profiel bepaald van alle leeftijdsgroepen in de vorige hyperendemische regio van Bakoye en Falémé. De seroprevalentie van Ov16 en Wb123 bij kinderen ≤14 jaar oud waren respectievelijk 0,1% en 2%, wat wijst op de

10 eliminatie van onchocercose transmissie en de eliminatie van LF als een volksgezondheid probleem sinds het stoppen van MDA in 2016.

We bestudeerden ook de distributie en de klinische presentatie van lymfoedeem in vroegere LF- endemische districten in de post ivermectin en albendazole MDA-periode. We stelden vast dat actieve opsporing van lymfoedeemgevallen (tijdens dorpsvergaderingen, oproepen, dorp bezoeken, enz) efficiënter was dan passieve rapportage (door basis gezondheidswerkers), de methode die door de meeste LF-programma’s wordt gebruikt, oa in Mali.

Ten slotte, werd onderzoek gedaan naar risicofactoren voor W. bancrofti-infectie in een omgeving waar LF co-endemisch is met Mansonella perstans. Na correctie voor leeftijd, geslacht en woonplaats, was de kans op W. bancrofti-infectie hoger bij met M. perstans microfilariae geïnfecteerde personen ondanks dat deze infecties door verschillende vectoren worden overgedragen. Deze bevinding is belangrijk voor het LF-eliminatieprogramma vanwege de mogelijkheid van vals-positieve LF-serologische testen door kruis reagerende antilichamen in gebieden waar M. perstans aanwezig is.

Als besluit, wij vonden geen bewijs voor een voortdurende LF-transmissie sinds het stoppen van

MDA met ivermectin en albendazole. Wat betreft onchocercose, suggereren onze resultaten dat het stoppen van MDA binnenkort mogelijk moet zijn. Mali zal echter de epidemiologische situatie van onchocercose en LF verder moeten opvolgen (inclusief met gebruik van entomologische indicatoren), om vroege tekens van heropleving of herintroductie te detecteren en erop te reageren.

Bovendien is er nood aan een sterker LF-morbiditeitsbeheerprogramma dat ook een actieve identificatie van lymfoedeemgevallen bevat.

11

Summary

Onchocerciasis and lymphatic filariasis (LF) are neglected tropical diseases (NTD) that affect millions of people in tropical settings, especially in remote and inaccessible areas. Onchocerciasis causes itching, skin lesions, blindness and epilepsy; whereas LF can result in lymphedema and hydrocele. These two diseases represent an important socio-economic burden due to permanent disabilities, including elephantiasis and blindness, that drive communities into extreme poverty.

After many years of successful mass drug administration (MDA), Mali, like several other countries, is eager to confirm the interruption of transmission of onchocerciasis and LF, stop the distribution of MDA and enter into an elimination and surveillance phase.

This thesis assessed preparedness for onchocerciasis and LF MDA interruption and lymphedema morbidity management in Mali. More specifically, we evaluated a new rapid diagnostic test (SD Bioline Onchocerciasis/LF IgG4 Rapid test) for detection of O. volvulus and W. bancrofti infection in children 6-7 years of age according to the LF TAS guidelines in two evaluation Units. This test appeared to be a useful tool for monitoring onchocerciasis and

LF exposure in co-endemic areas. We observed that all Ov16 antibody-positive subjects were residents of two previously known onchocerciasis meso- and hyperendemic districts.

Onchocerca volvulus antibody prevalence levels were 0.19% and 0.18 % in the two districts.

These values are above the 0.1% threshold proposed by World Health Organisation (WHO) for stopping ivermectin MDA. However, given the specificity of the Ov16 Elisa test, this threshold is now considered too low. The results of our study suggest that in many onchocerciasis hypo- endemic areas in Mali, ivermectin MDA can be stopped.

In a second field study, we determined the all age serological profile of onchocerciasis and LF in the previously onchocerciasis hyperendemic setting of Bakoye and Falémé. The Ov16 and

Wb123 seroprevalence in ≤14-year olds were less than 0.1% and 2% in the two areas

12 respectively, consistent with elimination of onchocerciasis transmission and elimination of LF as a public health problem since stopping treatment in 2016.

We also studied the distribution and clinical presentations of lymphedema in previously LF endemic health districts in the post ivermectin and albendazole MDA period. We found that active lymphedema case identification (through village meetings, calls, visits, etc.) was more efficient than passive case reporting (through community health workers), the method used by most LF elimination programs, including the one in Mali.

We studied risk factors for W. bancrofti infection in a setting where LF is co-endemic with M. perstans. After controlling for age, gender, and village of residence, the likelihood of being W. bancrofti infected was higher in individuals with detectable M. perstans microfilariae, despite the fact that these infections are transmitted by different vectors. This finding is important for the LF elimination program, because of the possibility of false positive LF serological testing caused by cross-reacting antibodies in areas where M. perstans is present.

In conclusion, we did not observe evidence of ongoing LF transmission after ivermectin and albendazole MDA was stopped. With respect to onchocerciasis, our study suggests that stopping of ivermectin should be considered in the near future. Regardless, Mali needs to continue periodic surveillance for both onchocerciasis and LF (including using entomological indicators) to detect and respond to early signs of resurgence/re-introduction. Moreover, the country needs a stronger LF morbidity management program, that includes identification of active lymphedema cases.

13

List of abbreviations

APOC African program of onchocerciasis control ArcGIS Environmental Systems Research Institute CA California Cag Circulating antigen CDDs Community drug distributors CDTi Community Directed Treatment with Ivermectin CFA Circulating Filarial Antigen CI Confidence Interval CMFL Community Microfilarial Filarial Load DEC Diethylcarbamazine DEC Diethylcarbamazine citrate Dr Doctor ELISA Enzyme-Linked Immunosorbent Assay. EOT elimination of transmission; EPHP: elimination as a public health problem EPHP elimination as a public health problem ESRI Environmental Systems Research Institute EU Evaluation units FMPOS Faculty of Medicine, Pharmacy and Odontostomatology FRU Filariasis Research Unit FTS Filariasis Test Strip GIS Geographic Information System GPELF Global Programme to Eliminate Lymphatic Filariasis GPS Global Positioning System ICT Immunochromatographic Card Test IgG4 Immunoglobulin G4 IRB Institutional Review Board IVM Ivermectin kg kilogram LF Lymphatic Filariasis LPS-like Lipopolysaccharide like M. perstans Mansonella perstans M&E Monitoring and Evaluation MDA Mass drug administration mf Microfilariae mff microfilariae Ml Milliliter MMDP Morbidity Management and Disability Prevention NA Not applicable NPELF National Programme for Elimination of Lymphatic Filariasis NTD Neglected Tropical Diseases O. volvulus Onchocerca volvulus OCP Onchocerciasis Control Progam OV16 Onchocerca volvulus specific antibodies PCR Polymerase Chain Reaction PCT Preventive Chemotherapy PMA Minimum Package of Activities PNLO National Onchocerciasis Control Programme

14

POC Point of care Prev Prevalence Prof Professor RDT rapid diagnostic test SCH Schistosomiasis; SD Standard Diagnostic Spp Standing for species pluralis ss skin snip SSB Sample Size Builder STH Soil-Transmitted Helminthiasis TAS transmission assessment survey TPE Tropical Pulmonary Eosinophilia TRA Trachoma USA United States of America USTTB University of Science, Technique and Technologies of Bamako VC vector control W. bancrofti Wuchereria bancrofti Wb123 Wuchereria bancrofti specific antibodies 123 WHO World Health Organization µg Microgram µl Microliter 95% CI ninety five percent confidence interval

15

16

Chapter 1 General introduction

17

1.1. Background

Elimination of onchocerciasis and lymphatic filariasis (LF) in endemic foci /districts depends on the performance of serological diagnostic tools and the implementation of integrated onchocerciasis and LF assessments in areas where this is possible [1]. It must also take into account morbidities such as lymphedema and coinfection with other minor filarial parasites such as Mansonella perstans which may play role in the context of elimination because of the possible cross reaction when using current available diagnostic tests [1,2].

Onchocerciasis and LF have a very similar biological life cycle, although the location of microfilariae (skin, eyes for onchocerciasis and blood for LF) and macrofilariae (in the skin for onchocerciasis and lymphatic vessels for LF) is different in the definitive human host [3,4].

The vectors transmitting Onchocerca volvulus are Simuliidae and Anopheles, Aedes, Culex and

Mansonia are those of LF [5,6]. In most tropical countries where these two parasites are present, they are geographically overlapping in term of endemicity [7,8].

Onchocerciasis is treated with ivermectin, for LF, in addition to ivermectin, albendazole is associated [9,10]. The vector competence and life span of macrofilariae determine the duration of mass drug administration (MDA) in each infection. Thus onchocerciasis requires 10 to 15 years of MDA while LF requires 5 to 6 years [9,10].

Evidence of the elimination of local transmission of onchocerciasis and LF has been reported in Latin America and in some African foci such as Mali where the principle of elimination has been reported by evaluating with skin biopsy and vector processing [11–13].

Faced with the reductions in the prevalence of onchocerciasis and LF, there is a motivation to stop most MDA distribution nationally [1]. However, there is a challenge in obtaining practical serological diagnostic tools as older diagnostic tools such as skin biopsy and night thick smears have become obsolete in an epidemiological context where the prevalence and intensity of the two infections have decreased significantly under drug pressure [1]. Also, the cost of

18 independent evaluations is enormous and difficult to be supported by national programmes and donors, hence the need to promote integrated evaluations [1]. Thus, the availability of diagnostic tools, particularly combined tests such as SD BIOLINE Oncho/LFIgG4 biplex rapid test (capable of simultaneously detecting exposure to Ov16 and Wb123 antigens for onchocerciasis and LF respectively), opens up the opportunity to conduct integrated assessments of onchocerciasis and LF [14,15]. Hence in a co-endemic country of Mali, onchocerciasis and LF integrated elimination evaluation using SD BIOLINE Oncho/LF

IgG4 biplex rapid test was conducted and reported here. Also, post-MDA epidemiology of lymphoedema and the role of Mansonella perstans were studied. In Mali, all endemic areas received 25 years of ivermectin treatment for onchocerciasis and nine to ten years of treatment with ivermectin and albendazole for LF.

1.2. Onchocerciasis and lymphatic filariasis epidemiology

1.2.1. Onchocerciasis life cycle and epidemiology

The life cycle of Onchocerca volvulus (Figure 1-1) involves long-lived adult worms

(macrofilariae) living in subcutaneous nodules which have an average female reproductive life span of approximately ten years. The embryonic skin-dwelling microfilariae (Mf) has a mean life expectancy of 12-24 months. The mf develops into L1, L2 and L3 within the fly in approximately one week and attain infectivity for humans (these stages do not multiply within the vector). The immature stages (L4 and juvenile adults) get to sexual maturity in the human host and start producing detectable mf after approximately two years [16]. The only definitive host of the parasites are humans [17].

The number of people infected by onchocerciasis is estimated to 37 million in 36 countries and among them 99% live in 31 countries in sub-Saharan Africa. Approximatively, 500,000 suffer significant visual loss and among them about one half are blind [18]. The number of people at risk of onchocerciasis infection is estimated to 85.6 million [19].

19

Onchocerciasis due to filarial worms Onchocerca volvulus (O. volvulus) are transmitted from person to person by the repeated bites of infected black flies (Simulium spp.) mainly in tropical areas [20]. These black flies breed in fast-flowing rivers and streams, mostly in remote villages located near fertile land where people rely on agriculture [21].

After vector control intervention and the subsequent use of ivermectin during CDTI, many countries noticed a decrease of infection intensities and number of endemic countries. As result of Onchocerciasis Control Programme (OCP), onchocerciasis was passed under control in

West Africa through the spray of insecticides against black-fly larvae (vector control) by helicopters and airplanes [21]. Then subsequently, this strategy was supplemented by extensive distribution of ivermectin since 1989 by covering another extension zones more in the south part of the countries [21]. In 1995, Kim and Benton (1995) estimated that between 1974 and

2002, the overall operations of the OCP would have averted 593,440 cases of blindness [22].

Source: http://www.cdc.gov/parasites/onchocerciasis/biology.html

20

Figure 1-1 Life cycle of Onchocerca volvulus

1.2.2. Lymphatic filariasis life cycle and epidemiology

Arthropod vectors are necessary for the maturation and the transmission of filarial worms from one vertebrate to another [23,24]. The female adult worms live in the lymphatics and get to maturity before producing live microfilariae. The microfilariae produced are found in the host blood in the daytime (in large blood vessels) or at night (in peripheral blood vessels) depending on the filarial species. When the microfilariae are circulating in the peripheral blood or moving in the cutaneous tissue, they get taken up by mosquito vectors during a blood meal. Once in the mosquito vector, the microfilariae pass through the wall of the digestive tract into the mosquito haemocoel. Later, the microfilariae pass through three maturation stages: the first larval stage (L1), the second larval stage (L2) and the infective third larval stage (L3) (Figure

1-2). Each stage in the vector has specific anatomic locations, so the L1 and L2 are usually found in the thorax of the host vector. The L3 in the head move into the mosquito mouth before being deposited on the vertebrate host’s skin where it penetrates actively the lymphatic vessels under the skin through the biting wound and goes to the deeper lymphatic vessels and lymph nodes [25]. In a host, the infective larvae undertake a maturation route that lasts nine to 14 days to get to the larval stage 4 (L4) that will become an adult worm after approximately 16 days.

When the GPELF was launch in 2000, 120 million people were infected with lymphatic filariasis (LF) and the number of people at risk of acquiring the infection was estimated to more than 1.3 billion (~20% of world population) [26]. During this period, the estimation of people with incapacities or disfigurations due to lymphedema, or breasts, and/or hydrocele of the scrotum was 40 million [26]. Almost, 25 million men had genital disease (most commonly hydrocele) and almost 15 million, mostly women, had lymphedema or elephantiasis [27]. There are fewer than 10–20 million persons in these areas who are infected with B. malayi.

21

Contrasting W. bancrofti, B. malayi has feline and primate reservoirs. B. timori is only located on the islands of south-eastern Indonesia.

LF is endemic in following continents: Africa, Asia, Indian subcontinent, the Western Pacific

Islands, focal areas of Latin America, and the Caribbean (especially Haiti and the Dominican

Republic). Approximately 65% of those at risk live in south and Southeast Asia, 30% lives in sub-Saharan Africa and the rest in other parts of the tropical world [28]. In the Americas, endemic foci continue on the island of Hispaniola and in coastal areas of Guyana and north- eastern Brazil. Infection with B. malayi is restricted to India, Malaysia and in numerous western

Pacific island groups including Indonesia and the Philippines.

As one of the leading causes of global disability, LF accounts for at least 2.8 million DALYs; this does not include the significant co-morbidity of mental illness commonly experienced by patients and their caregivers [29]. The average annual economic burden per chronic case was

US$115, majority of which resulted from productivity costs. The entire economic burden of

LF was estimated at US$5.8 billion annually. These results demonstrate the magnitude of the

LF burden and highlight the continued need to support the GPELF. Patients with clinical disease support the majority of the economic burden but will not benefit much from the current

MDA programme aimed at reducing transmission and not the morbidities of those currently affected by lymphedema and or hydrocele [30].

More than 90% of infections are caused by W. bancrofti and the remaining by Brugia malayi

(B. malayi) and Brugia timori (B. timori) for which humans are the only natural host.

More than two decades after the launch Global Programme to Eliminate Lymphatic Filariasis

(GPELF), in 2015, as the results of synchronized effort of countries and the GPELF of the 1.3 billion at risk at the beginning, 315 million people have now been able to stop taking treatment representing major progress towards disease elimination [26]. Overall, according to GPELF, between 2000 and 2015, the number of endemic countries decreased from 83 to 73 [26]. Of the

22 total population requiring preventative chemotherapy, 57% live in the South-East Asia Region

(9 countries) and 37% live in the African Region (35 countries). Both China and the Republic of Korea were considered endemic, but have confirmed elimination of LF as a public health problem in 2007 and 2008, respectively [31]. Despite this effort, so far as many as 36 million cases of hydrocele and lymphoedema remain.

Source: https://www.cdc.gov/parasites/lymphaticfilariasis/biology_w_bancrofti.html

Figure 1-2 Life Cycle of Wuchereria bancrofti

23

1.2.3. Mansonella perstans epidemiology

M. perstans is a vector-borne human filarial nematode, transmitted by tiny blood-sucking flies known as Culicoides [2,32]. It is extensively distributed in many parts of Sub-Saharan Africa and also occurs in parts of Central and South America [2]. Globally, an excess of 100 million people may be infected by M. perstans. It is estimated that 600 million people live at high risk of contracting an infection in Africa only [2].

A low prevalence of mansonelliasis occurs, in tandem with the existence of sparse farmlands of banana and plantain. Old stems of these crops provide suitable habitats for Culicoides species, which transmit M. perstans [33]. The distribution of M. perstans in the southern part of Cameroon varied with bio-ecological zones and ivermectin MDA history. The zones with high prevalence and intensities are in forested areas while those with low endemicity are in the savanna areas. MDA with ivermectin induced significant reduction in the endemicity of mansonellosis in the deciduous equatorial rainforest. In contrast, the prevalence and intensity remained relatively high and stable in the dense humid equatorial rainforest zones even after a decade of MDA with ivermectin [34]. A first geostatistical risk map for M. perstans in Uganda was presented. This study confirmed a widespread distribution of M. perstans and identified important potential drivers of risk including diurnal temperature range, normalized difference in vegetation index, and cattle densities. The level of co-infection with W. bancrofti was low

(0.3%), due to limited geographic overlap. But, where the two infections did overlap geographically, a positive association was found. The results of this study provided new insight about the ecologic preferences of this poorly known filarial parasite in Uganda, which might be relevant for other settings in sub-Saharan Africa including Mali [35].

In another study, comparing single infections with dual M. perstans–L. loa infection induced very high total IgE titers. This co infection study suggested the necessity of correlating IgE

24 titer and clinical symptoms to confirm the involvement of this immunoglobulin in the pathological processes during filariasis [36].

1.3. Pathogenesis of onchocerciasis and lymphatic filariasis

1.3.1. Pathogenesis of onchocerciasis

After the bite of an infected female black fly (Simulidae), infective larvae migrate through the subcutaneous tissues and develop into adult worms [37]. Two different features contribute to the pathogenesis of onchocerciasis: the adult worms and the microfilariae, both across results of the host immune response with the recent known contribution of Wolbachia in the pathogenesis [38,39]. Again, lipopolysaccharides (LPS-like) molecules from these intracellular bacteria are responsible for effective inflammatory responses from macrophages in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being discharged into the blood following the death of the parasite (Figure 1-3). Recent studies in animal models even implicated Wolbachia in the onset of blindness [40]. Patients with generalized onchocerciasis represent the vast majority of infected subjects and are characterized by having weak or no skin inflammation despite high parasite loads, while patients with severe chronic dermatitis suffer from severe symptoms and present low microfilaria and adult loads [38]. A third, small subgroup of people living in areas of endemicity do not acquire detectable evident infection notwithstanding exposure to infective vector bites. In Africa, O. volvulus nodules are most frequently observed in the pelvic area, with a few along the spine, chest, and knees [38]. In the Venezuelan O. volvulus strains, the symptomatology is much like the Africa ones, but in Central America nodules are mostly above the waist, especially at the level of the neck and head. These nodules are relatively benign, causing some disfigurement but no pain or ill health. The number of nodules may differ from one to well over 100 [37]. They comprise essentially of collagen fibers surrounding one to

25 several adult worms. Occasionally, the nodule will degenerate to form abscess or worms will become calcified.

Before treatment After treatment

Figure 1-3 Image of filaria worm with Wolbachia before and after treatment with doxycycline [40]

1.3.2. Pathogenesis of lymphatic filariasis

The infective larvae (L3) of W. bancrofti is transmitted to humans during the blood meal of infected mosquitoes. The larvae escape from the proboscis of the mosquito and penetrate the puncture wound actively. In endemic areas, exposure starts in early childhood and many bites are required to acquire the infection [41,42]. The development of the disease in human host is still unclear. While the infection is generally acquired during early childhood, clinical symptoms are generally expressed many years later. The role of Wolbachia, the endosymbiont bacteria, may also be involved in the pathogenesis and clinical manifestations of LF [43]. In filarial nematodes, Wolbachia endobacteria are compressed in intracytoplasmic vacuoles inside the hypodermal lateral cords of parasites worms (male and female) and reproductive organs of

26 the female. The endosymbiont can be detected using immunohistochemistry in oocysts and microfilariae (Figure 1-3) [44]. Lipopolysaccharide (or LPS-like molecules) from these intracellular bacteria are responsible for inflammatory responses from macrophages and in the animal models of filarial disease[40]. Severe inflammatory reactions to filarial chemotherapy have been also observed at the presence of Wolbachia or being released into the blood following the death of the parasite.

1.3.3. Pathogenesis of Mansonella perstans

Studies have shown that M. ozzardi and M. perstans parasites occur in sympatry; the observed close relationship between M. perstans in Africa and Brazil suggest that the origins of M. perstans is from the New World [45]. Wolbachia have been identified in M. perstans and its detection was associated with higher-level microfilaremia (Figure 1-3). Moreover to date significant sequencing work has been done and the finding support the presence of Wolbachia in Mansonella species [46,47].Therefore, the use of antibiotics for mansonellosis can generalized for the treatment [48].

For the vectors transmitting M. perstans, a wide spectrum of Culicoides spp. was observed. In

Cameroon, Culicoides milnei was demonstrated to be the major vector of M. perstans [32]. In

Senegal, among 1,159 Culicoides specimens studied no positive for M. perstans was observed requiring further identification of M. perstans vector in Senegal [49]. In Mali, different species of Culicoides were inventoried in two villages known endemic for M. perstans of different eco climatic zones. The test to find M. perstans DNA was not successful (Diallo, AA Unpublished data).

27

1.4. Clinical manifestations of onchocerciasis and lymphatic filariasis

1.4.1. Clinical manifestations of onchocerciasis

After the bite of flies on the susceptible host, the larvae are transmitted and matured into adults within the skin and subcutis [50] and the incubation period between 10 to 15 months coincides with the production of mf. They formed nodules that typically are not painful. The adult worms live inside the skin for years and yield microfilariae. The mf migrate through the body and cause visual impairment, blindness or pruritic popular rash [50,51]. The signs of river blindness or onchocerciasis are linked to the location of the parasites in the body especially in the skin and eyes. Subcutaneous nodules enclosing adult worms known as onchocercomata commonly located above bony prominences is the presenting sign. The clinical syndrome of onchocerciasis includes a variety of skin manifestations with intermittent severe pruritus, such as papular rash. In chronic onchocerciasis, the skin becomes thickened and lithified dermatitis, hyperpigmentation or depigmentation (lizard skin), and atrophy; lymphadenopathy, forming large masses, particularly in the inguinal region; obstruction of the scrotal lymphatic drainage, leading to enlargement and progressive spreading under the effect of gravity (hanging scrotum syndrome); and corneal opacities and anterior uveitis caused by microfilarial invasion [51].

The eyes can be affected with sclerosing keratitis, posterior eye disease such as chorioretinitis and optic neuritis [51].

In Africa, skin involvement tends to be greatest on the lower extremities as opposed to Central

America where the upper body is more generally affected. Vision loss is more frequent in

African savanna population compared to South America populations [52]. In a study from

Ghana the most frequent clinical manifestation observed among all participants was dermatitis

(25.4%), followed by visual impairment & nodules (7.9% each) and then by blindness (4.4%)

[53]. More recently another condition known as nodding syndrome was associated with onchocerciasis clinical features. This condition is characterized by bouts of repetitive head

28 nodding. Symptoms develop in children with previously normal development aged 3 to 18 years. Head nodding is the pathognomonic feature of nodding syndrome. The head nodding often occurs in association with feeding, a cold breeze, or cold weather [54,55]. In the last few years number of studies were conducted to describe the onchocerciasis associated epilepsy

(OAE) in endemic countries and later on nodding syndrome has been described as particular clinical manifestation of OAE [56–58]. The OAE is described by an onset of seizures between the ages of 3 and 18 years. Persons with this condition may present with a spectrum of epilepsy types that include absences, atonic and myoclonic neck seizures, and Nakalanga features and living in onchocerciasis endemic areas or infected with onchocerciasis [56].

1.4.2. Clinical manifestations of lymphatic filariasis

LF presents a wide range of clinical manifestations. Most of the infected persons are clinically asymptomatic. In asymptomatic (often microfilaremic) subjects, adult filarial parasites commonly cause subclinical lymphatic dilatation and dysfunction [42]. Filarial lymphadenopathy is seen generally in infected children; before adolescence, adult worms could be detected by ultrasonography of the inguinal, crural, and axillary lymph nodes and vessels

[59]. Death of the adult worm produces an inflammatory response, that progresses distally

(retrograde) along the affected lymphatic vessel, usually in the limbs and is called acute filarial lymphangitis. If present, general symptoms, such as headache or fever, are generally minor

[59].

In young males, adult W. bancrofti organisms are discovered frequently in the intra-scrotal lymphatic vessels and can be visualized on ultrasound examination by filarial “dance sign”.

Inflammation, driven most likely from the death of an adult worm may present clinically with funiculitis, epididymitis or orchitis. At the site of the adult worms death, a tender granulomatous nodule may be palpable [42,60].

29

In approximately 30% of LF infected persons, the chronic manifestations such as lymphedema and/or hydrocele will develop. The lymphedema mostly affects the legs, but can also occur in the arms, breasts, and genitalia [61]. These symptoms develop in most people many years after the initial infection. Severe pain, fever and chills affected extremities due to recurrent secondary bacterial infections can accelerate the progression of lymphedema to its advanced stage, known as elephantiasis [62].

Filarial hydrocele is believed to be the consequence of lymphatic vessels destruction caused by adult worms. Chyluria, which results from rupture of enlarged lymphatics into the renal pelvis, can develop as a manifestation of bancroftian filariasis [42]. Microscopic hematuria and proteinuria are also found in LF infected patients.

The manifestations of the tropical pulmonary eosinophilia (TPE) syndrome include cough, fever, marked eosinophilia, high serum immunoglobulin E concentrations, and markedly elevated anti-filarial antibodies. During the course of TPE, peripheral microfilaremia is absent in most patients. Most of the TPE cases have been reported from South Asia and Brazil. The most frequently affected are men aged between 20 and 40 years old [63].

1.4.3. Clinical manifestation of Mansonella perstans

In most infected persons, M. perstans is not causing any pathology or the symptoms are caused by other filarial parasites [64,65]. During the last 5 years, studies on M. perstans were focused on the clinical manifestations, the immunobiology, the vectors, the epidemiology, co-infections with other parasites and treatment.

Concerning clinical manifestations, acute swelling in forearms, hands and/or face resembling

Calabar swellings, itching of skin with or without rash or ulceration, pain or ache in scrotum and/or joint synovia, pain or ache in serous cavities, pain or ache in the liver region, neurological and psychic symptoms have been reported and attempted to be linked with M.

30 perstans infection [2]. M. perstans nematodes should be included in the differential diagnosis for patients with eosinophilia from disease-endemic countries [66]. The age group of >30 years have had the highest prevalence [49]. Symptomatic M. perstans infections of the pericardial

[67] and pleural cavities have also been described [68]. A remarkable severe case of edema potentially caused by a M. perstans was described from Nigeria [69]. A study from Gabon proposed that M. perstans infection could result in hormonal disorders such as signs of hypogonadism [70]. Eosinophilia is a classic feature of many M. perstans infections [71–73], but in a recent study from Chad [74] only 41% of patients with M. perstans mf were found with more than 7% blood eosinophilia. Nodules in the conjunctiva, swelling of eyelids and proptosis

[75] and intraocular infections [76] are other conditions that have been attributed to M. perstans.

1.5. Current control and prevention strategies

1.5.1. Drugs used for onchocerciasis and lymphatic filariasis treatment

The primary objective of the treatment of people infected with onchocerciasis or lymphatic LF is to reduce peripheral blood microfilariae (for LF) and tissue microfilariae, especially nodules

(for onchocerciasis). This treatment has a double objective including the reduction of complications and the prevention of transmission in the community (basic principle of mass drug treatment with different drugs). However, it is important to point out the existence of drugs that have an action on adult worms of Onchocerca volvulus and Wuchereria bancrofti,

Brugia malayi or Brugia timori. In this therapeutic approach, antifilarial drugs can be grouped into two groups: microfilaricidal and macrofilaricidal.

Diethylcarbamazine was initiated in the late 1940s. this drug is primarily a microfilaricide touching the neuromuscular system of the parasites and also promotes cellular cytotoxicity mediated by immune factors.

31

Notwithstanding its efficacy demonstrated in numerous clinical trials and field studies, the adverse effects are frequent. Among them are the accelerated impairment of visual capacity because of aggravation of chorioretinal lesions. Definitely, this inflammatory reaction may accelerate optic nerve disease, leading to permanent loss of visual function [77].

Diethylcarbamazine leads to the Mazzotti reaction in 2 hours after administration and can be very disagreeable. Therefore, it is impossible to treat patients on a community-wide scale using this drug. Diethylcarbamazine is therefore no longer advocated for the treatment of onchocerciasis by the WHO [19]. It is microfilaricidal for W. bancrofti, B. malayi and B. timori.

It appears to be macrofilaricidal (kills adult worms) for these species as well and is considered to be the drug of choice for these three infections [78].Diethylcarbamazine is effective in killing microfilariae of O. volvulus in the skin and eye and there considered as microfilaricidal for onchocerciasis treatment [78].

Ivermectin

Ivermectin commonly known as Mectizan' is the current drug of choice, and is provided free of charge by the manufacturer, not only in multinational health programmes but also for compassionate use in the field and in developed countries [79].

Ivermectin is a synthetic derivative of a macrocyclic lactose produced by the actinomycete

Streptomyces avermitilis. It has broad spectrum anti parasitic activity against nematodes [80].

It appears to be effective via its action as an agonist at gamma- aminobutyric acid (GABA) receptors, impairing the neuromuscular function of the parasites and leading to paralysis [81].

Ivermectin does not kill the adult worm but appears to worsen the release of mf from the gravid female adult worm, which occurs after the first dose [82,83]. However, after multiple doses at

3-month intervals, a one-third excess mortality of female worms has been demonstrated in one study [84]. The drug also affects embryonic development in O. volvulus, which occurs after multiple doses [85].

32

The optimal dose of ivermectin is 150 ug/kg, despite the fact that studies have demonstrated no significant difference in terms of efficacy when comparing doses of 100, 150 and 200 ug/kg

[86]. The frequency of administration is nevertheless very debatable. Several different schemes of administration are advocated: a single dose of 150 /lg/kg given annually [87] ; therapy given

6-monthly [88]; a single dose of ivermectin, repetitive every 3 to 6 months as necessary.

Most authors recommend administration once or twice a year, at least in endemic countries. In hyperendemic zones, treatment every 6 months may be necessary as ivermectin does not kill the adult worm, which goes on producing microfilariae throughout its life [89]. Consequently, long term treatment is expected to be necessary until the success of control of the vector and/or until a cheaper and safer macrofilaricidal drug becomes available. Noticeably, the availability of a macrofilaricidal drug would shorten the duration of treatment.

Albendazole

As a vermicide, albendazole causes degenerative alterations in the intestinal cells of the worm by binding to the colchicine -sensitive site of β-tubulin, thus inhibiting its polymerization or assembly into microtubules. This drug has been added to LF treatment after a study showing it effect in reducing mf and its long term effect on adult worm reproduction [10]. In LF MDA, albendazole is associated with ivermectin at the dose of 400mg single dose in person up to 5 years and above. But in Cochrane review it effect in mf and adult worm came with uncertainty

[90].

Moxidectin

More recently, moxidectin a macrofilaricidal drug was tested to see whether it is safe enough for use in mass treatment and in the purpose that it can reduce disease transmission faster than ivermectin. Moxidectin could be particularly valuable in post-conflict countries in Africa where CDTI has not yet been implemented or only recently [91,92].

33

Doxycycline

Taken the opportunities that Onchocerca volvulus and Wuchereria bancrofti are endosymbiont with Wolbachia for the vitality, reproduction and that doxycycline is effective on Wolbachia, this drug has been successfully tested on the treatment of both onchocerciasis and LF [93,94].

However, the treatment course is too long (min 3 weeks at the dose of 200mg tablet per day) to be deployed in mass treatment.

1.5.2. Treatment of onchocerciasis and lymphatic filariasis

1.5.2.1. Onchocerciasis

Patients currently infected with the parasite

The treatment regimen is based on ivermectin every six months for approximatively 10 to15 years corresponding to the life span of the adult worms or for as long as the infected person has evidence of skin or eye infection [75]. Ivermectin kills the microfilariae and prevents them from causing damage but it does not kill the adults [96]. There is also treatment with doxycycline that kills the adult worms by depleting the endosymbiont Wolbachia on which the adult worms depend to survive. Before ivermectin treatment is begun, co-infection with Loa loa need to be checked in those from Loa-endemic countries (primarily central Africa), because

Loa loa can be responsible for severe side effects to the medication used to treat onchocerciasis

[97]. Most recently moxidectin has been tested as major microfilaricidal drug to accelerate the elimination process [92].

1.5.2.2. Lymphatic filariasis

Patients infected with the parasite

Diethylcarbamazine (DEC) is the drug of choice for the treatment of LF and kills the microfilaria and the adult worms. DEC has been employed worldwide for more than 50 years

[98] typically, at 6 mg/kg/day for 12 days. Single dose treatment generally has the same effect as the 12-day regimen in terms of microfilarial clearance. The number of microfilariae is

34 associated with adverse effects, but these, in general, are limited. Dizziness, nausea, fever, headache, or pain in muscles or joints are the most common side effects reported [99].

Patients with LF and also having onchocerciasis, should not take DEC because it can worsen onchocercal eye disease. Serious adverse reactions, including encephalopathy and death, may occur in patients with loiasis (not present in Mali). The Loa loa mf densities in the infected person will determine the severity of the side effects. The higher the mf load, the more intense are the side effects [100]. The ivermectin drug kills only the microfilariae, but not the adult worm. Lymphedema and hydrocele are due to the adult worm death and subsequent inflammatory processes [101]. Despite DEC efficacy, in many African countries, including

Mali LF is treated by ivermectin because of the overlap between onchocerciasis and LF infection and possibility of severe adverse effects when treated by DEC. Some studies have shown adult worm killing with doxycycline treatment (200mg/day for 4–6 weeks) [93].

Patients with clinical symptoms

Lymphedema and elephantiasis (sequelae or complication of LF infection) are not indications for DEC treatment because most people with lymphedema are not actively infected with the filarial parasite [98]. To prevent the lymphedema from getting worse, patients should ask their physician for a referral to a lymphedema therapist so they can be informed about some basic principles of care such as hygiene, exercise and wound treatment [102,103]. Patients with hydrocele may have evidence of active infection, but typically do not improve clinically following treatment with DEC and should be treated with surgery.

1.5.3. Prevention 1.5.3.1. Prevention of onchocerciasis

The World Health Assembly adopted Resolution WHA47.32 invited the member states to initiate steps to control onchocerciasis as a public health problem. Ivermectin is the treatment of choice for onchocerciasis. In 1987, Merck & Co authorized ivermectin for the treatment of

35 human onchocerciasis and began a major effort to control the disease with a guarantee to deliver the drug at no cost for as long as necessary [104]. Most recently the paradigm for onchocerciasis prevention changed from control to elimination of the disease as public health problem like LF, based on some studies suggesting that elimination is possible using ivermectin treatment annually or bi-annually. Ivermectin typically is administered as a single yearly dose of 150mcg/kg of body weight (Figure 1-4). Some countries have adopted a twice-yearly treatment regimen to accelerate elimination. Ivermectin is highly active as a microfilaricide. It does not kill the adult worms; however, it may reduce the fertility of the mature female worm.

DEC is also effective at killing the microfilariae but may lead to severe side effects, particularly in heavy infections. Surgical excision of adult worms is recommended when nodules appear on the head.

Figure 1-4 Onchocerciasis elimination framework [105]

The second prevention component is vector control. It involves killing the larvae of the black fly vectors through cautious use of environmentally harmless insecticides. In the 1970s, OCP

36 achieved vector control by weekly aerial spraying of insecticides over fast-flowing rivers and streams in West Africa [106]. It took more than 14 -15 years to break the life-cycle of the parasite through aerial spraying, combined in 1989 with ivermectin treatment of eligible populations in endemic regions [106].

Onchocerciasis Control Programme in West Africa (1974 -2002)

The Onchocerciasis Control Program (OCP) begun as a vector control programme with the intention of eliminating the public health problem of blinding onchocerciasis in savannah areas of West Africa. From the primary set of foci, 654,000 km2 in nine countries, it was expanded to 1,300,000 km2 in 11 countries to prevent reinvasion by flies from none-controlled areas

[106,107]. The OCP originally aimed at stopping transmission by reducing vector populations using weekly aerial distribution of larvicide insecticides in the blackfly breeding sites for long enough to restrict gaining of new infections and let the adult worm population die naturally[108].

Near the end of the 1980s, the microfilaricidal drug ivermectin was licensed for human use after a series of clinical trials. A standard dose of ivermectin (150 ug/kg of body weight, orally) produces 98-99% reduction in skin microfilarial load within 1 to 2 months after treatment, with new mf progressively repopulating the skin (as female worms continue the production of live mf) from the third month after treatment. In addition to reducing transmission from humans to vectors, the microfilaricidal effect of ivermectin ameliorates most of the morbidity linked with onchocerciasis, for which mf are essentially responsible. Consequently, ivermectin is a drug in the arsenal of the so-called preventive chemotherapy treatment (PCT), a strategy validated by

WHO to challenge some of its prioritized NTDs [21]. The OCP piloted community trials of mass distribution of ivermectin in Ghana, showing that it was practicable and safe to administrate treatment at a large scale [109,110]. The OCP approved an annual treatment

37 strategy by 1989, which was used to supplement vector control in some areas and as the only intervention in most of the extension areas [106]. In some of these extension areas, mass ivermectin distribution was provided biannually (6-monthly) [11]. Ivermectin mass treatment was originally distributed by mobile teams and later through community-based distribution, supervised by trained nurses and/or technicians. Finally, this changed into CDTI, the selected mode of drug distribution an approach established by APOC to enhance its long-term sustainability [106].

African Programme for Onchocerciasis Control (1995-2015)

Convinced by ivermectin’s effectiveness for onchocerciasis control if high levels of geographic and therapeutic coverage could be achieved in countries. The African Programme for

Onchocerciasis Control (APOC) was launched in 1995 under the sponsorships of the WHO to cover the remaining endemic African countries external to previous OCP areas [111]. Countries and areas in need of treatment were mapped through rapid epidemiological mapping of onchocerciasis (REMO) [112,113]. APOC’s original objective was to establish, within an initial period of 12 years, effective and self-sustainable CDTI projects in order to control onchocerciasis morbidity [114]. CDTI was applied in 16 of 20 mapped countries, covering endemic areas inhabited by roughly 71.5 million people in 1995 [115]. APOC delivered its first treatments through CDTI in 1997 (80,000 treatments). The programme was gradually scaled up to extend an overall therapeutic coverage of about 73% in 2010 (75.8 million treatments).

In 2014, the number of people receiving ivermectin had increased to nearby 112.5 million people, despite the fact that no treatments happened in Liberia and Sierra Leone, due to the

Ebola outbreak, with an estimated coverage of 78% by 2015, the final year of the programme

[116].

In 2010, and inspired by the successful elimination of the infection in some foci of Mali and

Senegal [11,12] inside the western extension of the OCP, APOC changed its targets from the

38 control of onchocerciasis morbidity to elimination of the parasite reservoir, where feasible, with annual (or biannual) ivermectin MDA [105]. Other illustrations of onchocerciasis elimination in APOC projects offered more evidence to support the feasibility of this change in programme objectives [117,118].

Modelling to support for decision making for onchocerciasis

Onchocerciasis represent an ideal situation among human helminthiases whereby mathematical models have been used to inform control policy (e.g., for intervention strategy, the duration of the programme, the frequency of intervention application, the health impact of the diseases and the intervention) since the initial stages of their implementation [119]. Two main models were developed (EPIONCO and ONCHOSIM) for modelling to inform and to understand the onchocerciasis prevention/elimination strategies [120]. Metanalyses using mathematical modelling by Basáñez et al. was used to quantify the effect of ivermectin in mass treatment [9].

1.5.3.2. Prevention of lymphatic filariasis and Global Programme to

Eliminate LF

World Health Organization classified LF as eradicable or potentially eradicable infection after advances in diagnosis and treatment of LF in 1997. The same year, the World Health

Assembly adopted Resolution WHA 50.29, inviting the member states to initiate steps to eliminate LF as a public health problem. In the context of this call, WHO launched the

Global Programme to Eliminate Lymphatic Filariasis (GPELF) in 2000 [121]. The LF elimination strategy is based in two pillars: community-based mass drug administration

(MDA) to interrupt transmission through clearance of microfilaremia in the blood of infected person over the lifespan of the adult worms (~ 6 years) and morbidity management

(hydrocele and lymphedema) to improve the suffering of affected populations (persons affected by lymphedema and hydrocele) [122].

39

The MDA for LF elimination is implemented using albendazole and ivermectin distribution once a year for at least five consecutive years in five years and above persons in endemic communities. The objective of the MDA is to cover 65% (epidemiological coverage) of the total population at risk of LF using 6 mg/kg of body weight diethylcarbamazine citrate

(DEC) + 400 mg albendazole; or 150 µg/kg of body weight ivermectin + 400 mg albendazole (in areas where onchocerciasis is co-endemic including Mali) (Figure 1-5) and

400 mg albendazole preferably twice per year in areas where Loa loa is co-endemic [122].

WHO and its partners in the context of global LF elimination also established a disease elimination framework (Figure 1-6) [123].

< 1% mf Mapping < 0.1% ICT Additional TDM LQAS 3000 rounds Mass drug administration infants

Yes Stop No Stop Mf > 1%? TDM? TDM

Evaluation of MF in Ou Surveillance sentinels sites and i controle sites ICT ADN Anticorps

Revised criteria for MDA interruption (Source: TaskForce for Global Health) Number of MDA ≥ 5 with coverage rate >65%

Figure 1-5 lymphatic filariasis framework for MDA implementation from mapping to

MDA interruption and surveillance period [124]

MDA: mass drug administration, MMDP: morbidity management and disability prevention

Figure 1-6 Lymphatic filariasis elimination strategies [122]

40

1.5. Onchocerciasis and lymphatic filariasis guidelines for Mass drug

administration stopping and diagnostic tools

1.5.1. Onchocerciasis guidelines for Ivermectin mass drug

administration stopping and diagnostic tools

1.5.1.1. Serodiagnostics for onchocerciasis

The World Health Organization guideline recommends that for the discontinuation of mass treatment with ivermectin, to test a total of 2000 children under ten years of age and a seropositivity (using Ov16-based IgG4 immunoassays) threshold of less than 0.1% should be measured, for this purpose, it recommends an ELISA test. However, in practice for programmes, there are several ELISA test platforms with different procedures and the test procedures require equipment and a laboratory for this type of test, which is hard for programmes in endemic countries [125].

Recently rapid serological tests have been developed but face a validation problem from WHO, and also their performance in field conditions is sometimes questioned. Despite these technical problems, the use of the serological test in the guide is highly recommended with a low degree of certainty evidence. Surveys are conducted using a community-based approach by testing children in villages [125]. More recently there is international will to establish many quality assured laboratories’ to collect essential data to support countries in the process of onchocerciasis elimination [126].

1.5.1.2. Skin Snip test

The guidelines also discuss the possibility of performing skin biopsies under certain conditions as part of the evaluation of transmission elimination. In particular, during the transition to the introduction of Ov16 serology into the program, and this must be done in parallel with the use of Ov16 for the confirmation of positive serology cases detected with serological tests [125].

However, it must be acknowledged that this test has become obsolete because of its low

41 sensitivity, which is about 20% in a context where under drug pressure the prevalence and intensities of onchocerciasis have decreased drastically [127]. Also, this test suffers from acceptance in communities because it is invasive.

1.5.1.3.O-150 PCR in Simuliidae

The WHO guide makes O-150 PCR a strong recommendation with unambiguous evidence.

The O-150 PCR test is to be performed on batch of Simuliidae for the detection of Onchocerca volvulus DNA (L3 stage larvae). For the test to be valid, at least 6000 Simuliidae collected and processed must be in a transmission site. The upper bound of the confidence interval at 95% of the prevalence of infectious Simuliidae must be less than one infected Simulium per 1000 PCR- treated pares (1/1000= 0.1%) or one infected Simulium for 2000 if all Simuliidae are treated

(1/2000=0.05%). On a practical level, the collection of Simuliidae is tedious and faces ethical problems for the people who is involved for the collection, particularly their exposure to

Simulium bites [125].

1.5.2. Lymphatic filariasis guidelines for Ivermectin and albendazole

mass drug administration stopping and diagnostic tools

In the guideline for evaluating the interruption of LF transmission, it is recommended after the

5-6-year treatment phases that transmission be stopped when testing children aged 6-7 years on the basis that these children were born immediately after the first treatment sessions and that they were not exposed to W. bancrofti antigens. There is an Excel software that allows each evaluation unit to determine the sample size and also the survey approach, either school- based when the school enrolment rate reaches 75% or community-based when the rate is lower than this value. It also allows random selection of villages where the survey should be conducted [124].

42

1.5.2.1. Night thick smear

Night thick smear, which was a valuable test during the mapping and monitoring and evaluation phase of the programmes, has lost its usefulness in the evaluation phase of transmission cessation because of low sensitivity and unacceptability by the community members [124].

1.5.2.2. Immunochromatographic test (ICT)

This test was used both during the LF mapping phase and transmission interruption for its excellent sensitivity. However, during the monitoring and evaluation phases and the evaluation of transmission elimination phase, it was also used but was criticised, in particular on the fact that studies showed overestimations of antigenemia rates and some possible cross-reactions with other parasites [128–130]. More recently, this test has been replaced in most programs by the FTS, which gives more conclusive results in the evaluation of transmission failure.

1.5.2.3. Filariasis Test Strip (FTS)

Most recently, a new test known as the Filariasis Test Strip has been developed and tested in more than 15 locations in 11 countries to strengthen the capacity of programs to conduct evaluations for transmission cessation. This WHO-recommended test can be used for mapping, monitoring and evaluation, but also for transmission elimination surveys to make decisions to interrupt mass drug distribution [124]. Since 2015, FTS has replaced ICT in LF control programme in countries where W. bancrofti is present for transmission assessment surveys.

However, the utility of FTS in Loa loa- and M. perstans-endemic areas has been problematic because of antigenic cross-reactivity leading to some false positive results [131].

1.5.2.4. Wb123 antibodies test

A new serology test has recently been made available to the scientific community, the Wb123 test for the detection of antibodies against the Wb123 antigen of W. bancrofti. The test advantage is its ability to detect recent exposures to LF. The test is not still recommended by

43

WHO for use in LF elimination programmes. It has good sensitivity and specificity

[14,132,133].

The advantage is the availability of the test in Biplex format with a window for both Ov16

(IgG4 antibodies) and Wb123 (IgG4 antibodies) allowing for integrated surveillance activities for co endemic areas or countries. The test can be run safety in the field or process in the laboratory with dried blood spots collected in the field. Therefore, it is practical for programmes in countries endemic to LF and onchocerciasis with limited resources [14]. It reduces the costs of assessments by allowing an integrated assessment. However, the test lacks guidance about the threshold and sample size to use for programmatic decision making.

1.6. Mali country profile

1.6.1. Geography and climate

Mali is a landlocked country located in West Africa between latitudes 10° and 25° North, and longitudes 13° West. Mali is eighth-largest country in Africa with a total area of over 1,241,238 squares kilometers. The country shares 7,420 km of borders with seven countries [134].

The Niger river with a total distance of 2,400 km (with 1,700 km in Mali and Senegal) and the

Senegal river with a full length of 1,700 km with 700 km in Mali are the two main rivers taking their source in Guinea Conakry. Most of the country is in the southern Sahara Desert part.

Late June to early December is the rainy season in the southern part of the country. This period also corresponds to the period of transmission of many vector-borne diseases, including malaria, schistosomiasis, and those caused by filarial parasites [134]. The central area has a hot semi-arid climate with very high temperatures with a long, intense dry season and a short, irregular rainy season [134]. The figure 1-7 shows the map of Mali with the main cities and the main rivers.

44

Source : https://fr.wikipedia.org/wiki/Mali#Géographie Figure 1-7 Map of Mali showing the main rivers and cities

45

1.6.1. Demography

The Malian population has increased from 9,810,911 Million in 1998 to 19, 972, 000 inhabitants according to the last population census conducted in the country in 2009. The population of Mali is very young, with 48.8% of the total population aged less than 15 years old. The economically active population of 15 to 64 years old was reported to be 67.3% in 2014

[134].

1.6.2. Economic context

The economy of Mali is based on agriculture, with a mostly rural population engaged in subsistence agriculture [134]. The country is among the ten poorest nations of the world and is one of the 37 heavily indebted developing countries. Mali depends in majority on foreign aid from many sources, including multilateral organizations and bilateral programs. Mali's great potential wealth is based on mining (gold production mainly) and production of agricultural commodities, livestock, and fishing. The most productive agricultural area is along the river of

Niger, the Inner Niger Delta and the southwestern region around Sikasso [134].

Agriculture faces recurring difficulties: recurring droughts since the 1970s, lower prices of raw materials produced, such as cotton, higher production costs (inputs and fuels). As the industrial sector is not very developed, Mali imports a large part of consumer goods [134]. The real growth of the gross domestic product (GDP) is estimated at 5.3% in 2006 against 6.1% in 2005 and inflation was contained in 2006 at 1.5% against 6.4% in 2005. While the poverty rate has declined over the 2001-2006 period, it remains very high, with a national average of 47.4% in

2006 [134].

46

1.6.3. Health system organization

Mali sectoral health and population policy was adopted in 1990 by the Government based on the decentralization of care and community participation. The health system has three levels of care (Figure 1-8):

- The central level with its five specialized hospitals in Bamako providing the third reference;

- The intermediate level consisting of 7 specialized hospitals at regional level provide the second reference;

- The operational level at the district level has two levels which are: the first level, composed of 1086 functional Community health centers in 2011 offers the minimum package of activities

(PMA) as well as para public health structures, confessional, army health services, clinics and other private health facilities. The PMA includes curative, preventive (reproductive health, child survival, vaccination) and promotional care.

- The second level or first reference level consists of 75 reference health districts. It provides

support for the reference from the first degree.

47

Source: H. Dolo, 2014, Master Dissertation, Institute of Tropical Medicine Figure 1-8 Description of Malian health system organization

1.6.4. Lymphatic filariasis and onchocerciasis in Mali

1.6.4.1. Epidemiology of onchocerciasis in Mali

The public health importance of onchocerciasis in Mali was reported as early as the 1970′s

[135]. Onchocerciasis has initially been prevalent in five regions in the country, including

Kayes, Koulikoro, Sikasso, Segou and Mopti. The national onchocerciasis control programme was established in 1986 to control the disease as public health problem. The eastern part of the endemic regions (including Koulikoro Rive Droite, Sikasso, Ségou and Mopti) was included in the first program area of the OCP. In 2006, onchocerciasis was declared eliminated as a public health program in large parts of these areas with only epidemiological and entomological surveillance [11,12]. The western part of the endemic regions including Kayes and Koulikoro

48 rive gauche was included in the western extension of OCP in 1987 with ivermectin distribution

(IVM, donated by Merck & Co.) and later with CDTI with support from the African Program of Onchocerciasis Control (APOC) and using the community drug distributors (CDDs).

According to the National Onchocerciasis Control Programme (in French Programme National de Lutte contre l’Onchocercose au Mali (PNLO)), 34 health districts out 75 in the country were endemic for onchocerciasis between 1971 and 1987 with a total of 4,589,963 population at risk of onchocerciasis. The pre-control mapping identified 9 health districts with prevalence above

60% (hyperendemic), 19 health districts with prevalence between 30.1% to 60%

(mesoendemic), 2 health districts with prevalence ranging from 15.1% to 30 % (hypoendemic),

1 health district with prevalence ranging from 5.1 to 15% (hypoendemic) and 2 health districts with prevalence between 0.1% to 5% (hypoendemic) (Figure 1-9). Onchocerciasis control efforts have begun in early 1974 with Onchocerciasis Control Program (OCP) in West Africa, which ended in 2002. The main strategy implemented by OCP was vector control with the spraying of larvicidal agent. After the end of OCP, the next phase was taken by PNLO with the support of the African Programme for Onchocerciasis Control (APOC) until 2015.

49

Source: B. Guindo Hellen Keller International, NTD programme Figure 1-9 Pre-control mapping of onchocerciasis between 1974- 1987 in Mali

After that, as LF is endemic throughout the country, ivermectin treatment continued de facto still to 2018. From 1990 to present, non-governmental organizations (NGO) have been supporting the programme's activities through support mainly for MDA and monitoring and evaluation. As results of the different operations; currently, two health districts stopped ivermectin MDA, 20 health districts were under treatment. Eleven health districts were under surveillance, and the remaining health districts were district not deemed endemic for onchocerciasis. During the last onchocerciasis prevalence assessment using skin snips, only two health districts were found to have a prevalence between 0.1% and 5%. In 29 health districts, the last prevalence reported was 0% (Figure 1-10).

50

Source: B. Guindo Hellen Keller International, NTD programme Figure 1-10 Onchocerciasis endemicity in the different districts between 2008 and 2015 evaluation in Mali

As the elimination goal of onchocerciasis changed from control to elimination, the next planning for onchocerciasis will be black fly’s breeding’s sites assessment, the pre-stop MDA assessment in endemic districts to identify current hotspots and the stop MDA activities to know the communities(districts) eligible for ivermectin MDA interruption as described in figure 1-11.

51

Source: B. Guindo Hellen Keller International, NTD programme

Figure 1-11 Planning of onchocerciasis activities in elimination context

1.6.4.2. Epidemiology of lymphatic filariasis in Mali

In Mali, in spite of the public health importance of LF was noted as early as the 1970′s [136] the prevalence and distribution were not studied again until 2002. This was due to the prioritization given to diseases that are associated with higher mortality rates such as malaria.

Using the Immunochromatographic card test (ICT) for LF antigen detection, all eight administrative regions of Mali were demonstrated to be endemic for LF (antigenemia prevalence more than equal or more than 1%) with approximately 10 million people at risk of the disease [8]. Figure 1-12 shows the mapping of LF in the different health districts of Mali.

According to the National Lymphatic Filariasis Elimination Programme (in french Programme

National d’Elimination de la Filariose Lymphatique (PNEFL)) data in 2004, all the eight

52 regions and the district of Bamako were LF endemic. The LF national prevalence was 7.07% ranging from 1% in the North part of the country to 18.6% in the south region.

Africa

The Mapping survey data showed Mali an overall prevalence of 7.07% in the country. The different regions prevalence are reported below: region of Sikasso :………….18.6% region of Mopti :……………15.4% region of Kayes :…… ……..8.6% region of Kidal :……………….7.3% region of Gao :……………...…5.1% region of Ségou : …………….4.0% region of Koulikoro…………3.8% region of Tombouctou :……1.0% District of Bamako overall ..1.5%

Source: Helen Keller International NTD programme, 2017 Figure 1-12 Pre-control mapping of Lymphatic filariasis in 2004

More than one thousand cases of chronic LF clinical manifestations were reported by the census survey undertaken by the National LF elimination program (Dembele M, personal communication, June 2014). In Mali, in addition to the presence of onchocerciasis and LF, there are schistosomiasis, soil-transmitted helminth and trachoma that are targeted for preventive chemotherapy (figure 1-13) [8].

53

LF: lymphatic filariasis; ONCH: onchocerciasis; SCH: schistosomiasis; STH: soil-transmitted helminthiasis;

TRA: trachoma. In Kidal region, the endemicity level of schistosomiasis in each district is not yet clear and further mapping is planned.

Figure 1-13 Mapping neglected tropical diseases targeted for preventive chemotherapy in Mali in 2012 [8]

LF elimination efforts begun in 2004 -2005 with diseases mapping followed by the first administration of Albendazole and Ivermectin in two LF endemic health district in 2005.

Progressively the MDA was scaled up to all the health districts in 2009. From 2004 to present,

NON-governmental organization (NGO) have been supporting the programme's activities mainly MDA and hydrocele surgery and limited morbidity management of lymphedema. As

54 of 2016, all 22 Evaluation unit (EU) (combination of one or more health districts for TAS of

LF treatment interruption) of the country passed the TAS (with less than 2% seroprevalence in children of 6 to 7 years old). As part of the post-MDA surveillance, in 2015, the first two districts that stopped the MDA passed their TAS2 using ICT test in the 6-7 years old children three years after their last MDA (Figure 1-14). The figure 1-13 shows the co endemicity of different neglected tropical diseases that are targeted by preventive chemotherapy [8] .

As of 2019, all the 75 endemic health districts (results of redistricting original 65 health districts) have passed the transmission assessment survey (TAS) because less than 2% of antigenemia prevalence in 6 to 7 years old children reported and therefore stopped MDA and entered the program surveillance phase.

Source: Helen Keller International NTD programme, 2017

Figure 1-14 Lymphatic filariasis elimination status in Mali as of 2017

55

1.7. Rationale

The progress against onchocerciasis and LF as results of the national programme activities, show significant reductions in the prevalence and intensity of the two NTD [11,12,137,138].

With regard to this progress, the stakeholders are willing to stop ivermectin and albendazole

MDA based in evidence-criteria to prepare the elimination surveillance phase. However, the diagnostic tools (skin snip and thick smear) used for mapping and monitoring and evaluation have become obsolete in a context where the prevalence of onchocerciasis and LF are drastically decreasing. The lack of performance of the previously used diagnostic tool requires the testing of new performant diagnostic tools, particularly serological ones [1]. The co- endemicity of onchocerciasis and LF in tropical countries including Mali, supports the importance to test the feasibility of integrated assessments of NTD at the operational level [1].

Also, in the context of the preparation for the elimination phase, emphasis must be placed on epidemiology and the management of morbidities such as lymphedema. It is also essential to consider the role of other existing parasitic infection in these settings and their interaction with onchocerciasis, LF, schistosomiasis, geohelminthiases, and trachoma that are targeted during chemoprevention. They can play a role in the deployment of serological tests with potential cross-reaction [34,139,140].

The recent point of care format of the rapid Oncho/LF IgG4 Biplex (SD, South Korea) that detects antibodies against the antigens Wb123 and OV16 respectively for LF and onchocerciasis opens new prospects for surveillance of onchocerciasis and LF [14,141].

However, to date, they have not been used for stopping decisions because of current WHO

Guidelines Currently, three ELISA platforms exist (PATH/CDC ELISA, OEPA/TCC ELISA and SD-ELISA (commercial prototype kit) using different protocols for processing and they require laboratory for processing the samples and the rapid test format is criticized for their low field performance [142,143]. Hence the work of this thesis is to address these concerns to

56 inform the onchocerciasis and LF programs to support the preparedness of elimination phase in Mali.

1.7. Scientific Objectives

Chapter 1 Study the serologic prevalence of onchocerciasis and lymphatic filariasis with SD

Bioline Biplex Wb123/ov16 in elimination context in different co-endemic areas of Mali;

Chapter 2 Study the burden and clinical presentation of lymphedema in previous LF endemic health districts during post mass drug administration phase; and

Chapter 3 Determine risk factors for W. bancrofti microfilaremia in an endemic area of Mali where M. perstans is co endemic.

1.8. Organization of the thesis

Chapter 1.

General introduction. This chapter provides an overview of onchocerciasis and lymphatic flariasis epidemiology, disease burden, control and elimination strategies. We presented the country profile including socio economic and the health system description of Mali and the context specific epidemiology of onchocerciasis and lymphatic filariasis in the country and the current and prospected activities to fight against the two infection. We also give in this chapter the rationale, specific objectives and organization of the thesis.

Chapter 2.

This chapter presents the results of an integrated serological assessment in 6-7-year-old children using SD Bioline Biplex wb123/ov16 to evaluate the elimination of transmission of onchocerciasis and lymphatic filariasis in two lymphatic filariasis evaluation units of Mali.

Chapter 3.

57

This chapter presents the results of serological evaluation of onchocerciasis and lymphatic filariasis elimination in the Bakoye and Falémé foci in Mali in 2017 -2018 under more than 24 years of ivermectin.

Chapter 4.

This chapter describes lymphedema in three previously Wuchereria bancrofti-endemic health districts in Mali after cessation of albendazole ivermectin mass drug administration for lymphatic filariasis.

Chapter 5.

In this chapter, we describe factors associated with W. bancrofti microfilaremia in Mali. Factors assessed include village of residence, age, gender and co-infection with M. perstans. In this chapter we are focused on understanding the interaction between M. perstans infection and W. bancrofti infection.

Chapter 6.

This chapter focuses on the general discussion of the thesis, the conclusion and further perspectives for research in onchocerciasis and LF. We highlighted most important findings of our work and their implication for onchocerciasis and lymphatic filariasis elimination programme in Malian context and for their similar programme in Africa. We discussed the diagnostic tests used currently, the feasibility of integrated evaluation of onchocerciasis and lymphatic filariasis. We also reported on the morbidities related to lymphedema and the finding related to M. perstans in the context of LF elimination framework.

58

1.9. Studies team contribution

This work was conducted in a very difficult environment in Mali because of the security and political crisis that has been ongoing since 2012. I benefited from the assistance of Dr. Yaya

Ibrahim Coulibaly for supervision of the field data collection and for assistance writing the articles and the thesis.

Drs. Siaka Yamoussa Coulibaly, Salif Doumbia, Moussa Brema Sangare, Ilo Dicko, and

Abdallah Amadou Diallo, Mr. Lamine Soumaoro, Mr. Michel Emmanuel Coulibaly, Dr.

Fatoumata dite Nene Konipo and all of the students of our Research and Training Unit on

Filariasis participated in the field data collection in Mali. In addition, they were of great support in the design and implementation of the different protocols under my supervision.

This work was also made possible by logistic and academic involvement of Prof. Robert

Colebunders from Global Health Institute of the Universiteit of Antwerpen and Dr. Thomas

Nutman from National Institute of Health (NIH) who validated our research ideas and also corrected the final versions of the different manuscripts and the structuring and writing of the thesis. They were promotors of the thesis.

Prof. Maria Gloria Basáñez and Dr. Martin Walker from Imperial College and the Royal

Veterinary College, respectively, also helped and supported our scientific stays where they actively participated in the writing of our article on serology and the drafting of the final version of the thesis. They also played an important role as co promotors of the thesis

Myself as PhD student, I have been involved in the protocol writing, data collection in the field, data analysis and manuscript writing under the supervision of my mentors and supervisors in

Mali, Belgium, United States of America and United Kingdom

Dr Amy D. Klion from the National Institute of Health (NIH) contributed also by revising a number of the thesis chapter for English correction.

59

Myself, as a PhD student, I have been involved in the protocol writing, data collection in the field, data analysis and manuscript writing under the supervision of my mentors and supervisors in Mali, Belgium and United Kingdom

1.10. References

1. World Health Organization . Accelerating work to overcome the global impact of neglected tropical diseases . 2012.

2. Simonsen PE, Onapa AW, Asio SM. Mansonella perstans filariasis in Africa. Acta Trop. 2011; 120 Suppl 1:S109-20.

3. CDC - Lymphatic Filariasis - Biology - Life Cycle of Wuchereria bancrofti. Available at: https://www.cdc.gov/parasites/lymphaticfilariasis/biology_w_bancrofti.html. Accessed 7 October 2019.

4. CDC - Onchocerciasis - Biology. Available at: https://www.cdc.gov/parasites/onchocerciasis/biology.html. Accessed 22 August 2019.

5. Duke BO, Lewis DJ, Moore PJ. Onchocerca-Simulium complexes. I. Transmission of forest and Sudan-savanna strains of Onchocerca volvulus, from Cameroon, by Simulium damnosum from various West African bioclimatic zones. Ann Trop Med Parasitol 1966; 60:318–326.

6. Coulibaly YI, Dao S, Traore AK, Diallo A, Sacko M, Traoré SF. [Presence and risk of transmission of Wuchereria bancrofti is a reality in rural Mali: the case of the town of Bariambani in the Cirle of Kati]. Mali Med. 2006; 21:12–17.

7. Cano J, Basáñez M-G, O’Hanlon SJ, et al. Identifying co-endemic areas for major filarial infections in sub-Saharan Africa: seeking synergies and preventing severe adverse events during mass drug administration campaigns. Parasit. Vectors 2018; 11:70.

8. Dembélé M, Bamani S, Dembélé R, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl. Trop. Dis. 2012; 6:e1574.

9. Basáñez M-G, Pion SDS, Boakes E, Filipe JAN, Churcher TS, Boussinesq M. Effect of single- dose ivermectin on Onchocerca volvulus: a systematic review and meta-analysis. Lancet Infect. Dis. 2008; 8:310–322.

10. Gyapong JO, Kumaraswami V, Biswas G, Ottesen EA. Treatment strategies underpinning the global programme to eliminate lymphatic filariasis. Expert Opin. Pharmacother. 2005; 6:179– 200.

11. Diawara L, Traoré MO, Badji A, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl. Trop. Dis. 2009; 3:e497.

12. Traore MO, Sarr MD, Badji A, et al. Proof-of-principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl. Trop. Dis. 2012; 6:e1825.

60

13. Dadzie Y, Neira M, Hopkins D. Final report of the Conference on the eradicability of Onchocerciasis. Filaria J 2003; 2:2.

14. Steel C, Golden A, Stevens E, et al. Rapid Point-of-Contact Tool for Mapping and Integrated Surveillance of Wuchereria bancrofti and Onchocerca volvulus Infection. Clin. Vaccine Immunol. 2015; 22:896–901.

15. Dieye Y, Storey HL, Barrett KL, et al. Feasibility of utilizing the SD BIOLINE Onchocerciasis IgG4 rapid test in onchocerciasis surveillance in Senegal. PLoS Negl. Trop. Dis. 2017; 11:e0005884.

16. Basáñez MG, Boussinesq M. Population biology of human onchocerciasis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354:809–826.

17. Bradley JE, Whitworth J, Basáñez MG. Chapter 39: Onchocerciasis. In: microbiology and microbial infections. London: Edward Arnold Publishers Ltd, 2005: pp 781-801.

18. Organization WH. Working to overcome the global impact of neglected tropical diseases: first WHO report on neglected tropical diseases. 2010;

19. Onchocerciasis and its control. Report of a WHO Expert Committee on Onchocerciasis Control. World Health Organ. Tech. Rep. Ser. 1995; 852:1–104.

20. Traoré S, Wilson MD, Sima A, et al. The elimination of the onchocerciasis vector from the island of Bioko as a result of larviciding by the WHO African Programme for Onchocerciasis Control. Acta Trop. 2009; 111:211–218.

21. W H O. WHO | Prevention, control and elimination of onchocerciasis. 2016. Available at: http://www.who.int/onchocerciasis/control/en/. Accessed 27 April 2017.

22. Kim, A, Benton B. Cost-benefit Analysis of the Onchocerciasis Control Program (OCP). World Bank Technical Paper No. 282. Series on River Blindness Control in West Africa. The World Bank, Washington DC, USA. Work Bank, 1995. Available at: http:// www- wds.worldbank.org/external/default/WDSContentServer/WDSP/IB/1995/ 05/01/000009265_3970311123047/Rendered/PDF/multi0page.pdf. Accessed 8 August 2019.

23. Sasa M. Human filariasis: a global survey of epidemiology and control. Human filariasis: a global survey of epidemiology and control 1976;

24. Schacher JF. Laboratory models in filariasis: a review of filarial life-cycle patterns. Southeast Asian Journal of Tropical Medicine and Public Health, 4 1973; 4:336–49. 25. Beaver PC, Jung RC, Cupp EW, Craig CF. Clinical parasitology. Clinical parasitology 1984;

26. Global programme to eliminate lymphatic filariasis: progress report, 2015. Wkly Epidemiol Rec 2016; 91:441–455.

27. Addiss D. Lymphatic filariasis. Bull. World Health Organ. 1998; 76 Suppl 2:145–146.

28. Organization WH. Global programme to eliminate lymphatic filariasis: Progress report on mass drug administration in 2008. Weekly Epidemiological Record= Relevé épidémiologique hebdomadaire, 84 2009; 84:437–444.

29. Ton TGN, Mackenzie C, Molyneux DH. The burden of mental health in lymphatic filariasis. Infect Dis Poverty 2015; 4:34.

61

30. Mathew CG, Bettis AA, Chu BK, et al. The health and economic burden of lymphatic filariasis prior to mass drug administration programmes. Clin. Infect. Dis. 2019;

31. Global programme to eliminate lymphatic filariasis: progress report, 2016. Wkly Epidemiol Rec 2017; 92:594–607.

32. Wanji S, Tayong DB, Ebai R, et al. Update on the biology and ecology of Culicoides species in the South-West region of Cameroon with implications on the transmission of Mansonella perstans. Parasit. Vectors 2019; 12:166.

33. Akpan SS, Mbah M, Achi E. The occurrence of Mansonella perstans among residents of Calabar metropolis in Cross River State of Nigeria. Ann. Parasitol. 2015; 61:17–20.

34. Wanji S, Tayong DB, Layland LE, et al. Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin. Parasit. Vectors 2016; 9:311.

35. Stensgaard A-S, Vounatsou P, Onapa AW, et al. Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria. PLoS Negl. Trop. Dis. 2016; 10:e0004319.

36. Bouyou-Akotet MK, Moussavou Boussougou MN, Ovono-Abessolo F, Owono-Medang M, Kombila M. Influence of Mansonella perstans microfilaraemia on total IgE levels in Gabonese patients co-infected with Loa loa. Acta Trop. 2014; 131:11–15.

37. Cross JH. Filarial Nematodes. In: Baron S, ed. Medical Microbiology. Galveston (TX): University of Texas Medical Branch at Galveston, 1996.

38. Tamarozzi F, Halliday A, Gentil K, Hoerauf A, Pearlman E, Taylor MJ. Onchocerciasis: the role of Wolbachia bacterial endosymbionts in parasite biology, disease pathogenesis, and treatment. Clin. Microbiol. Rev. 2011; 24:459–468.

39. Essentials of Human Parasitology - Judith Stephenson Heelan, Frances W. Ingersoll - Google Livres.

40. Taylor MJ. A new insight into the pathogenesis of filarial disease. Curr. Mol. Med. 2002; 2:299– 302.

41. Dreyer G, Figueredo-Silva J, Carvalho K, Amaral F, Ottesen EA. Lymphatic filariasis in children: adenopathy and its evolution in two young girls. Am. J. Trop. Med. Hyg. 2001; 65:204–207.

42. Nutman TB. Insights into the pathogenesis of disease in human lymphatic filariasis. Lymphat Res Biol 2013; 11:144–148.

43. Taylor MJ, Cross HF, Ford L, Makunde WH, Prasad GB, Bilo K. Wolbachia bacteria in filarial immunity and disease. Parasite Immunol 2001; 23:401–409.

44. Kramer LH, Passeri B, Corona S, Simoncini L, Casiraghi M. Immunohistochemical/immunogold detection and distribution of the endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a polyclonal antiserum raised against WSP (Wolbachia surface protein). Parasitol. Res. 2003; 89:381–386.

45. Tavares da Silva LB, Crainey JL, Ribeiro da Silva TR, et al. Molecular Verification of New

62

World Mansonella perstans Parasitemias. Emerging Infect. Dis. 2017; 23:545–547.

46. Keiser PB, Coulibaly Y, Kubofcik J, et al. Molecular identification of Wolbachia from the filarial nematode Mansonella perstans. Mol. Biochem. Parasitol. 2008; 160:123–128.

47. Coulibaly YI, Dembele B, Diallo AA, et al. A randomized trial of doxycycline for Mansonella perstans infection. N. Engl. J. Med. 2009; 361:1448–1458.

48. Gehringer C, Kreidenweiss A, Flamen A, Antony JS, Grobusch MP, Bélard S. Molecular evidence of Wolbachia endosymbiosis in Mansonella perstans in Gabon, Central Africa. J. Infect. Dis. 2014; 210:1633–1638.

49. Bassene H, Sambou M, Fenollar F, et al. High Prevalence of Mansonella perstans Filariasis in Rural Senegal. Am. J. Trop. Med. Hyg. 2015; 93:601–606.

50. Puri PK. Onchocerciasis. Cutis 2015; 95:131, 139–40.

51. Cooper PJ, Nutman TB. Onchocerciasis. In: Hunter’s tropical medicine and emerging infectious disease. Elsevier, 2013: 827–834.

52. Clifford Kwan‐Gett TS, Kemp C, Kovarik C. Infectious and tropical diseases: A handbook for primary care. J Travel Med 2007; 14:359–360.

53. Otabil KB, Gyasi SF, Awuah E, et al. Prevalence of onchocerciasis and associated clinical manifestations in selected hypoendemic communities in Ghana following long-term administration of ivermectin. BMC Infect. Dis. 2019; 19:431.

54. Winkler AS, Friedrich K, König R, et al. The head nodding syndrome--clinical classification and possible causes. Epilepsia 2008; 49:2008–2015.

55. Nyungura JL, Akim T, Lako A, Gordon A, Lejeng L, William G. Investigation into the nodding syndrome in Witto Payam, Western Equatoria state, 2010. South Sudan Medical Journal 2011; 4:3–6.

56. Colebunders R, Nelson Siewe FJ, Hotterbeekx A. Onchocerciasis-Associated Epilepsy, an Additional Reason for Strengthening Onchocerciasis Elimination Programs. Trends Parasitol. 2018; 34:208–216.

57. Colebunders R, Njamnshi AK, van Oijen M, et al. Onchocerciasis-associated epilepsy: From recent epidemiological and clinical findings to policy implications. Epilepsia Open 2017; 2:145–152.

58. Geelhand de Merxem D, Siewe Fodjo JN, Menon S, Hotterbeekx A, Colebunders R. Nodding syndrome research, lessons learned from the NSETHIO project. Glob Ment Health (Camb) 2019; 6:e26.

59. Dreyer G, Norões J, Figueredo-Silva J, Piessens WF. Pathogenesis of lymphatic disease in bancroftian filariasis: Parasitology Today 2000; 16:544–548.

60. Figueredo-Silva J, Dreyer G. Bancroftian filariasis in children and adolescents: clinical- pathological observations in 22 cases from an endemic area. Ann Trop Med Parasitol 2005; 99:759–769.

61. Eberhard ML, Walker EM, Addiss DG, Lammie PJ. A survey of knowledge, attitudes, and perceptions (KAPs) of lymphatic filariasis, elephantiasis, and hydrocele among residents in an

63

endemic area in Haiti. Am. J. Trop. Med. Hyg. 1996; 54:299–303.

62. Dreyer G, Norões J, Figueredo-Silva J, Piessens WF. Pathogenesis of lymphatic disease in bancroftian filariasis: a clinical perspective. Parasitol. Today 2000; 16:544–548.

63. Ottesen EA, Nutman TB. Tropical pulmonary eosinophilia. Annu. Rev. Med. 1992; 43:417– 424.

64. Mediannikov O, Ranque S. Mansonellosis, the most neglected human filariasis. New Microbes New Infect. 2018; 26:S19–S22.

65. Crainey JL, Ribeiro da Silva TR, Luz SLB. Historic accounts of Mansonella parasitaemias in the South Pacific and their relevance to lymphatic filariasis elimination efforts today. Asian Pac. J. Trop. Med. 2016; 9:205–210.

66. Gobbi F, Beltrame A, Buonfrate D, et al. Imported Infections with Mansonella perstans Nematodes, Italy. Emerging Infect. Dis. 2017; 23:1539–1542.

67. Foster DG. Filariasis, a rare cause of pericarditis. J Trop Med Hyg 1956; 59:212–214.

68. Kahn JB. Pleural effusion associated with Dipetalonema perstans (Acanthocheilonema perstans). J. Infect. Dis. 1983; 147:166.

69. Olumide YM, Obembe BM. Dipetalonema perstans in a patient with chronic lymphoedema. Case report. East Afr. Med. J. 1983; 60:186–189.

70. Lansoud-Soukate J, Dupont A, De Reggi ML, Roelants GE, Capron A. Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis. Acta Trop. 1989; 46:249– 256.

71. Gelfand M, Bernberg H. Tropical eosinophilic syndrome. A clinical description of the disorder as seen in S. Rhodesia. Cent. Afr. J. Med. 1959; 5:405–411.

72. Baker JEP, B Nm. A study of eosinophilia and A. perstans infestation in African patients. Central African Journal of Medicine 1967;

73. Clarke VV, Harwin RM, MacDonald DF. Filariasis: Dipetalonema perstans infections in Rhodesia. Central African Journal … 1971;

74. Bregani ER, Balzarini L, Mbaïdoum N, Rovellini A. Prevalence of filariasis in symptomatic patients in Moyen Chari district, south of Chad. Trop Doct 2007; 37:175–177.

75. Baird JK, Neafie RC, Connor DH. Nodules in the conjunctiva, bung-eye, and bulge-eye in Africa caused by Mansonella perstans. Am. J. Trop. Med. Hyg. 1988; 38:553–557. 76. Bregani ER, Ceraldi T, Rovellini A, Ghiringhelli C. Case report: intraocular localization of Mansonella perstans in a patient from south Chad. Trans. R. Soc. Trop. Med. Hyg. 2002; 96:654.

77. Taylor HR, Greene BM. Ocular changes with oral and transepidermal diethylcarbamazine therapy of onchocerciasis. Br. J. Ophthalmol. 1981; 65:494–502.

78. Kuhlmann FM, Fleckenstein JM. Antiparasitic Agents. In: Infectious Diseases. Elsevier, 2017: 1345–1372.e2.

79. Dull HB. Mectizan donation and the Mectizan Expert Committee. Acta Leiden. 1990; 59:399– 403.

64

80. Van Laethem Y, Lopes C. Treatment of onchocerciasis. Drugs 1996; 52:861–869.

81. Soboslay PT, Newland HS, White AT, et al. Ivermectin effect on microfilariae of Onchocerca volvulus after a single oral dose in humans. Trop. Med. Parasitol. 1987; 38:8–10.

82. Schulzkey H. Ivermectin in the treatment of onchocerciasis. ISI ATLAS OF SCIENCE- PHARMACOLOGY 1987; 1:246–249.

83. Albiez EJ, Walter G, Kaiser A, et al. Effects of high doses of diethylcarbamazine on adult Onchocerca volvulus examined by the collagenase technique and by histology. Tropical medicine and parasitology: official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) 1988; 39:87–92.

84. Duke BO, Zea-Flores G, Castro J, Cupp EW, Munoz B. Effects of three-month doses of ivermectin on adult Onchocerca volvulus. Am. J. Trop. Med. Hyg. 1992; 46:189–194.

85. Chavasse DC, Post RJ, Davies JB, Whitworth JA. Absence of sperm from the seminal receptacle of female Onchocerca volvulus following multiple doses of ivermectin. Tropical medicine and parasitology: official organ of Deutsche Tropenmedizinische Gesellschaft and of Deutsche Gesellschaft fur Technische Zusammenarbeit (GTZ) 1993; 44:155–158.

86. Newland HS, White AT, Greene BM, et al. Effect of single-dose ivermectin therapy on human Onchocerca volvulus infection with onchocercal ocular involvement. Br. J. Ophthalmol. 1988; 72:561–569.

87. Glover M, Murdoch M, Leigh I. Subtle early features of onchocerciasis in a European. J. R. Soc. Med. 1991; 84:435.

88. Somo RM, Ngosso A, Dinga JS, Enyong PA, Fobi G. A community-based trial of ivermectin for onchocerciasis control in the forest of southwestern Cameroon: clinical and parasitologic findings after three treatments. Am. J. Trop. Med. Hyg. 1993; 48:9–13.

89. Dadzie KY, Remme J, Alley ES, de Sole G. Changes in ocular onchocerciasis four and twelve months after community-based treatment with ivermectin in a holoendemic onchocerciasis focus. Trans. R. Soc. Trop. Med. Hyg. 1990; 84:103–108.

90. Macfarlane CL, Budhathoki SS, Johnson S, Richardson M, Garner P. Albendazole alone or in combination with microfilaricidal drugs for lymphatic filariasis. Cochrane Database Syst. Rev. 2019; 1:CD003753.

91. Kuesel AC. Research for new drugs for elimination of onchocerciasis in Africa. Int. J. Parasitol. Drugs Drug Resist. 2016; 6:272–286.

92. Opoku NO, Bakajika DK, Kanza EM, et al. Single dose moxidectin versus ivermectin for Onchocerca volvulus infection in Ghana, Liberia, and the Democratic Republic of the Congo: a randomised, controlled, double-blind phase 3 trial. The Lancet 2018; 392:1207–1216.

93. Taylor MJ, Hoerauf A, Townson S, Slatko BE, Ward SA. Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis. Parasitology 2014; 141:119–127.

94. Wan Sulaiman WA, Kamtchum-Tatuene J, Mohamed MH, et al. Anti-Wolbachia therapy for onchocerciasis & lymphatic filariasis: Current perspectives. Indian J. Med. Res. 2019; 149:706– 714.

65

95. Ejere HOD, Schwartz E, Wormald R, Evans JR. Ivermectin for onchocercal eye disease (river blindness). Cochrane Database Syst. Rev. 2012; :CD002219.

96. Lariviere M, Vingtain P, Aziz M, et al. Double-blind study of ivermectin and diethylcarbamazine in African onchocerciasis patients with ocular involvement. The Lancet 1985; 2:174–177.

97. Turner JD, Tendongfor N, Esum M, et al. Macrofilaricidal activity after doxycycline only treatment of Onchocerca volvulus in an area of Loa loa co-endemicity: a randomized controlled trial. PLoS Negl. Trop. Dis. 2010; 4:e660.

98. CDC - Lymphatic Filariasis - Treatment. 2018. Available at: https://www.cdc.gov/parasites/lymphaticfilariasis/treatment.html. Accessed 10 May 2018.

99. Yongyuth P, Koyadun S, Jaturabundit N, Jariyahuttakij W, Bhumiratana A. Adverse reactions of 300 MG diethylcarbamazine, and in a combination of 400 MG albendazole, for a mass annual single dose treatment, in migrant workers in Phang Nga province. J Med Assoc Thai 2007; 90:552–563.

100. Carme B, Boulesteix J, Boutes H, Puruehnce MF. Five cases of encephalitis during treatment of loiasis with diethylcarbamazine. Am. J. Trop. Med. Hyg. 1991; 44:684–690.

101. de Souza DK, Ahorlu CS, Adu-Amankwah S, et al. Community-based trial of annual versus biannual single-dose ivermectin plus albendazole against Wuchereria bancrofti infection in human and mosquito populations: study protocol for a cluster randomised controlled trial. Trials 2017; 18:448.

102. Yahathugoda TC, Weerasooriya MV, Samarawickrema WA, Kimura E, Itoh M. Impact of two follow-up schemes on morbidity management and disability prevention (MMDP) programme for filarial lymphedema in Matara, Sri Lanka. Parasitol. Int. 2018; 67:176–183.

103. Douglass J, Mableson HE, Martindale S, Kelly-Hope LA. An Enhanced Self-Care Protocol for People Affected by Moderate to Severe Lymphedema. Methods Protoc. 2019; 2.

104. Colatrella B. The Mectizan Donation Program: 20 years of successful collaboration - a retrospective. Ann Trop Med Parasitol 2008; 102 Suppl 1:7–11.

105. WHO A. Conceptual and Operational Framework of Onchocerciasis Elimination with Ivermectin Treatmen. 2010. Available at: https://apps.who.int/iris/bitstream/handle/10665/275466/JAF16.6(II)-eng.pdf? Accessed 21 June 2019.

106. Boatin B. The onchocerciasis control programme in west africa (OCP). Ann Trop Med Parasitol 2008; 102 Suppl 1:13–17.

107. Boatin BA, Richards FO. Control of onchocerciasis. Adv Parasitol 2006; 61:349–394.

108. Sékétéli A, Guillet P, Coloussa B, Philippon B, Quillévéré D, Samba EM. [National entomological teams of the western extension zone of the Onchocerciasis Control Program (OCP) in west Africa from 1986 to 1990]. Bull. World Health Organ. 1993; 71:737–753.

109. Remme J, De Sole G, van Oortmarssen GJ. The predicted and observed decline in onchocerciasis infection during 14 years of successful control of Simulium spp. in west Africa. Bull. World Health Organ. 1990; 68:331–339.

66

110. Alley ES, Plaisier AP, Boatin BA, et al. The impact of five years of annual ivermectin treatment on skin microfilarial loads in the onchocerciasis focus of Asubende, Ghana. Trans. R. Soc. Trop. Med. Hyg. 1994; 88:581–584.

111. Remme JHF. The African programme for onchocerciasis control: Preparing to launch. Parasitology Today 1995; 11:403–406.

112. Ngoumou P, Walsh JF, Mace JM. A rapid mapping technique for the prevalence and distribution of onchocerciasis: a Cameroon case study. Ann Trop Med Parasitol 1994; 88:463–474.

113. Noma M, Nwoke BEB, Nutall I, et al. Rapid epidemiological mapping of onchocerciasis (REMO): its application by the African Programme for Onchocerciasis Control (APOC). Ann Trop Med Parasitol 2002; 96 Suppl 1:S29-39.

114. Amazigo UV, Obono M, Dadzie KY, et al. Monitoring community-directed treatment programmes for sustainability: lessons from the African Programme for Onchocerciasis Control (APOC). Ann Trop Med Parasitol 2002; 96 Suppl 1:S75-92.

115. Coffeng LE, Stolk WA, Zouré HGM, et al. African Programme For Onchocerciasis Control 1995-2015: model-estimated health impact and cost. PLoS Negl. Trop. Dis. 2013; 7:e2032.

116. World Health Organization/African Programme for Onchocerciasis Control,. World Health Organization/African Programme for Onchocerciasis Control, 2015. Report of the Consultative Meetings on Strategic Options and Alternative Treatment Strategies for Accelerating Onchocerciasis Elimination in Africa. WHO/MG/15.20. . 2015.

117. Tekle AH, Elhassan E, Isiyaku S, et al. Impact of long-term treatment of onchocerciasis with ivermectin in Kaduna State, Nigeria: first evidence of the potential for elimination in the operational area of the African Programme for Onchocerciasis Control. Parasit. Vectors 2012; 5:28.

118. Higazi TB, Zarroug IMA, Mohamed HA, et al. Interruption of Onchocerca volvulus transmission in the Abu Hamed focus, Sudan. Am. J. Trop. Med. Hyg. 2013; 89:51–57.

119. Basáñez M-G, McCarthy JS, French MD, et al. A research agenda for helminth diseases of humans: modelling for control and elimination. PLoS Negl. Trop. Dis. 2012; 6:e1548.

120. Basáñez MG, Walker M, Turner HC, Coffeng LE, de Vlas SJ, Stolk WA. River blindness: mathematical models for control and elimination. Adv Parasitol 2016; 94:247–341.

121. Ottesen EA. The global programme to eliminate lymphatic filariasis. Trop. Med. Int. Health 2000; 5:591–594.

122. Ottesen EA. Editorial: The Global Programme to Eliminate Lymphatic Filariasis. Trop. Med. Int. Health 2000; 5:591–594.

123. Ichimori K, King JD, Engels D, et al. Global programme to eliminate lymphatic filariasis: the processes underlying programme success. PLoS Negl. Trop. Dis. 2014; 8:e3328.

124. WHO. Transmission assessment surveys in the Global Programme to Eliminate Lymphatic Filariasis: WHO position statement. Wkly Epidemiol Rec 2012; 87:478–482.

125. Guidelines for stopping mass drug administration and verifying elimination of human

67

onchocerciasis: criteria and procedures. Geneva: World Health Organization, 2016.

126. Shott J, Ducker C, Unnasch TR, Mackenzie CD. Establishing quality assured (QA) laboratory support for onchocerciasis elimination in Africa. Int. Health 2018; 10:i33–i39.

127. Weiss N, Karam M. Evaluation of a specific enzyme immunoassay for onchocerciasis using a low molecular weight antigen fraction of Onchocerca volvulus. Am. J. Trop. Med. Hyg. 1989; 40:261–267.

128. Dorkenoo AM, Sodahlon YK, Bronzan RN, et al. [Lymphatic filariasis transmission assessment survey in schools three years after stopping mass drug treatment with albendazole and ivermectin in the 7 endemic districts in Togo]. Bull Soc Pathol Exot 2015; 108:181–187.

129. Dorkenoo MA, Bronzan R, Yehadji D, et al. Surveillance for lymphatic filariasis after stopping mass drug administration in endemic districts of Togo, 2010-2015. Parasit. Vectors 2018; 11:244.

130. Pion SD, Montavon C, Chesnais CB, et al. Positivity of Antigen Tests Used for Diagnosis of Lymphatic Filariasis in Individuals Without Wuchereria bancrofti Infection But with High Loa loa Microfilaremia. Am. J. Trop. Med. Hyg. 2016; 95:1417–1423.

131. Chesnais CB, Awaca-Uvon N-P, Bolay FK, et al. A multi-center field study of two point-of- care tests for circulating Wuchereria bancrofti antigenemia in Africa. PLoS Negl. Trop. Dis. 2017; 11:e0005703.

132. Weil GJ, Steel C, Liftis F, et al. A rapid-format antibody card test for diagnosis of onchocerciasis. J. Infect. Dis. 2000; 182:1796–1799.

133. Golden A, Stevens EJ, Yokobe L, et al. A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus. PLoS Negl. Trop. Dis. 2016; 10:e0004292.

134. Mali - Wikipedia. 2018. Available at: https://en.wikipedia.org/wiki/Mali. Accessed 28 May 2018.

135. Prost A. [Situation in a focus of onchocerciasis in Mali after 13 years of Simulium control. I. Parasitological aspects]. Ann Soc Belg Med Trop 1977; 57:569–75.

136. Toure YT. Bio-Écologie Des Anophèles (Diptera, Culcidae) Dans Une Zone Rurale de Savane Soudanienne Au Mali (Village de Banambani). Incidence Sur La Transmission Du Paludisme et de La Filariose de Bancroft. 1979;

137. Walker M, Stolk WA, Dixon MA, et al. Modelling the elimination of river blindness using long- term epidemiological and programmatic data from Mali and Senegal. Epidemics 2017; 18:4– 15.

138. Coulibaly YI, Dembele B, Diallo AA, et al. The Impact of Six Annual Rounds of Mass Drug Administration on Wuchereria bancrofti Infections in Humans and in Mosquitoes in Mali. Am. J. Trop. Med. Hyg. 2015; 93:356–360.

139. Wanji S, Amvongo-Adjia N, Koudou B, et al. Cross-Reactivity of Filariais ICT Cards in Areas of Contrasting Endemicity of Loa loa and Mansonella perstans in Cameroon: Implications for Shrinking of the Lymphatic Filariasis Map in the Central African Region. PLoS Negl. Trop. Dis. 2015; 9:e0004184.

68

140. Bloch P, Simonsen PE, Weiss N, Nutman TB. The significance of guinea worm infection in the immunological diagnosis of onchocerciasis and bancroftian filariasis. Trans R Soc Trop Med Hyg 1998; 92:518–21.

141. Golden A, Steel C, Yokobe L, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS One 2013; 8:e69231.

142. Ov-16 Meeting Notes | Neglected tropical diseases support center. Available at: http://www.ntdsupport.org/resources/ov-16-meeting-notes. Accessed 13 July 2018.

143. WHO | Report of the Second Meeting of the WHO Onchocerciasis Technical Advisory Subgroup. Available at: https://www.who.int/onchocerciasis/resources/WHO-CDS-NTD-PCT- 2018.11/en/. Accessed 27 August 2019.

69

70

Chapter 2 Integrated seroprevalence-based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test

Dolo H, Coulibaly YI, Dembele B, Guindo B, Coulibaly SY, Dicko I, Doumbia SS,

Dembele M, Traore MO, Goita S, Dolo M, Soumaoro L, Coulibaly ME, Diallo AA, Diarra

D, Zhang Y, Colebunders R, Nutman TB. Integrated seroprevalence-based assessment

of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis

evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test.

PLoS Negl Trop Dis. 2019 Jan 30;13(1):e0007064.

71

Abstract

Background: Mali has become increasingly interested in the evaluation of transmission of both

Wuchereria bancrofti and Onchocerca volvulus as prevalences of both infections move toward their respective elimination targets. The SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used in 2 evaluation units (EU) to assess its performance as an integrated surveillance tool for elimination of lymphatic filariasis (LF) and onchocerciasis.

Methods: A cross sectional survey with SD Bioline Onchocerciasis/LF IgG4 Rapid Test was piggy-backed onto a transmission assessment survey (TAS) (using the immunochromatographic card test (ICT) Binax Filariasis Now test for filarial adult circulating antigen (CFA) detection) for

LF in Mali among 6-7-year-old children in 2016 as part of the TAS in two EUs namely Kadiolo-

Kolondieba in the region of Sikasso and Bafoulabe -Kita-Oussoubidiagna-Yelimane in the region of Kayes.

Results: In the EU of Kadiolo- Kolondieba, of the 1,625 children tested, the overall prevalence of

W. bancrofti CFA was 0.62% (10/1,625) [CI= 0.31-1.09]; while that of IgG4 to Wb123 was 0.19%

(3/1,600) [CI= 0.04-0.50]. The number of positives tested with the two tests were statistically comparable (p=0.09). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of W. bancrofti CFA was 0% (0/1,700) and that of Wb123 IgG4 antibody was 0.06%

(1/1,700), with no statistically significant difference between the two rates (p=0.99).

In the EU of Kadiolo- Kolondieba, the prevalence of Ov16-specific IgG4 was 0.19% (3/1,600)

[CI= 0.04-0.50]. All 3 positives were in the previously O. volvulus-hyperendemic district of

Kolondieba. In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of

Ov16-specific IgG4 was 0.18% (3/1,700) [CI= 0.04-0.47]. These 3 Ov16 IgG4 positives were from previously O.volvulus mesoendemic district of Kita.

Conclusions: The SD Bioline Onchocerciasis/LF IgG4 Rapid test appears to be a good tool for integrated exposure measures of LF and onchocerciasis in co-endemic areas.

72

2.1. Introduction

Approaches to onchocerciasis control and lymphatic filariasis (LF) elimination have proceeded along parallel but independent courses in Mali. For LF, annual mass drug administration

(MDA) with albendazole and ivermectin began in 2004 and continued for 5 to 9 consecutive

MDA campaigns. Onchocerciasis was originally highly endemic in five regions of Mali

(Kayes, Koulikoro, Sikasso, Segou and Mopti [1]; the eastern most regions (Koulikoro rive droite, Sikasso, Ségou and Mopti) were included in the original onchocerciasis control programme (OCP) based on vector control with larvicides [2]. The western parts of the endemic regions (Kayes and Koulikoro rive gauche) were part of the western extension of OCP in which ivermectin administration was used in MDA compaigns and later community directed therapeutic intervention (CDTi) under the umbrella of African Programme for Onchocerciasis

Control (APOC) using community drug distributors (CDDs).

LF and onchocerciasis are overlapping in many districts of Mali [1]. Both the LF and onchocerciasis control programs were implemented based on precontrol mapping data for each of diseases [1]. For meso and hyperendemic onchocerciasis, the use of skin snips and eye examination for mapping demonstrated that 5 of the 8 administrative regions of Mali were endemic for O. volvulus [1,3]; whereas all of the 8 administrative units (encompassing 75 health districts) were endemic for LF based on a 2004 survey using the immunochromatographic test (ICT) known commercially as the Binax Filariasis Now test

(Alere, Portland, ME) where circulating filarial antigen (CFA) prevalences were greater than

1% [1,4].

In 2005, the results of longitudinal studies from 3 formerly O. volvulus hyperendemic foci in

Mali and Senegal provided evidence that onchocerciasis elimination could be achieved based only on mass drug administration of ivermectin [5]. The evaluations used skin snips for the detection of microfilaridermia and blackfly dissection [5,6]. At the same time, when LF was

73 mapped and reported to be endemic throughout the country, all the districts in Mali were treated using MDA (ivermectin with albendazole) for LF.

LF transmission assessment surveys (TAS) as recommended by World Health Organization

(WHO) were initiated in 2012 and are currently being performed in 22 evaluation units (EU).

An EU includes one or several endemic districts based on geographic location, treatment coverage and population size [7].

The Binax Filariasis Now ICT cards and more recently the Filariasis Test Strip (FTS) [8] have been used for LF mapping and for the TAS, but these tests may have their limitations because of their slow kinetics of disappearance and their potential cross-reaction in cases of Loa loa infection, a filarial parasite that is absent in Mali [9]. For onchocerciasis, post-treatment surveillance based on positivity in children with Ov16-based immunoassays is the current gold standard, but the challenge remains in the definition of prevalence cutoffs using the various forms of the Ov16 ELISA [10] or the SD Bioline’s Ov16-containing RDTs [11].

Initially, O. volvulus infection mapping in Mali was conducted using skin snip and eye examination that left many hypo-endemic areas excluded from the various control programs and from further consideration for CDTi. As a consequence of “redistricting” in 2016, the number of onchocerciasis-endemic districts increased from 17 to 34. Among these 34, only 2 have stopped CDTi. Hence, re-mapping is needed in many potentially O. volvulus -endemic areas. Mass drug administration of ivermectin is still ongoing in 20 districts, and 12 are under surveillance. These 12 districts (under surveillance for onchocerciasis) had previously been part of the OCP vector control program; however, they have received albendazole and ivermectin for LF for at least 5 MDA rounds. Beyond these 34 districts, there may be a need for re-mapping potentially O. volvulus-hypo-endemic regions if elimination goals are to be achieved in the near future [12].

In this study, the SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used concurrently with 74 the ICT in two LF evaluation units (EU) in Mali consisting of districts with different endemicity levels for LF and onchocerciasis prior to MDA/CDTi to assess its performance as an integrated surveillance tool for LF and onchocerciasis elimination.

2.2. Methods

Ethics statement

As this work was conducted as part of the National Neglected Tropical Diseases control program activity, it was deemed exempt from IRB approval by the ethical committee of the

University of Science, Techniques and Technologies of Bamako. However, a protocol related to the evalution of SD Bioline Onchocerciasis/LF IgG4 Rapid Test in Mali was approved by the ethical committee (2017/199/CE/FMPOS). Participation in this study was entirely voluntary. The study was clearly explained to the community leaders and health authorities and their permission obtained before any activities were undertaken. Oral consent was obtained from the legal guardians of all the 6- to 7-year old children due to low literacy level in communities.

Study area

In the Sikasso region, the study was conducted in the EU of the district of Kadiolo and

Kolondièba. In this EU, the district of Kadiolo had been endemic for W. bancrofti (24.4% pre- control) but hypoendemic for O. volvulus (20% pre-control), while the district of Kolondieba had been co-endemic for these two parasites ( 37% and 60% pre-control prevalences for LF and onchocerciasis respectively) [1,13] (Table 2-1).

75

Table 2-1 Lymphatic filariasis and onchocerciasis pre-control endemicity and current status of mass drug distribution per district

Evaluation Unit Districts Precontrol Number Last Pre-control Number

Endemicity prevalence Ivermectin/ year LF prevalence ivermectin

LF Albendazole MDA onchocerciasis CDTi LF Onchocerciasis MDA

Kadiolo Endemic Hypoendemic 24.4 8 2016 20 NA Kadiolo -Kolondieba Kolondieba Endemic Hyperendemic 37 9 2016 60 25

Bafoulabe Endemic Mesoendemic 9.6 7 2016 42 25 Bafoulabe -Kita- Kita Endemic Mesoendemic 9.6 6 2016 40 24 Oussoubidiagna- Oussoubidiagna Endemic Hyperendemic 9.6 6 2016 60 25 Yelimane Yelimane Endemic Hypoendemic 9.6 7 2016 33 NA

MDA= mass drug administration, LF= lymphatic Filariasis, Prev=prevalence %, CDTi= community directed treatment with ivermectin, NA= not applicable

76

In Kayes region, the entire EU of the district of Bafoulabe, Kita, Oussoubidiagna and Yelimane was known to be endemic for LF (with 9.6% pre-control prevalence in each district), but O. volvulus was found to be endemic in certain districts with pre-control prevalences as follows:

Kita (mesoendemic-40%), Bafoulabe (mesoendemic - 42%), Oussoubidiagna (hyperendemic-

60%), Yelimane (hypoendemic - 33%) (Table 2-1). In all these districts, 2016 was the last year of ivermectin and albendazole distribution for LF although mass drug administration of ivermectin continues for onchocerciasis [1,13].

Study design

The present study was piggy-backed onto TAS surveys (using the Binax Filariasis Now test for filarial adult circulating antigen detection) for LF across 2 EUs in Mali to demonstrate the utility of the SD Bioline Onchocerciasis/LF IgG4 Rapid Test for integrated assessment of W. bancrofti and O. volvulus transmission [14].

Sampling and participants

The sample size builder (SSB) was used to automate the calculations for determining appropriate survey strategy and sample size calculations based on TAS sampling strategy. The design of the surveys is flexible in order to best fit the local situation and depends upon factors such as the primary school enrolment rate, the populations size, the number of schools or enumeration areas, and the cost of different survey methods [7]. For this current study community-based survey was conducted due to low school enrollment rate.

The number of villages and the number of households included in the study were determined by the SSB [7]. Children of 6 and 7-year old within the randomly selected households made the sample. The minimum size required for the TAS were 1,556 and 1,692 children 6-7-year- old (according to the SSB) respectively for EUs of Kadiolo-Kolondieba and Bafoulabe -Kita-

Oussoubidiagna-Yelimane. The estimated total population of 6 to 7-year-old children were

77

37,620 and 71,152 respectively in the EU of Kadiolo- Kolondieba and Bafoulabe-Kita-

Oussoubidiagna-Yelimane (Table 2-2).

78

Table 2-2 Study area description per evaluation unit and details on number of villages and mean number of expected children

Evaluation Unit Districts per Number of Number of Number children 6-7 Mean Number of

evaluation unit villages per villages per year of age per expected children 6-7

district evaluation unit evaluation unit year of age per village

Kadiolo 11

Kadiolo -Kolondieba Kolondieba 19 322 37,620 117

Total 30

Bafoulabe 8

Bafoulabe -Kita- Kita 15

Oussoubidiagna- Oussoubidiagna 5 665 71,152 107

Yelimane Yelimane 3

Total 31

79

A multistage sampling technique was used for the sampling. In each EU, the villages and the backup villages were randomly selected using the SSB tool. The backup villages were chosen in addition to 30 villages that made the cluster for an EU. In the one case where the selected village was inaccessible or refused to participate in the survey, a backup village was used to replace it. At the village level, the list of the households was made available, and two tables of random numbers generated from computer using the SSB were also used to select randomly the households to be included in the study. In each selected household, all the children 6-7 years of age were included in the study. A household was defined as a group of persons living in the same house and sharing the same food.

Circulating filarial antigen and antibody measurement

Children aged 6-and 7-year-old were tested using Binax Filariasis Now™ test and retested ~ 6 months later with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test in the EU of Kadiolo –

Kolondieba at the point of care (POC) (Table 2-2). In the EU of Bafoulabe -Kita-

Oussoubidiagna-Yelimane, dried blood spots (DBS) were collected and cryopreserved at the time of the TAS as the SD Bioline Onchocerciasis/LF IgG4 Rapid Test were not available. All tests for CFA were performed at the POC using the Binax Filariasis Now™ test (Alere,

Portland, ME), using the manufacturer’s instructions. In the EU of Kadiolo –Kolondieba, the

SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used at the POC again exactly as recommended by the manufacturer (SD Diagnostics, Korea). For the dried blood spots used for antibody assessments in the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, the blood was collected on filter paper spots (TropBio, Townsville, Austrailia) and cryopreserved in liquid nitrogen dry shippers in the field before being transported to the Filariasis Research Unit laboratory in Bamako the capital city of Mali. The DBS were then stored at – 800C prior to elution as described in Kamgno et al, [15] and tested using the SD Bioline Onchocerciasis/LF

80

IgG4 Rapid Test. The SD Bioline Onchocerciasis/LF IgG4 Rapid Test measures simultaneously the presence of IgG4 antibodies to Wb123 and Ov16.

Statistical analysis

All data analyses were performed using SPSS Version 24 (Statistical package for Social

Sciences) and used the 5% level of significance. The Fisher's exact test was used when appropriate and the Clopper-Pearson 95% confidence interval around the prevalences were used for statistical comparisons. The geographic coordinates were measured for each village visited during the survey using mobile phones and thereafter used to map the ICT positive individuals as well as those positive for IgG4 antibodies against Wb123 and Ov16.

81

2.3. Results

In the EU of Kadiolo- Kolondieba, 1,625 children aged of 6-7-year-old were tested at the point of care (POC) using the Binax Filariasis Now™ ICT cards; whereas 1,600/1,625 (98.5%) were tested using the POC SD Bioline Onchocerciasis/LF IgG4 Rapid Test. In the EU of Bafoulabe-

Kita-Oussoubidiagna-Yelimane, 1,700 children aged of 6-7-year-old were tested at the POC using Binax Filariasis Now test and retested using elutions of DBS on the SD Bioline

Onchocerciasis/LF IgG4 Rapid Test (Table 2- 3).

82

Table 2-3 Study sites and population tested

Evaluation Unit Districts Number Number tested Endemicity level* Number tested

villages Binax Filariasis Onchocerciasis Ov16/WB123 RDT

/district Now™ test

Kadiolo 11 581 Hypoendemic 502

Kadiolo -Kolondieba Kolondieba 19 1,044 Hyperendemic 1,098

Total 30 1,625 NA 1,600

Bafoulabe 8 461 Mesoendemic 461

Bafoulabe -Kita- Kita 15 713 Mesoendemic 713

Oussoubidiagna- Oussoubidiagna 5 348 Hyperendemic 348

Yelimane Yelimane 3 178 Hypoendemic 178

Total 31 1,700 NA 1,700

*All districts previously LF endemic (>1% prevalence); Binax Filariasis Now test= Immunochromatographic cards test, NA= not applicable, LF= lymphatic filariasis; RDT=rapid diagnostic test

83

CFA and Wb123 IgG4 antibody prevalence

In the EU of Kadiolo- Kolondieba, an overall prevalence of W. bancrofti infection based on

CFA was found to be 0.62% [95% CI= 0.31-1.09]. When assessed at the district level, the CFA prevalence was 0.69 % [95% CI= 0.21-1.65] in Kadiolo and 0.57% [95% CI= 0.23-1.19] in

Kolondieba. In the same EU, the overall prevalence of Wb123 IgG4 was 0.19% [95% CI=

0.04-0.50]; when assessed per district, the prevalence of IgG4 to Wb123 was 0.20% [95% CI=

0.0-0.97] in Kadiolo and 0.18% [95% CI= 0.03-0.60] in Kolondieba (Table 2-4). The prevalences obtained using either of the two tests were statistically comparable (p=0.99). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of W. bancrofti infection based on CFA in chidren was 0% (0/1,700). For the Wb123 IgG4 antibody, only

1/1,700 (0.06% [95% CI= 0.0-0.28]) was found. This single positive was reported in the district of Bafoulabe. Thus, the local prevalence of Wb123 IgG4 was 0.21% [95% CI= 0.01-1.06]

(Table 2-4). There were no differences between the CFA positive cases and those positive for

Wb123 (p=0.99).

84

Table 2-4 Circulating filarial antigen and Wuchereria bancrofti seroprevalence per district and evaluation unit

Evaluation Unit Districts Number tested Number % Pos Binax Number tested Number % Positive

Binax Positive Filariasis test Biplex Positive Wb123

Filariasis test Binax [95% CI] Wb123 [95% CI]

Filariasis

Kadiolo 581 4 0.69 [0.21-1.65] 502 1 0.20 [0.00-0.97] Kadiolo - Kolondieba 1,044 6 0.57 [0.23-1.19] 1,098 2 0.18 [0.03-0.60] Kolondieba Total 1,625 10 0.62 [0.31-1.09] 1,600 3 0.19 [0.04-0.50]

Bafoulabe 461 0 0.00 [0.0-0.64] 461 1 0.21 [0.01-1.06]

Bafoulabe -Kita- Kita 713 0 0.00 [0.0-0.41] 713 0 0.00 [0.0-0.42]

Oussoubidiagna- Oussoubidiagna 348 0 0.00 [0.0-0.85] 348 0 0.00 [0.0-85]

Yelimane Yelimane 178 0 0.00 [0.0-1.66] 178 0 0.00 [0.0-1.66]

Total 1,700 0 0.00 [0.0-0.17] 1,700 1 0.06 [0.0-0.28]

%=percentage, Binax Filariasis test= immunochromatographique card test, CI= confidence interval, Pos= positive, Wb123: Wuchereria bancrofti specific antibodies 123

85

Ov16 IgG4 antibody prevalence

In the EU of Kadiolo –Kolondieba, 3/1,600 children were positive for Ov16-specific IgG4 with an overall antibody prevalence of 0.19% [95% CI= 0.04-0.50] being found. All 3 positives were in the district of Kolondieba, leading to a local prevalence of 0.27% [95% CI= 0.06-0.74] in this previously O. volvulus-hyperendemic district (Table 2-5). It should be noted that 0/502 children in Kadiolo (an O. volvulus-hypoendemic region) were IgG4 positive to Ov16, but the upper 95% confidence level around this 0% prevalence was 0.59%. There was no statistical difference between the number positive (0/502) in Kadiolo, an O. volvulus-hypoendemic district and Kolondieba a previously O. volvulus-hyperendemic district (3/1,095) (p=0.55). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, an overall prevalence of Ov16-specific

IgG4 of 0.18% [95% CI= 0.04-0.47] was recorded. These 3 individual Ov16 IgG4 positives were in the district of Kita among the 713 children tested, giving a prevalence of 0.42 % for this district [95% CI= 0.10-1.14] (Table 2-5).

86

Table 2-5 Onchocerca volvulus seroprevalence per district and evaluation unit according to pre-control endemicity

Evaluation Unit Districts O. volvulus Number tested Number Positive % Positive

endemicity Biplex Ov16 Ov16 [95% CI]

Kadiolo Hypoendemic 502 0 0.00 [0.0-0.59]

Kadiolo -Kolondieba Kolondieba Hyperendemic 1,098 3 0.27 [0.06-0.74]

Total NA 1,600 3 0.19 [0.04-0.50]

Bafoulabe Mesoendemic 461 0 0 [0.0-0.64]

Kita Mesoendemic 713 3 0.42 [0.10-1.14] Bafoulabe -Kita- Oussoubidiagna Hyperendemic 348 0 0.00 [0.0-0.85] Oussoubidiagna-Yelimane Yelimane Hypoendemic 178 0 0.00 [0.0-1.66]

Total Not applicable 1,700 3 0.18 [0.04-0.47]

Ov16= Onchocerca volvulus specific antibodies 16, CI= confidence interval, %=percentage,

87

Again, it should be noted that in the O. volvulus-hypoendemic district (Yelimane), although there were no Ov16 positive children, the upper limit of the 95% CI exceeded 1%, suggesting that cut off point of antibody positivity for children should be raised above the 0.1% threshold.

The comparison of positive cases of Ov16 between the previous O. volvulus-hypoendemic district (Yelimane) and the previous O. volvulus-mesoendemic district with 3 positives cases was similar (p=0.99).

LF and onchocerciasis geographic distribution

CFA positive children were found primarily in the EU of Kadiolo-Kolondieba with 10 CFA positives detected using Binax Filariasis Now™ test in five different villages (Figure 4- 1). No

CFA carriers were found in the 31 villages of the Bafoulabe-Kita-Oussoubidiagna-Yelimane

EU, but one child was found positive for Wb123 IgG4 in the district of Bafoulabe (Figure 2-

1). In the EU of Kadiolo Kolondieba, 3 positive Wb123 IgG4 children were observed in three different villages. In the same EU, 3 Ov16 IgG4 positive children were found in 3 different villages (Figure 2- 1). In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, 3 Ov16 IgG4 positive cases were detected in a single village of Kita district previously mesoendemic at the pre-control (Figure 2- 1). Overall, single Ov16 or Wb123 IgG4-positive children were spread across 5 different villages of the 30 villages screened in Kadiolo –Kolondieba EU (Figure 2-

1).

88

Source: map created by Dansine Diarra (geographer) specifically for this article. Figure 2-1 Distribution of circulating filarial antigen (orange dots) IgG4 antibodies to

Wb123 (red dots) and to Ov16 (blue dots) in the evaluation units of Kadiolo –

Kolondieba and Bafoulabe -Kita-Oussoubidiagna-Yelimane

89

2.4. Discussion

Mali has become increasingly interested in the evaluation of transmission of both O. volvulus and W. bancrofti as prevalences of both infections move toward their respective elimination targets. This reduction is due to the fact that the national LF elimination program, since its creation, has distributed the required number of mass treatment campaigns with at least 5 consecutive rounds of albendazole and ivermectin treatment; thus TAS is ongoing in the different EUs [1]. With regard to onchocerciasis, after periods of vector control, and ivermectin

MDA lasting more than 24 years in previously hyper and mesoendemic foci, the program needs to fulfill the criteria for the cessation of ivermectin distribution [1]. Additionally, elimination goals likely were reached in most endemic foci by 2006 [6]. In 2016, as part of the TAS for LF using the Binax Filariasis Now test by the national LF elimination program in two EUs, namely

Kadiolo-Kolondieba in the region of Sikasso and Bafoulabe-Kita-Oussoubidiagna-Yelimane in the region of Kayes, the SD Bioline Onchocerciasis/LF IgG4 Rapid Test was used to assess its performance as an integrated surveillance tool [14] to inform each of the two national elimination programs involved in LF and onchocerciasis elimination respectively.

For the assessment of W. bancrofti transmission, in the EU of Kadiolo- Kolondieba, W. bancrofti circulating filarial antigen (CFA) prevalence was 0.62% [95% CI= 0.31-1.09] while the seroprevalence (antibodies) targeting the same parasite was 0.19 % [95% CI= 0.04-0.50].

In the EU of Bafoulabe-Kita-Oussoubidiagna-Yelimane, the CFA prevalence was 0% [95%

CI= 0.0-0.17] while the antibody seroprevalence was 0.06% [95% CI= 0.0-0.28]. The prevalences of LF infection measured using these two tests were comparable and with all the tests, the upper bounds did not reach the cutoff point of 2% (Table4- 4).

Recent studies suggested that the Binax Filariasis Now test may overestimate the W. bancrofti infection prevalence [16,17]. Given that the specificity of the tests for CFA may be lower than previously thought in Loa-endemic areas [18], given that antibodies to Wb123 appear earlier 90 than CFA in longitudinally followed children [19] and given the excellent superimposition of

W. bancrofti prevalence results using either antibody (Wb123) or antigen (CFA) testing, we suggest that the measurement of IgG4 to Wb123 might be preferable in decisions to stop MDA or in TAS following cessation of MDA as it allows the measurement of recent exposure. The challenge remains, however, to determine an acceptable prevalence threshold that would guarantee interruption of W. bancrofti transmission particularly since vector monitoring is not part of most LF elimination programs. In the Gambia, the recent use of the Wb123 IgG4 test suggests that this serological tool could be used to decide to stop MDA [20]. Therefore, maybe the SD Bioline Onchocerciasis/LF IgG4 Rapid Test could be utilized for transmission assessment and as a decision tool for MDA cessation, should modelling studies help to corroborate these empiric findings.

Despite the application of the LF sampling strategy (not the recommended target age group less than 10-year-old and sample size of 3,000 children for onchocerciasis transmission), the level of exposure to O. volvulus, as determined by the detection of the IgG4 antibodies to Ov16, the current prevalence estimates overlaid quite nicely with previous epidemiological profiles of the different study districts (Table 4- 1, 4-3 and 4-5). All Ov16 antibody positive subjects were reported in the previously known onchocerciasis meso and/or hyperendemic districts [6].

Our results show that O. volvulus transmission level was above the current elimination threshold (0.1%) among the 6-7 year old, though the utility of this 0.1% target as a threshold is currently under debate [11,21]. The results of this study suggest that many of the hypoendemic areas are not in need of ivermectin distribution [22]. However, the sample size used in our study was calculated for LF transmission assessment and not for the evaluation of

O. volvulus transmission (typically 3,000 children per transmission focus) [21].

Within the context of onchocerciasis elimination, new data are needed to re-categorize O. volvulus transmission potentials in subregions of countries such as Mali where many areas have 91 undergone more than 24 years of CDTi with no or few epidemiological assessments [1].

Moreover, entomologic (blackfly) data are needed to confirm our findings that onchocerciasis transmission has been interrupted in these previously endemic district before to decide about

CDTi cessation as suggested in a previous study [11]. The serological profiles reported here are similar to what was observed in southern Mexico where a decision to interrupt CDTi was taken [23].

Overall, the SD Bioline Onchocerciasis/LF IgG4 Rapid Test Wb123/Ov16 test, has 84% sensitivity and 98-99% specificity [24]. As no antibody-based test is likely to have specificities of >99% (meaning 1 % false positive rates) the current threshold of elimination of onchocerciasis based on Ov16-specific antibodies (by whatever test available) must be increased and also results must be underscored by transmission assessment in pooled blackfly populations [25].

2.5. Conclusion

Based on the present study using the SD Bioline Onchocerciasis/LF IgG4 Rapid Test in previously LF- and onchocerciasis co-endemic regions in Mali, this POC test appears to be a good tool for integrated measurement of exposure to W. bancrofti and O. volvulus in children, with the potential to be used as a surrogate for interruption of transmission in a given focus as well as in post-MDA impact measures in local populations in countries such as Mali. These data suggest that there is likely little or no transmission of either O. volvulus or W. bancrofti.

Moreover, given the upper limits of the CI for Ov16 IgG4 in an O. volvulus-hypo-endemic region (and the fact that it does not differ from the hyper and mesoendemic regions) a 0.1% threshold based on Ov-16-based IgG4 immunoassays may certainly be too conservative.

Obviously, additional data underlying the utility of this POC test will be necessary if the elimination goals for both onchocerciasis and LF are to be achieved.

92

2.6.References

1. Dembélé M, Bamani S, Dembélé R, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl. Trop. Dis. 2012; 6:e1574.

2. Boatin B. The onchocerciasis control programme in west africa (OCP). Ann Trop Med Parasitol 2008; 102 Suppl 1:13–17.

3. Noma M, Nwoke BEB, Nutall I, et al. Rapid epidemiological mapping of onchocerciasis (REMO): its application by the African Programme for Onchocerciasis Control (APOC). Ann Trop Med Parasitol 2002; 96 Suppl 1:S29-39.

4. Coulibaly YI, Dao S, Traore AK, Diallo A, Sacko M, Traoré SF. [Presence and risk of transmission of Wuchereria bancrofti is a reality in rural Mali: the case of the town of Bariambani in the Cirle of Kati]. Mali Med. 2006; 21:12–17.

5. Diawara L, Traoré MO, Badji A, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl. Trop. Dis. 2009; 3:e497.

6. Traore MO, Sarr MD, Badji A, et al. Proof-of-principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl. Trop. Dis. 2012; 6:e1825.

7. World Health Organization. Lymphatic filariasis monitoring and epidemiological assessment of mass drug administration global programme to eliminate lymphatic filariasis a manual for national elimination programmes. 2011;

8. Weil GJ, Curtis KC, Fakoli L, et al. Laboratory and field evaluation of a new rapid test for detecting Wuchereria bancrofti antigen in human blood. Am. J. Trop. Med. Hyg. 2013; 89:11–15.

9. Gass KM. Rethinking the serological threshold for onchocerciasis elimination. PLoS Negl. Trop. Dis. 2018; 12:e0006249.

10. Guidelines for stopping mass drug administration and verifying elimination of human onchocerciasis: criteria and procedures. Geneva: World Health Organization, 2016.

11. Lont YL, Coffeng LE, de Vlas SJ, et al. Modelling Anti-Ov16 IgG4 Antibody Prevalence as an Indicator for Evaluation and Decision Making in Onchocerciasis Elimination Programmes. PLoS Negl. Trop. Dis. 2017; 11:e0005314.

12. Rebollo MP, Zoure H, Ogoussan K, Sodahlon Y, Ottesen EA, Cantey PT. Onchocerciasis: shifting the target from control to elimination requires a new first-step-elimination mapping. Int. Health 2018; 10:i14–i19.

13. Walker M, Stolk WA, Dixon MA, et al. Modelling the elimination of river blindness using long-term epidemiological and programmatic data from Mali and Senegal. Epidemics 2017; 18:4–15.

93

14. Steel C, Golden A, Stevens E, et al. Rapid Point-of-Contact Tool for Mapping and Integrated Surveillance of Wuchereria bancrofti and Onchocerca volvulus Infection. Clin. Vaccine Immunol. 2015; 22:896–901.

15. Kamgno J, Pion SD, Chesnais CB, et al. A Test-and-Not-Treat Strategy for Onchocerciasis in Loa loa-Endemic Areas. N. Engl. J. Med. 2017; 377:2044–2052.

16. Dorkenoo AM, Sodahlon YK, Bronzan RN, et al. [Lymphatic filariasis transmission assessment survey in schools three years after stopping mass drug treatment with albendazole and ivermectin in the 7 endemic districts in Togo]. Bull Soc Pathol Exot 2015; 108:181–187.

17. Coulibaly YI, Coulibaly SY, Dolo H, et al. Dynamics of antigenemia and transmission intensity of Wuchereria bancrofti following cessation of mass drug administration in a formerly highly endemic region of Mali. Parasit. Vectors 2016; 9:628.

18. Pion SD, Montavon C, Chesnais CB, et al. Positivity of Antigen Tests Used for Diagnosis of Lymphatic Filariasis in Individuals Without Wuchereria bancrofti Infection But with High Loa loa Microfilaremia. Am. J. Trop. Med. Hyg. 2016; 95:1417–1423.

19. Hamlin KL, Moss DM, Priest JW, et al. Longitudinal monitoring of the development of antifilarial antibodies and acquisition of Wuchereria bancrofti in a highly endemic area of Haiti. PLoS Negl. Trop. Dis. 2012; 6:e1941.

20. Won KY, Sambou S, Barry A, et al. Use of antibody tools to provide serologic evidence of elimination of lymphatic filariasis in the gambia. Am. J. Trop. Med. Hyg. 2018; 98:15–20.

21. Ov-16 Meeting Notes | Neglected tropical diseases support center. Available at: http://www.ntdsupport.org/resources/ov-16-meeting-notes. Accessed 13 July 2018.

22. Prost A, Hervouet JP, Thylefors B. [Epidemiologic status of onchocerciasis]. Bull. World Health Organ. 1979; 57:655–662.

23. Rodríguez-Pérez MA, Unnasch TR, Domínguez-Vázquez A, et al. Interruption of transmission of Onchocerca volvulus in the Oaxaca focus, Mexico. Am. J. Trop. Med. Hyg. 2010; 83:21–27.

24. Lipner EM, Dembele N, Souleymane S, et al. Field applicability of a rapid-format anti-Ov- 16 antibody test for the assessment of onchocerciasis control measures in regions of endemicity. J. Infect. Dis. 2006; 194:216–221.

25. Golden A, Faulx D, Kalnoky M, et al. Analysis of age-dependent trends in Ov16 IgG4 seroprevalence to onchocerciasis. Parasit. Vectors 2016; 9:338.

94

Chapter 3 Serological Evaluation of Onchocerciasis and Lymphatic

Filariasis Elimination in the Bakoye and Falémé foci, Mali

Housseini Dolo, Yaya I Coulibaly, Moussa Sow, Massitan Dembélé, Salif S Doumbia, Siaka

Y Coulibaly, Moussa B Sangare, Ilo Dicko, Abdallah A Diallo, Lamine Soumaoro, Michel E

Coulibaly, Dansine Diarra, Robert Colebunders, Thomas B Nutman, Martin Walker, Maria-

Gloria Basáñez. Serological Evaluation of Onchocerciasis and Lymphatic Filariasis

Elimination in the Bakoye and Falémé foci, Mali. Clinical Infectious Diseases. 2020 Mar 24.

95

Abstract

Background. In Mali, ivermectin-based onchocerciasis elimination from the Bakoye and

Falémé foci, reported in 2009–2012, was a beacon leading to policy shifting from morbidity control to elimination of transmission (EOT) goals. These foci are also endemic for lymphatic filariasis (LF) and in 2007–2016 mass administration of ivermectin plus albendazole was implemented. We report Ov16 (onchocerciasis) and Wb123 (LF) seroprevalence in these foci after 24–25 years of treatment to evaluate if onchocerciasis EOT and LF elimination as a public health problem (EPHP) have been achieved.

Methods. The SD Bioline Onchocerciasis/LF IgG4 Rapid Diagnostic Test was used to evaluate

2,186 children aged 3–10 years in 13 villages (plus two hamlets) in Bakoye, and 2,270 children in 15 villages (plus one hamlet) in Falémé. In Bakoye, all-age serosurveys were conducted in three historically hyperendemic villages, testing 1,867 individuals aged 3–78 years.

Results. In Bakoye, IgG4 seropositivity was 0.27% (95% CI = 0.13–0.60%) for both Ov16 and

Wb123 antigens. In Falémé, Ov16 and Wb123 seroprevalence was, respectively, 0.04%

(95%CI = 0.01–0.25%) and 0.09% (95%CI = 0.02–0.32%). Ov16-seropositive children were from historically meso- and hyperendemic villages. Ov16 positivity was less than 2% in age groups ≤14 years, increasing to 16% in those ≥40 years. Wb123 seropositivity was lower than

2%, reaching 3% in those aged ≥40 years.

Conclusions. Ov16 and Wb123 seroprevalence among children in the Bakoye and Falémé foci in Mali is consistent with EOT (onchocerciasis) and EPHP (LF) since stopping treatment in

2016. The few Ov16 -seropositive children should be skin -snip PCR tested and followed up

96

3.1.Background

Onchocerciasis and lymphatic filariasis (LF) are endemic in Mali [1] but large-scale interventions have progressed towards elimination of transmission (EOT) for onchocerciasis and elimination as a public health problem (EPHP) for LF. The Onchocerciasis Control

Programme in West Africa (OCP) began vector control in Mali in 1977, identifying and larviciding Simulium (blackfly) breeding sites [2]. Some endemic parts of Mali were included in the OCP western extension, with ivermectin mass drug administration (MDA) starting in

1987. MDA was initially delivered by mobile teams and later by community-directed treatment with ivermectin (CDTI) assisted by the African Programme for Onchocerciasis Control

(APOC) [3]. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) started in

Mali in 2004, supporting ivermectin and albendazole distribution [1].

In 2010, APOC launched a conceptual and operational framework for onchocerciasis elimination with ivermectin treatment [4], spurred by promising findings in foci of Mali and

Senegal using this strategy. In 2012, following the World Health Organization (WHO) roadmap on neglected tropical diseases ( NTDs) [5], the target for onchocerciasis changed from morbidity control to EOT, contrasting with the LF goal of EPHP [6].

Mali became one of the first African countries to demonstrate the principle of onchocerciasis elimination by ivermectin MDA as the sole intervention when elimination was documented in the Bakoye and Falémé foci, firstly in 2009 and in 2012 [7,8], following 15 (Bakoye) and 16

(Falémé) years of annual treatment. Treatment duration corresponds to first–last year when all first-line villages were treated, 1992–2006 for Bakoye and 1991–2006 for Falémé [7]. Because the LF programme started in 2007 in the same river basins, ivermectin distribution continued de facto for 9 years after 2006 (LF MDA stopped in 2016), bringing treatment duration to 24–

25 years. Since 2011, a bednet distribution programme for malaria (which would also impact

Anopheles-transmitted LF [9], the type occurring in Mali) was implemented.

97

For onchocerciasis, a serological threshold of <0.1% seropositivity to the Onchocerca volvulus

Ov16 antigen in children aged <10 years is currently recommended by WHO for stopping ivermectin MDA [10]. For Anopheles -transmitted LF, the WHO guidelines recommend (for

Anopheles-transmitted LF) a threshold of <2% seropositivity to Wuchereria bancrofti circulating filarial antigen (CFA) with the filariasis test strip (FTS) (which replaced the immunochromatographic card test, ICT) in 6–7-year olds before ivermectin plus albendazole

MDA may be stopped [11].

LF transmission assessment surveys (TAS) were conducted in 2016 and treatment stopped because all the health districts of the two foci passed the TAS using FTS, i.e. W. bancrofti antigenemia prevalence after 9 years of treatment was <2% at health district level [12].

Documenting onchocerciasis elimination in Bakoye and Falémé was based on skin snip microscopy for detection and quantification of O. volvulus microfilaridermia, and PCR- based pool screening of blackfly samples for detection of infective, L3 larvae [7,8]. These data, alongside historical infection trends and treatment coverage information were later modelled using EPIONCHO and ONCHOSIM to estimate the risk of resurgence in Bakoye and the neighbouring River Gambia focus in Senegal [13] (data from Falémé were not available). Both models captured adequately the temporal prevalence trends and suggested a very low risk of resurgence in Bakoye, although EPIONCHO indicated a more substantive risk, particularly in historically hyperendemic communities [13]. Since the publication in 2016 of the World Health

Organization (WHO) guidelines for stopping MDA and verifying onchocerciasis elimination

[10], serological evaluation has become an important tool for transmission assessment.

Here, we use a rapid diagnostic test [14,15] to assess onchocerciasis and LF seroprevalence in the Bakoye and Falémé foci, 10 years after the first evidence of onchocerciasis elimination in

98 these foci [7] and 3 years after passing TAS for LF in an onchocerciasis–LF co-endemic area in Mali.

3.2. Material and methods

Ethical Approval

The study protocol (no. 2017/199/CE/FMPOS) was approved by the Ethical Committee of the

University of Sciences, Techniques and Technologies of Bamako (USTTB), Faculty of

Medicine, Pharmacy and Odontostomatology (FMPOS). The objectives, procedure and methodology were explained to village elders and residents, and informed consent was obtained from parents/guardians of children and participants aged ≥18 years.

Study Sites and Baseline Infection Indicators

The study was conducted between November 2017 and January 2018 in the Bakoye and Falémé former onchocerciasis foci [7,8]. In 2017, the Bakoye focus had a total of 92,707 inhabitants; 13 villages (plus two hamlets) were studied, with baseline O. volvulus microfilarial

(mf) prevalence ranging from 31% to 70%. In the Falémé focus, with a total of 22,551 inhabitants in 2017, 15 villages (plus one hamlet) were studied, with baseline mf prevalence ranging from 20% to 57%. Figure 3-1 shows the locations of the foci and study villages in

Mali. Tables 5-1 and 5-2 summarize the pre-control parasitological data (mf prevalence standardized using the OCP reference population [16], and community microfilarial load

(CMFL), the geometric mean number of microfilariae (mff) per skin snip (ss) in those aged

≥20 years [16], for the study villages in Bakoye and Falémé, respectively. In Bakoye, 3 (23%) communities had been hypoendemic (mf prevalence <40%); 5 (38.5%) mesoendemic (mf prevalence ≥40% but <60%), and 5 (38.5%) hyperendemic (≥60%). In Falémé, 6 (43%) had been hypoendemic and 8 (57%) mesoendemic. (Endemicity levels are as used in [12]).

99

Censuses of the village populations for 2017 were provided by the Kita (Bakoye focus) and

Kéniéba (Falémé focus) health districts.

100

Figure 3-1 Map of the study area and the location of the study villages in the Kayes region of Mali

(A) Map of Mali showing the location of the Bakoye and Falémé foci (inset indicates location of Mali within Africa). (B) The Bakoye focus is in the Kita cercle, with most the 15 study communities (13 villages and two hamlets) located along the Bakoye river. (C) The Falémé focus is in the Kéniéba cercle, with the 16 study communities (15 villages and one hamlet) located along the Falémé river (bordering Senegal). Cercles are the second administrative unit in Mali (the first being regions). Villages (indicated by solid circles) are colored by their baseline endemicity level: yellow (hypoendemic), orange (mesoendemic) and red (hyperendemic); green circles denote locations for which no baseline data (collected in 1985–1986) were available and therefore their initial endemicity status is unknown.

101

Table 3-1 Prevalence and Intensity of Onchocerca volvulus Microfilariae in the Pre-Ivermectin MDA Period (1985–1988) for 13 Villages in Bakoye, Mali

Village Year of Census Positive/ Prevalence (%)a (95% CMFL Endemicity b survey (% xam) examined CI) (mff/ss)c leveld Fataba-Kouroubala 1988 284 (75.4) 57/ 214 30.6 (24.5–36.7) 3.22 Hypoendemic Kokounkoutouba 1987 173 (70.5) 28/ 122 30.7 (22.6–38.8) 2.29 Hypoendemic Baniangafata 1988 208 (75.5) 57/ 157 39.7 (32.1–47.3) 4.36 Hypoendemic Dianga-Foula 1985 214 (47.2) 48/ 101 43.5 (34.1–53.3) 16.91 Mesoendemic Fatafing 1987 335 (78.5) 106/ 263 45.5 (39.7–51.7) 6.17 Mesoendemic Badougou 1988 160 (68.1) 45/ 109 48.4 (39.4–58.0) 4.37 Mesoendemic

Tieourou-Santankoto 1988 340 (86.2) 142/ 293 48.6 (42.8–54.2) 10.20 Mesoendemic

Madila 1987 204 (83.3) 82/ 170 50.1 (42.5–57.5) 8.17 Mesoendemic Kantila 1985 171 (83.6) 84/ 143 60.1 (52.0–67.9) 17.25 Hyperendemic Keniefeto 1988 127 (78.0) 51/ 99 62.4 (52.9–71.7) 14.92 Hyperendemic Nioumala 1985 98 (72.5) 52/ 71 64.9 (53.3–75.2) 33.94 Hyperendemic Kibi 1988 205 (72.2) 90/ 148 67.5 (59.8–74.8) 21.62 Hyperendemic Galé 1985 410 (62.9) 173/ 258 70.0 (64.4–75.5) 31.13 Hyperendemic

aStandardised prevalence according to OCP reference population [16] ; bWilson 95% confidence intervals [17]; cCMFL: geometric mean no. of microfilariae per skin snip (mff/ss) in those aged ≥20 years according to Remme et al. [16]; dendemicity levels as in [13].

102

Table 3-2 Prevalence and Intensity of Onchocerca volvulus Microfilariae in the Pre-Ivermectin MDA Period (1986–1990) for 14 Villages in Falémé, Mali

Village Year of Census Positive/ Prevalence (%)a CMFL Endemicity survey (% exam) examined (95% CI) (mff/ss)c leveld Madina-Mandinga 1990 414 (81.9) 53/ 339 19.9 (15.9–24.3) 2.30 Hypoendemic Koffing 1987 244 (84.4) 46/ 206 23.3 (18.1–29.5) 1.55 Hypoendemic Yatia-Berola 1986 217 (81.6) 45/ 177 24.3 (18.6–31.1) 1.29 Hypoendemic Sakola-Loulo 1986 343 (70.3) 52/ 241 26.6 (21.4–32.5) 1.68 Hypoendemic Djoulafoundouni 1986 220 (74.6) 43/ 164 30.3 (24.0–37.9) 2.88 Hypoendemic Koutila 1986 227 (77.1) 55/ 175 33.0 (26.6–40.4) 5.37 Hypoendemic Djidian-Kenieba 1986 133 (89.5) 48/ 119 40.1(32.0–49.3) 6.23 Mesoendemic Moussala 1989 214 (80.4) 65/ 172 42.2 (35.3–49.9) 5.18 Mesoendemic Sanoukou 1986 164 (82.9) 55/ 136 42.2 (34.0–50.3) 5.18 Mesoendemic Satadougou-Tintiba 1986 170 (82.4) 55/ 140 43.3 (35.6–51.9) 4.36 Mesoendemic Sely 1986 231 (71.4) 61/ 165 43.5 (36.3–51.3) 6.20 Mesoendemic Fadougou 1986 200 (76.0) 65/ 152 47.5(39.6–55.3) 6.41 Mesoendemic Kounda-Mahina 1986 271 (80.1) 72/ 217 47.7 (41.4–54.6) 7.02 Mesoendemic Mankouke 1986 290 (74.8) 125/ 217 56.8 (50.0–63.1) 20.93 Mesoendemic

aStandardised prevalence according to OCP reference population [16]; bWilson 95% confidence intervals [17]; cCMFL: geometric mean no. of microfilariae per skin snip (mff/ss) in those aged ≥20 years according to Remme et al. [16]; dendemicity levels as in [13].

103

Temporal Trends of Onchocerciasis Under Mass Drug Administration

Temporal records of census population, numbers examined and positive, crude and standardized mf prevalence, and CMFL, were extracted from the OCP database, data from the

Bakoye focus analysed in Walker et al. ,2017 [13], and national onchocerciasis control programme reports. Although [7,8] presented baseline and post-MDA (14–15 years) evaluation

(pie chart) snapshots of the data for Bakoye and Falémé, and temporal trends of mf prevalence were presented for Bakoye in [13], the latter did not include data for Falémé or CMFL. For

Bakoye, reported treatment coverage for 1992–2006 ranged from 75 to 83% [13]; for Falémé, coverage for 1991–2006 was 60–82%. For 2007–2016, coverage ranged from 79 to 84% in

Bakoye (dropping to 24% in 2016), and from 75 to 83% in Falémé. LF was endemic in the health district of Kita (Bakoye focus) and Kéniéba (Falémé focus) with a baseline antigenemia prevalence (in those aged ≥15 years) of 8.61% (13/151; 95% CI = 5.1–14.2%) using ICT to detect CFA in 2004.

Study Populations

Serum samples were collected from children aged 3‒10 years born/resident in 13 villages (plus two hamlets) in Bakoye and 15 villages (plus one hamlet) in Falémé (December 2017). In addition, during the second phase (January 2018), and because modelling [13] had suggested a potential risk of resurgence in previously hyperendemic villages in Bakoye (see above), serosurveys were also performed in the remaining population (covering those aged 3–78 years) in Kantila, Nioumala and Galé, which had had mf prevalences above 60% (Table 3-1). The objective was to analyse all-age serological profiles in these villages and ascertain if EOT had truly been achieved. This was deemed not necessary in villages of lower baseline endemicity in both the Bakoye and Falémé foci.

104

Serology Test

The SD Bioline Onchocerciasis/LF IgG4 Rapid Diagnostic Test (RDT) was used at the point- of-care as recommended by the manufacturer (SD Diagnostics, Korea). The test result was read after 30 minutes. During the serology testing participants and or their parents/guardians were asked if they were long-term residents (born in the village) or migrants in the communities.

Sample Sizes

Sample sizes were calculated based on updated census data obtained in 2016–2017, provided by the Kita and Kéniéba health districts. It was estimated that the 3‒10-year olds comprised approximately 22% of the population based on empirical data from onchocerciasis-endemic communities in Africa used to inform EPIONCHO’s demographic structure [19], in agreement with the OCP reference population [16]. The proportional representation of different age groups in the three historically hyperendemic communities in the Bakoye focus was calculated in an analogous manner.

The study was powered on the basis of our a priori expectation that onchocerciasis EOT had taken place in Bakoye and Falémé since the last rounds of CDTI for onchocerciasis were distributed in 2006‒2007 [7,8] and for a further 9 years for LF. Therefore, the ‘true’ underlying seroprevalence in 3‒10-year olds would be 0%. We further assumed that the specificity of the

RDT was approximately 97.5% [14,15] and, therefore, we calculated sample sizes required to measure an RDT seroprevalence of 2.5% with a +/-0.5% precision. This yielded, for the first phase of the study, sample sizes of 3,508 in Bakoye and of 2,739 in Falémé. According to [10], a sample size of 2,000 children is needed to detect an Ov16 seroprevalence <0.1% (upper 95% confidence limit).

105

For the second phase (all-age serosurvey) of the study, sample sizes for all other age groups

(≥11 years) in the three selected historically hyperendemic communities in Bakoye were based on estimating 50% seroprevalence (by RDT) with a precision of +/-5%. The value of 50% provided the most conservative sample size estimate of 1,257. In all cases, sample size calculations included a finite population size correction (i.e. sampling without replacement).

Data Analysis

The standardized mf prevalence at baseline reported in Tables 5-1 and 5-2 are accompanied by

95% confidence intervals (95% CIs) calculated according to Wilson score interval [17]. Point seroprevalence estimates and associated 95% CIs were calculated using the same method by focus, village, and age group.

106

3.3. Results

Study Population Characteristics

In the Bakoye focus, 2,186 children (aged 3–10 years were tested for Ov16 and Wb123 IgG4 positivity (62% of the target sample size). The median age was 7 years and 53.6% were boys.

For the three villages in which all-age serological surveys were conducted, 825 individuals aged ≤10 years and 1,042 individuals (aged ≥11 years) were tested (93% of the target); of the latter, the median age was 19 years, and 47.6% were men. In the Falémé focus, 2,270 children aged 3–10 years were included in the study (83% of the target). The median age was 6 years, and 51.9% were boys. Table 3-3 describes the population tested.

107

Table 3-3 Description of the Study Population Tested with Onchocerciasis/LF IgG4

Rapid Diagnostic Test in Bakoye and Falémé, Mali, 2017–2018

Bakoye Falémé

N (% target) % or [range] N (% target) % or [range]

Age group

Children aged ≤10 years 2,186 (62.3%) 2,270 (82.9%)

3–6 yr 1,003 45.9 1,415 62.3

7–10 yr 1,183 54.1 855 37.7

Median age (years) 7 [3–10] 6 [3–10]

Male/ Female 1,172/ 1,014 53.6/ 46.4 1,178/ 1,092 51.9/ 48.1

Children aged ≤10 years 825 (110.4%) – – (Kantila, Nioumala, Galé)

Persons aged >10 years 1,042 (82.9%) – – (Kantila, Nioumala, Galé)

11–14 yr 309 (149.3%) 29.7 – –

15–19 yr 232 (124.7%) 22.3 – –

20–24 yr 114 (69.1%) 10.9 – –

25–29 yr 70 (47.6%) 6.7 – –

30–39 yr 134 (72.0%) 12.9 – –

40–49 yr 71 (48.6%) 6.8 – –

≥50 yr 112 (50.9%) 10.8 – –

Median age (years) 19 [11–78] – –

Male/ Female 496/ 546 47.6/ 52.4 – –

Onchocerciasis Mf Prevalence and CMFL Trends in Bakoye and Falémé

108

Figures 3-2 and 3-3 depict the decrease in mf prevalence and CMFL for the two foci, respectively, during 15–16 years of annual ivermectin MDA (MDA started in 1989 in both foci, but it was only in 1991 for Falémé and 1992 for Bakoye that all first-line villages were treated [13]). In Bakoye, levels of initial endemicity were higher than in Falémé. By 2010 (the last skin snip-based parasitological evaluation following the last ivermectin MDA round for onchocerciasis in 2006), most reported values are zero in both foci, with 95% CIs reflecting uncertainty due to sampling.

109

Figure 3-2 Trends in Onchocerca volvulus microfilarial infection in Bakoye from 1985 to 2010

(A) Microfilarial (Mf) prevalence. (B) Community Microfilarial Load (CMFL), as defined in Table 1 [16]. Error bars for prevalence denote (Wilson score interval) 95% CIs [17]. The baseline data correspond to 1985–1989. In 1989 annual ivermectin MDA started with an initial coverage of 59–62% which improved to 73–

83% in 1998–2006 [13].

110

Figure 3-3 Trends in Onchocerca volvulus microfilarial infection in Falémé from 1986 to 2010

A) Microfilarial (Mf) prevalence. (B) Community Microfilarial Load (CMFL), as defined in Table 1 [16].

Error bars for prevalence denote (Wilson score interval) 95% CIs [17]. The baseline data correspond to 1986–1990. In 1990 annual ivermectin MDA started with an initial coverage of 63%

which improved to 75–82% in 2000–2006.

111

Ov16 and Wb123 Seroprevalence Among Children in Bakoye and Falémé

In Bakoye, 6/2,186 children aged 3–10 years were positive for Ov16 IgG4 (0.27%, 95% CI =

0.13–0.60%), and 6 children were also positive for Wb123 IgG4 antibodies (same prevalence and 95% CIs but not necessarily the same children). The children who were Ov16-positive were from the initially mesoendemic village of Badougou (a girl aged 10 years), the hyperendemic village of Kibi (a boy, aged 10, who was also positive for Wb123), and the highly hyperendemic village of Galé, where four children were seropositive (one girl aged 4 and another aged 8, positive for Ov16 only, plus one boy aged 7 and a girl aged 10, positive for both Ov16 and Wb123). The children who were only positive for Wb123 came from the villages of Kokounkoutouba (a boy aged 3), Kantila (a girl aged 9), and Galé (a boy aged 4).

Table 3-4 summarises the rates of Ov16 and Wb123 positivity per village in Bakoye.

In Falémé, Ov16 positivity was 1/2,270 (0.04%, 95%CI = 0.01–0.25%) in the 3–10-year-olds, the positive child originating from the mesoendemic community of Sely (one boy aged 7).

Wb123 seropositivity was 2/2,270 (0.09%, 95%CI = 0.02–0.32%), the positive children being from the villages of Madina-Mandinga (a girl aged 7) and Mahinamine (a boy aged 10). Table

5-5 summarises the rates of Ov16 and Wb123 positivity per village in Falémé. Table 3-5 summarises the rates of Ov16 and Wb123 positivity per village in Falémé.

112

Table 3-4 . Ov16 and Wb123 IgG4 Seroprevalence by Village in 3–10-year Old Children in Bakoye, Mali, 2017–2018

Village Census Target Tested (% Ov16 Ov16 Wb123 Wb123 population sample target) positive prevalence (%) positive prevalence (%) size (95% CI) (95% CI) Fataba-Kouroubala 202 197 158 (80%) 0 0.0 (0.0–2.4) 0 0.0 (0.0–2.4) Kokounkoutouba 240 233 137 (59%) 0 0.0 (0.0–2.7) 1 0.7 (0.1–4.0) Baniangafata 134 132 259(196%) 0 0.0 (0.0–1.5) 0 0.0 (0.0–1.5) Dianga-Foula 122 121 154(127%) 0 0.0 (0.0–2.4) 0 0.0 (0.0–2.4) Fatafing 500 470 36 (8%) 0 0.0 (0.0–9.6) 0 0.0 (0.0–9.6) Badougou 70 70 244(349%) 1 0.4 (0.1–2.3) 0 0.0 (0.0–1.6) Tieourou-Santankoto 170 167 86 (52%) 0 0.0 (0.0–4.3) 0 0.0 (0.0–4.3) Madila 324 311 112 (36%) 0 0.0 (0.0–3.3) 0 0.0 (0.0–3.3) Kantila 147 145 114 (79%) 0 0.0 (0.0–3.3) 1 0.9 (0.2–4.8) Keniefeto 569 530 19 (4%) 0 0.0 (0.0–16.8) 0 0.0 (0.0–16.8) Nioumala 54 54 48 (89%) 0 0.0 (0.0–7.4) 0 0.0 (0.0–7.4) Kibi 569 530 79 (15%) 1 1.3 (0.2–6.8) 1 1.3 (0.2–6.8) Galé 590 548 663(121%) 4 0.6 (0.2–1.5) 3 0.5 (0.2–1.3) Madina* NA NA 34 0 0.0 (0.0–10.2) 0 0.0 (0.0–10.2) Kouroudioula* NA NA 43 0 0.0 (0.0–8.2) 0 0.0 (0.0–8.2) Total 3,691 3,508 2,186 (62%) 6 0.3 (0.1–0.6) 6 0.3 (0.1–0.6) * Madina and Kouroudioula are hamlets of Galé. NA = Not available at the time of sample size calculation.

113

Table 3-5. Ov16 and Wb123 IgG4 Seroprevalence by Village in 3–10-year Old Children in Falémé, Mali, 2017–2018

Village Target Ov16 Wb123 Census Tested (% Ov16 Wb123 sample prevalence (%) prevalence (%) population target) positive positive size (95% CI) (95% CI) Madina-Mandinga 131 127 156 (123%) 0 0.0 (0.0–2.4) 1 0.6 (0.1–3.5) Koffing 142 140 173 (124%) 0 0.0 (0.0–2.2) 0 0.0 (0.0–2.2) Yatia-Berola 102 101 118(117%) 0 0.0 (0.0–3.2) 0 0.0 (0.0–3.2) Sakola-Loulo 290 280 171 (61%) 0 0.0 (0.0–2.2) 0 0.0 (0.0–2.2) Djoulafoundouni 123 122 191 (157%) 0 0.0 (0.0–2.0) 0 0.0 (0.0–2.0) Koutila 259 251 108 (43%) 0 0.0 (0.0–3.4) 0 0.0 (0.0–3.4) Djidian-Kenieba 194 190 170 (90%) 0 0.0 (0.0–2.2) 0 0.0 (0.0–2.2) Moussala 140 138 89 (65%) 0 0.0 (0.0–4.1) 0 0.0 (0.0–4.1) Sanoukou 387 347 178 (51%) 0 0.0 (0.0–2.1) 0 0.0 (0.0–2.1) Satadougou-Tintiba 101 100 70 (70%) 0 0.0 (0.0–5.2) 0 0.0 (0.0–5.2) Sely 203 198 98 (50%) 1 1.0 (0.2–5.6) 0 0.0 (0.0–3.8) Fadougou 86 86 148 (172%) 0 0.0 (0.0–2.5) 0 0.0 (0.0–2.5) Kounda-Mahina 353 338 110 (33%) 0 0.0 (0.0–3.4) 0 0.0 (0.0–3.4) Mankouke 237 230 167 (73%) 0 0.0 (0.0–2.3) 0 0.0 (0.0–2.3) Mahinamine 93 91 257 (282%) 0 0.0 (0.0–1.5) 1 0.4 (0.1–2.2) Berindina NA NA 66 0 0.0 (0.0–5.5) 0 0.0 (0.0–5.5) Total 2,841 2,739 2,270 (83%) 1 0.04 (0.01–0.25) 2 0.1 (0.02–0.32) * Berindina is a hamlet of Kenieba. NA = Not available at the time of sample size calculation.

114

Age-Specific Seroprevalence of Ov16 and Wb123 in Bakoye

In the 3 historically hyperendemic villages selected for full age-range serological sampling,

Ov16 IgG4 antibodies were detected in all 3 villages. Overall seroprevalence estimates (for individuals aged 3 to ≥50 years) were: 0.63% (95% CI = 0.17–2.26%) in Kantila (2 positives/319 tested); 7.0% (95% CI = 4.22–11.41%) in Nioumala (14 positives/200 tested), and 2.97% (95% CI = 2.19–4.02%) in Galé (40 positives/1,348 tested).

Wb123 IgG4 antibodies were also detected in all 3 villages. Overall seroprevalence estimates

(for individuals aged 3 to ≥50 years) were: 0.31% (95% CI = 0.06–1.75%) in Kantila (1 positive/319 tested); 1.0% (95% CI = 0.27–3.57%) in Nioumala (2 positives/200 tested), and

1.19% (95% CI = 0.73–1.92%) in Galé (16 positives/1,348 tested). Figure 3-4 presents the age profiles of Ov16 and Wb123 seropositivity in the three villages combined. Table 3-6 presents the results of Ov16 and Wb123 serology by age group in each of the three villages. The figure

3-5 is the comparaison of serology trend of Mali data with that of the neighboring Senegal.

115

Figure 3-4 Age-specific Ov16 and Wb123 seroprevalence profiles in the villages of

Kantila, Nioumala and Galé combined

Ov16 (white bars) and Wb123 (grey bars) seropositivity by age group with 95% (Wilson score) confidence intervals.

116

Table 3-6 Ov16 and Wb123 IgG4 Seroprevalence by Age Group in Three Bakoye Villages Hyperendemic for Onchocerciasis at Baseline,

Mali, 2017–2018

Village Census Target Tested (% Ov16 Ov16 Wb123 Wb123 Age group population sample target) positive prevalence (%) positive prevalence (yr) size (95% CI) (%) (95 % CI) Kantila 600 483 319 (66%) 2 0.6 (0.2–2.3) 1 0.3 (0.1–1.8) 3–10 147 145 114 (79%) 0 0.0 (0.0–3.3) 1 0.9 (0.2–4.8) 11–14 82 58 63 (109%) 0 0.0 (0.0–5.8) 0 0.0 (0.0–5.8) 15–19 67 50 38 (76%) 1 2.6 (0.5–13.5) 0 0.0 (0.0–9.2) 20–24 55 43 28 (65%) 0 0.0 (0.0–12.1) 0 0.0 (0.0–12.1) 25–29 45 37 18 (49%) 0 0.0 (0.0–17.6) 0 0.0 (0.0–17.6) 30–39 67 50 28 (56%) 0 0.0 (0.0–12.1) 0 0.0 (0.0–12.1) 40–49 45 37 14 (38%) 0 0.0 (0.0–21.5) 0 0.0 (0.0–21.5) ≥ 50 92 63 16 (25%) 1 6.3 (1.1–28.3) 0 0.0 (0.0–19.4) Nioumala 222 201 200 (100%) 14 7.0 (4.2–11.4) 2 1.0 (0.3–3.6) 3–10 54 54 48 (89%) 0 0.0 (0.0–7.4) 0 0.0 (0.0–7.4) 11–14 30 26 17 (65%) 0 0.0 (0.0–18.4) 0 0.0 (0.0–18.4) 15–19 25 22 22 (100%) 1 4.6 (0.8–21.8) 0 0.0 (0.0–14.9) 20–24 20 18 26 (144%) 0 0.0 (0.0–12.9) 0 0.0 (0.0–12.9) 25–29 17 15 12 (80%) 1 8.3 (1.5–35.4) 0 0.0 (0.1–24.3)

117

30–39 25 22 32 (145%) 3 9.4 (3.2–24.2) 1 3.1 (0.6–15.7) 40–49 17 15 12 (80%) 2 16.7 (4.7–44.8) 1 8.3 (1.5–35.4) ≥ 50 34 29 31 (107%) 7 22.6 (11.4–39.8) 0 0.0 (0.0–11.0) Galé 2,416 1,320 1,348(102%) 40 3.0 (2.2–4.0) 16 1.2 (0.7–1.9) 3–10 590 548 663 (121%) 4 0.6 (0.2–1.5) 3 0.5 (0.2–1.3) 11–14 331 123 229 (186%) 1 0.4 (0.1–2.4) 3 1.3 (0.5–3.8) 15–19 271 114 172 (151%) 5 2.9 (1.3–6.6) 3 1.7 (0.6–5.0) 20–24 222 104 60 (58%) 3 5.0 (1.7–13.7) 2 3.3 (0.9–11.4) 25–29 182 95 40 (42%) 1 2.5 (0.4–12.9) 0 0.0 (0.0–8.9) 30–39 270 114 74 (65%) 7 9.5 (4.7–18.3) 0 0.0 (0.0–4.9) 40–49 181 94 45 (48%) 4 8.9 (3.5–20.7) 1 2.2 (0.4–11.6) ≥ 50 369 128 65 (51%) 15 23.1 (14.5–34.6) 4 6.2 (2.4–14.8)

118

Figure 3-5 Age-specific Ov16 Seroprevalence in Three Historically Hyperendemic

Villages of the Bakoye Focus, Mali, and Three River Basins in the Kédougou Region,

Senegal.

The SD Bioline Onchocerciasis/LF IgG4 Rapid Diagnostic Test was used to evaluate, in 2018, 1,867 individuals aged 3–78 years in the villages of Kantila, Nioumala and Galé of the Bakoye focus (Mali), which had ≥60% microfilarial prevalence in 1985–1989 prior to ivermectin treatment [13]. The Luminex® multiplex-bead assay was used to test, in 2014, 1,131 individuals aged 5–100 years in the river basins of Gambia, Falémé and Koalakabe, in the Kédougou Region of Senegal [24]. The Bakoye focus had received 24 years of (annual) treatment by 2016. The Kédougou Region had received a maximum of 20 years of (biannual since 1992) treatment by 2014 [24]. Seroprevalence in Bakoye, Mali (white bars) and Kédougou, Senegal (grey bars) with 95% (Wilson score) confidence intervals.

119

3.4. Discussion

The proof-of-principle elimination of onchocerciasis from the Bakoye, Falémé and River

Gambia foci in Mali and Senegal [7,8] provided seminal evidence that elimination in Africa could be achieved using ivermectin alone. This was pivotal in driving change in the

WHO/APOC goals for onchocerciasis from control to elimination [4]. Elimination was assessed based on epidemiological (skin snipping to detect/enumerate mf) and entomological

(PCR to detect O. volvulus L3 larvae in blackflies) evidence [7,8]. Since 2016, the WHO has advocated more stringent serological criteria for the safe stopping of MDA. These include the

(statistically significant) demonstration of an Ov16 (by IgG4 ELISA) seroprevalence of <0.1% in children aged <10 years as measured [10]. For bancroftian (Anopheles-transmitted) LF, the

FTS test is recommended for TAS and post-elimination surveillance, with a threshold of <2%

[11]. The serological data presented here were obtained using the SD Bioline

Onchocerciasis/LF IgG4 RDT test and so cannot be used for direct comparison with recommended thresholds indicative of elimination (either EOT for onchocerciasis or EPHP for

LF). However, these data do provide important empirical information on the status of filariases transmission in Mali.

Robust interpretation of serological data critically depends on the assay’s diagnostic performance. The SD Bioline biplex test has a (manufacturer)-reported sensitivity of 92– 98% for onchocerciasis and 81–95% for LF. Specificity estimates are, respectively, 97– 100% and

96–99% [20]. Notwithstanding uncertainty in the RDT field performance [14,15,21–23], the

Ov16 seroprevalence of 0.27% and 0.04% in children aged 3–10 years in, respectively, Bakoye and Falémé is broadly consistent with EOT . The Wb123 seroprevalence of 0.27% in Bakoye and 0.09% in Falémé is also likely consistent with EPHP for LF, although there is no operational guidance on the interpretation of Wb123 serology in the context of LF transmission

(but see [23]). Therefore, at the focus level and as of 2017–2018, we find no substantive

120 evidence of onchocerciasis or LF resurgence since MDA cessation in 2016. However, the few

Ov16 seropositive children identified here should be PCR-tested on skin snips to distinguish between parasite exposure and infection; if found negative, they could be omitted from the seroprevalence calculation but re-examined 1–1.5 years later to determine if they have become patent and should be treated [10].

The serological results in Bakoye contribute to validate previous modelling projections, indicating sustained elimination when EPIONCHO was fitted to the entire longitudinal data series (Fig. 3c of (Fig. 3c of [13]). By contrast, likely resurgence was predicted by EPIONCHO

(but not ONCHOSIM) in the neighbouring and more highly endemic River Gambia focus in

Senegal (which was treated biannually) (Fig. 3g of [13]). Indeed, ivermectin MDA was resumed by the Senegalese National Onchocerciasis Control Program in the River Gambia focus in 2013 [24]. In 2014, an Ov16/Wb123 serological evaluation (using Luminex® multiplex-bead assay) across three river basins of the Kédougou Region (containing areas and some communities of [7,8]), found an Ov16 seroprevalence of 2.5% (7/279) in <10-year-olds, including three positives in the River Gambia focus and four positives in the Senegalese part of the Falémé focus [24]. It is unclear whether these children were found in the same communities previously reported free of onchocerciasis transmission [7,8], but concerns were raised that transmission had either not been interrupted or had resurged [24]. All-age Ov16 serological profiles in the Bakoye focus of Mali [this work] are compared with those of the

Kédougou Region of Senegal [24] in Figure 3-5. Resurgence of onchocerciasis has been reported elsewhere in West Africa [25,26] highlighting the importance of robust epidemiological and entomological surveillance following cessation of interventions [27].

The three villages in Bakoye and one in Falémé that had children aged ≤10 years who were

Ov16 IgG4 antibody positive were meso- or hyperendemic at baseline. The village of Galé—

121 one of the most hyperendemic villages before MDA (Figure 3-2)—had four seropositive children (0.6%, Table 3-4). Although, arguably, these may all be false positives, variation in seropositivity among villages highlights the importance of spatial scale when designing monitoring, evaluation and surveillance sampling protocols. There currently exists no explicit guidance on how onchocerciasis elimination surveys should be implemented, nor on the appropriate spatial unit. For LF, an online survey-design tool can be used for school-based or community-based cluster randomised surveys within ‘evaluation units’ [11]. More guidance is needed on how best to implement onchocerciasis serological (and entomological) surveys.

Spatially-explicit protocols, which have been developed for onchocerciasis elimination mapping (to identify previously untreated hypoendemic areas) [21] may prove useful.

The certainty of evidence indicative of onchocerciasis elimination for the <0.1% serological threshold in children aged <10 years is low [10]. Recent modelling work has suggested that a higher threshold of 2% may be safe for stopping MDA and that children aged 5–14 years may be more informative [28]. Yet, considerable uncertainty remains on both the technical threshold for elimination which is confounded by limited understanding of O. volvulus transmission dynamics and population biology at low transmission levels [29] and whether a particular threshold can be measured using current diagnostic tools [30]. The WHO recommends Ov16

ELISA serology, but different ELISA protocols vary in performance characteristics, and laboratory capacity in Africa must be strengthened [31,32]. The development and manufacture of standardized and quality-assured ELISA kits with sensitivity/specificity compatible with measuring (revised) serological thresholds is a priority.

Inspection of all-age serological profiles in the previously hyperendemic villages of Kantila,

Nioumala and Galé analysed together provides an overview of historical exposure trends in

Bakoye. Seroprevalence was very low in the ≤14-year olds, with upper 95% confidence limits

122 less than 2% in 3–6, 7–10 and 11–14 (Figure 3-4), suggesting pronounced transmission suppression. Four of the five seropositive children aged ≤10 years originated from the historically highly hyperendemic village of Galé. Seroprevalence increases slowly with age from 15–19, only reaching 16% in those aged ≥40 years. These individuals would have been

≥15–20 years old when ivermectin distribution began and, under intense pre-treatment transmission, their age-specific mf prevalence would already have reached >60% [33]. These results are consistent with those of [34], indicating that Ov16 seropositivity likely declines over time after prolonged treatment (although mortality rates in these populations would have also to be considered). The all-age seroprevalence profiles for Wb123 also increased somewhat with age, reaching c.3% in those aged ≥40 years but remaining below 2% in all those aged <40. A recent study in Mali found a strong correlation between ICT and Wb123 seropositivity in children aged 6–7 years, suggesting that evaluating IgG4 to Wb123 might be preferable in stop-

MDA decisions or in TAS following MDA cessation, as it allows measuring recent exposure

[35]. Such study also reported Ov16 seropositivity in children from previously onchocerciasis meso- and hyperendemic communities and none from hypoendemic villages. These findings, together with those presented here and reported in Senegal for children aged <10 years, where the seven seropositive individuals were found in the River Gambia and Fálémé foci [24], indicate that not all Ov16 seropositive results should be dismissed as false positives, as a trend is clearly emerging associating Ov16 seropositivity with pre-MDA endemicity status.

It is hoped that current LF antigenemia and mf prevalence thresholds for EPHP will also be sufficient to lead to EOT, albeit empirical evidence for this is limited [23] and LF may persist despite passing TAS [36]. In reality, thresholds indicative of interrupted transmission will vary with transmission conditions and local vector biting rates [37]. Hence, while mathematical modelling [28,37] can help decision-makers define thresholds based on acceptable levels of risk, elimination will ultimately be demonstrated using robustly-sampled surveillance data.

123

Surveillance will come at a cost that the global health community must recognise and prepare for to sustain the great progress already made along the path to eliminating onchocerciasis and

LF.

3.5. Conclusion

The antibody seroprevalence against Ov16 and Wb123 antigens among children in the Bakoye and Falémé foci in Mali is very low and consistent with both onchocerciasis and LF having been likely eliminated since stopping MDA in 2016 (pending skin snip PCR- testing of the few

Ov16 seropositive children identified). Periodic (including entomological) surveillance should be continued in this region to detect and respond to any early signs of resurgence/re- introduction. This will help to sustain elimination and maintain the Bakoye and Falémé foci as an example of a global health intervention success.

124

3.6.References

1. Dembélé M, Bamani S, Dembélé R, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl. Trop. Dis. 2012; 6:e1574.

2. Akpoboua KL, Hougard JM, Agoua H, Seketeli A, Quillevere D. [Importance and role of spreading larvicides on the soil in river beds for the control program against onchocerciasis in west Africa]. Bull Soc Pathol Exot 1994; 87:278–82.

3. Boatin BA, Richards FO. Control of onchocerciasis. Adv Parasitol 2006; 61:349–394.

4. World Health Organization/African Programme for Onchocerciasis Control. Conceptual and Operational Framework of Onchocerciasis Elimination with Ivermectin Treatment. 2010. Available at: https://www.who.int/apoc/oncho_elimination_report_english.pdf. Accessed 4 October 2019.

5. World Health Organization. Accelerating Work to Overcome the Impact of Neglected Tropical Diseases: A Roadmap for Implementation. 2012. Available at: https://www.who.int/neglected_diseases/NTD_RoadMap_2012_Fullversion.pdf. Accessed 4 October 2019.

6. Global programme to eliminate lymphatic filariasis: progress report, 2016. Wkly Epidemiol Rec 2017; 92:594–607.

7. Diawara L, Traoré MO, Badji A, et al. Feasibility of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: first evidence from studies in Mali and Senegal. PLoS Negl Trop Dis, 2009; 3(7):e497.

8. Traore MO, Sarr MD, Badji A, et al. Proof-of-principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl. Trop. Dis. 2012; 6:e1825.

9. Kelly-Hope LA, Molyneux DH, Bockarie MJ. Can malaria vector control accelerate the interruption of lymphatic filariasis transmission in Africa; capturing a window of opportunity? Parasit. Vectors 2013; 6:39.

10. World Health Organization/Department of Control of Neglected Tropical Diseases. Guidelines for stopping mass drug administration and verifying elimination of human onchocerciasis. Criteria and procedures (Ukety T, Ed.), 44 pp. 2016. Available at: https://apps.who.int/iris/bitstream/handle/10665/204180/9789241510011_eng.pdf;jsessionid=85 ADE0FAAE6368A0802984B4A3810DCF?sequence=1. Accessed 4 October 2019.

11. World Health Organization/Department of Control of Neglected Tropical Diseases. Lymphatic filariasis: monitoring and epidemiological assessment of mass drug administration. A manual for national elimination programmes (Ichimori K, Ed.), 79pp. 2011. Available at: https://www.who.int/lymphatic_filariasis/resources/9789241501484/en/. Accessed 4 October 2019.

12. Mali National Health Direction. National Lymphatic Filariasis Elimination Program. 2016 Report. Bamako: Ministere de la Santé et des Affaires Sociales.

125

13. Walker M, Stolk WA, Dixon MA, et al. Modelling the elimination of river blindness using long-term epidemiological and programmatic data from Mali and Senegal. Epidemics 2017; 18:4– 15.

14. Steel C, Golden A, Stevens E, et al. Rapid Point-of-Contact Tool for Mapping and Integrated Surveillance of Wuchereria bancrofti and Onchocerca volvulus Infection. Clin. Vaccine Immunol. 2015; 22:896–901.

15. Golden A, Steel C, Yokobe L, et al. Extended result reading window in lateral flow tests detecting exposure to Onchocerca volvulus: a new technology to improve epidemiological surveillance tools. PLoS One 2013; 8:e69231.

16. Moreau JP, Prost A, Prod’hon J. [An attempt to normalize the methodology of clinico parasitologic surveys of onchocerciasis in West-Africa (author’s transl)]. Med Trop (Mars) 1978; 38:43–51.

17. Brown LD, Cat TT, DasGupta A. Interval estimation for a proportion. Stat Sci, 2001; 16 (2): 101–133.

18. Remme J, Ba O, Dadzie KY, Karam M. A force-of-infection model for onchocerciasis and its applications in the epidemiological evaluation of the Onchocerciasis Control Programme in the Volta River basin area. Bull. World Health Organ. 1986; 64:667–681.

19. Filipe JAN, Boussinesq M, Renz A, et al. Human infection patterns and heterogeneous exposure in river blindness. Proc. Natl. Acad. Sci. USA 2005; 102:15265–15270.

20. SD BIOLINE Onchocerciasis/LF IgG4 Rapid Test - Alere is now Abbott. Available at: https://www.alere.com/en/home/product-details/sd-bioline-onchocerciasis-LF-igg4-rapid- test.html. Accessed 4 October 2019.

21. Health OW. Report of the 1st meeting of the WHO onchocerciasis technical advisory subgroup, Varembé Conference Centre, Geneva, Switzerland, 10-12 October 2017. Report of the 1st meeting of the WHO onchocerciasis technical advisory subgroup, Varembé Conference Centre, Geneva, Switzerland, 10-12 October 2017 2018;

22. Unnasch TR, Golden A, Cama V, Cantey PT. Diagnostics for onchocerciasis in the era of elimination. Int. Health 2018; 10:i20–i26.

23. Won KY, Sambou S, Barry A, et al. Use of antibody tools to provide serologic evidence of elimination of lymphatic filariasis in the gambia. Am. J. Trop. Med. Hyg. 2018; 98:15–20.

24. Wilson NO, Badara Ly A, Cama VA, et al. Evaluation of Lymphatic Filariasis and Onchocerciasis in Three Senegalese Districts Treated for Onchocerciasis with Ivermectin. PLoS Negl. Trop. Dis. 2016; 10:e0005198.

25. Koala L, Nikiema A, Post RJ, et al. Recrudescence of onchocerciasis in the Comoé valley in Southwest Burkina Faso. Acta Trop. 2017; 166:96–105.

26. Koala L, Nikièma AS, Paré AB, et al. Entomological assessment of the transmission following recrudescence of onchocerciasis in the Comoé Valley, Burkina Faso. Parasit. Vectors 2019; 12:34.

126

27. Cantey PT, Roy SL, Boakye D, et al. Transitioning from river blindness control to elimination: steps toward stopping treatment. Int. Health 2018; 10:i7–i13.

28. Coffeng LE, Stolk WA, Golden A, de Los Santos T, Domingo GJ, de Vlas SJ. Predictive value of ov16 antibody prevalence in different subpopulations for elimination of african onchocerciasis. Am. J. Epidemiol. 2019; 188:1723–1732.

29. Hamley JID, Milton P, Walker M, Basáñez MG. Modelling exposure heterogeneity and density dependence in onchocerciasis using a novel individual-based transmission model, EPIONCHO-IBM: implications for elimination and data needs. PLoS Negl Trop Dis . PLoS Negl. Trop. Dis. 2019;

30. Gass KM. Rethinking the serological threshold for onchocerciasis elimination. PLoS Negl. Trop. Dis. 2018; 12:e0006249.

31. Ov-16 Meeting Notes | Neglected tropical diseases support center. Available at: http://www.ntdsupport.org/resources/ov-16-meeting-notes. Accessed 13 July 2018.

32. Shott J, Ducker C, Unnasch TR, Mackenzie CD. Establishing quality assured (QA) laboratory support for onchocerciasis elimination in Africa. Int. Health 2018; 10:i33–i39.

33. Basáñez MG, Boussinesq M. Population biology of human onchocerciasis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354:809–826.

34. Paulin HN, Nshala A, Kalinga A, et al. Evaluation of onchocerciasis transmission in tanzania: preliminary rapid field results in the tukuyu focus, 2015. Am. J. Trop. Med. Hyg. 2017; 97:673–676.

35. Dolo H, Coulibaly YI, Dembele B, et al. Integrated seroprevalence-based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test. PLoS Negl. Trop. Dis. 2019; 13:e0007064.

36. Rao RU, Nagodavithana KC, Samarasekera SD, et al. A comprehensive assessment of lymphatic filariasis in Sri Lanka six years after cessation of mass drug administration. PLoS Negl. Trop. Dis. 2014; 8:e3281.

37. Davis EL, Reimer LJ, Pellis L, Hollingsworth TD. Evaluating the evidence for lymphatic filariasis elimination. Trends Parasitol. 2019;

127

128

Chapter 4 Lymphedema in three previously Wuchereria bancrofti-endemic health districts in Mali after cessation of mass drug administration

Housseini Dolo, Yaya Ibrahim Coulibaly, Fatoumata Nene Konipo, Siaka Yamoussa

Coulibaly, Salif Seriba Doumbia, Moussa Brema Sangare, Lamine Soumaoro, Michel

Emmanuel Coulibaly, Abdallah Amadou Diallo, Yaye Diarra, Modibo Sangare, Seydou

Doumbia, Robert Colebunders, Thomas B. Nutman. Lymphedema in three previously

Wuchereria bancrofti-endemic health districts in Mali after cessation of mass drug administration. BMC Infectious Diseases. 2020 Dec;20(1):1-8.

129

Abstract

Background

Lymphedema is a public health problem in countries with lymphatic filariasis (LF) including Mali. We studied the epidemiology and clinical presentation of lymphedema in three previously LF-endemic health districts of Mali after at least five consecutive rounds of mass drug administration (MDA) with albendazole and ivermectin.

Methods

From 2016 to 2018, we used passive and active case finding methods to identify lymphedema cases in three health districts with high pre-MDA LF prevalence: Kolondieba (66%), Bougouni (44%) and

Kolokani (34%).

Results

Three hundred and thirty-nine cases of lymphedema were identified, 235 (69.32%) through active case finding. Their median age was 56 years (range 2-90) and 286 (84.36%) were women. Lymphedema was reported in 226 (78.5%) people aged 41 years and older compared to 73 (21.5%) people below the age of 41 years (Chi2 =17.28, df=5, p=0.004). One hundred and seventy-five cases of lymphedema were found in Kolondieba (66 per 100,000 people), 116 in Bougouni (19 per 100,000) and 48 in Kolokani

(16 per 100,000). Stage III lymphedema was observed in 131 (38.64%), stage II in 108 (31.86%), stage

IV in 46 (13.57%), stage I in 23 (6.78%), stage V in 21 (6.19%) and stage VI in ten (2.95%). In the three study districts, lymphedema affected the legs in 281 (82.89%), the arms in 42 (12.39%) and both in 16 (4.72%) (Chi2=13.63, p=0.008).

Conclusion

Health districts in Mali with the highest pre-MDA LF prevalences had the highest prevalence of lymphedema. Efforts to actively identify lymphedema cases should be scaled up in previous LF- endemic areas and should be supplemented by a morbidity management and disability prevention plan at the peripheral health system level.

Keywords: Lymphedema, distribution, clinical investigation, Mali, active and passive case detection

130

4.1. Introduction

Lymphedema or elephantiasis results from the accumulation of interstitial fluid in the affected anatomic compartment causing localized swelling [1, 2]. Lymphatic filariasis (LF)-related lymphedema is caused by three filarial species, namely Wuchereria bancrofti, Brugia malayi and Brugia timori [3]. Podoconiosis is another condition in Africa causing lymphedema.

However, it occurs only in regions with both high altitude and significant rainfall and therefore, is considered not to occur in Mali. Reported evidence suggests that podoconiosis is the result of a genetically determined abnormal inflammatory reaction to mineral particles in irritant red clay soils derived from volcanic deposits [4, 5].

LF-related lymphedema affects over 15 million people worldwide [3]. In Mali, no lymphedema national surveillance system exists, but LF is considered to be implicated in most lymphedema cases. In Bamako, 0.58% of outpatients in the dermatology clinic at Gabriel Touré teaching hospital were reported to have lymphedema in 2018 [6]. As part of the country's certification process for the elimination of lymphatic filariasis as Mali's long-term objective, it is important to understand the burden of lymphedema and to manage this concern with the second goal of

GPELF (MMDP) [3].

In LF-related lymphedema, the lymphatic system is damaged because the host either fails to modulate the inflammatory response towards either the filarial parasite or its endosymbiont

Wolbachia or because secondary infections by bacteria and/or fungi drive the ongoing inflammatory processes [7]. Lymphedema often leads to social stigmatization, mental health problems, loss of income and increased medical expenses for patients and their caregivers [3].

LF prevalence mapping in 2004 showed that all 8 administrative regions of Mali were endemic for W. bancrofti, with an overall nationwide prevalence of 7.07% (1% in the north and 18.6% in the south of Mali) [8]. From 2005 to 2015, MDA with albendazole plus ivermectin decreased the prevalence of W. bancrofti infection to zero in Mali in adults tested for microfilaremia

131

(Dembele, 2016 Unpublished). LF transmission assessment surveys (TAS) performed in the 3 health districts between 2010 and 2015 found antigenemia rates < 2% in 6-7 year old children suggesting the interruption of LF transmission according to World Health Organization guidelines [9].

The control of LF morbidities, particularly hydrocele and lymphedema, should be an essential component of LF elimination programme. However, in Mali, morbidity management, more particularly that of lymphedema, is not considered as a priority within a national program that focusses on the MDA strategy. Therefore, the burden of disease caused by LF morbidities in

Mali is unknown. We hypothesised that the highest burden of disease would be present in districts of Mali with previously high LF prevalence.

The control of LF morbidities, particularly hydrocele and lymphedema, should be an essential component of LF elimination programme. However, in Mali, morbidity management, more particularly that of lymphedema, is not considered as a priority within a national program that focusses on the MDA strategy. Therefore, the burden of disease caused by LF morbidities in

Mali is unknown. We hypothesised that the highest burden of disease would be present in districts of Mali with previously high LF prevalence.

In preparation of a multi-site clinical trial (NCT02927496) to investigate the impact of doxycycline on the regression of early stages of lymphedema, we screened three health districts of Mali (Kolondieba, Bougouni and Kolokani) with previously high LF prevalences, to identify lymphedema cases. In this paper, we describe the characteristics and epidemiological distribution of the lymphedema cases identified.

4.2. Material and Methods

Study design and population

132

A cross sectional study was performed in the health districts of Kolondieba, Bougouni and

Kolokani from August 2016 to August 2018 (Figure 4- 1).

Source: Filariasis Unit, 2019; modify with Paint 3D software

Figure 4-1 Map of Mali showing the three study districts (Kolondieba, Bougouni and

Kolokani in Read)

LF mapping in 2004 had documented a LF prevalence of 66% in Kolondieba, 48% in Bougouni and 34% in Kolokani [10]. Bougouni and Kolondieba are very large districts characterized by difficult geographic accessibility and relatively little knowledge (by the inhabitants) of the underlying causes of lymphedema. We screened for lymphedema cases using both passive and

133 active case detection methods. With the passive case detection method, heads of community health centers and community health workers were asked to report people living with lymphedema. With the active case detection method, a research team identified lymphedema cases through villages meetings, mobile phone calls and scheduled visits to remote villages.

We defined lymphedema as any non-traumatic progressive and evolving swelling of at least one upper or lower limb associated with a history of adenolymphangitis (ADL) episodes. For swelling of the lower limbs we used the Dreyer classification [11] to determine the stage of lymphedema as follows: stage I: reversible swelling that disappears spontaneously at night; stage II: non-reversible swelling that does not disappear spontaneously at night; stage III: presence of shallow skin folds; stage IV: presence of buds; stage V: presence of deep skin folds; stage VI: presence of mossy lesions; stage VII: inability to perform normal daily activities correctly and independently.

For swelling of the upper limbs, we adapted the G Dreyer's classification as follows: Stage I: swelling of an arm reported by the affected person with history of an adenolymphagitis crisis but not necessarily observed by the investigator. Stage II any swelling of an arm without visible skin folds, Stage III any swelling of an arm with at least one skin fold. When more than one member was affected, we considered the most advanced stage to classify lymphedema of the person. For both legs and arms, stage I assignments were based on history.

To estimate the prevalence of lymphedema per health district we divided the number of lymphedema cases identified by all the methods (active and passive) in each health district by the 2017 population size of the district multiplied by 100,000.

Data collection and analysis

Each case identified passively or actively was geopositioned using handheld GPS devices. We used medians to measure central tendency and chi-square test to determine statistical differences in lymphedema prevalence across categorical variables known to have a potential

134 influence on lymphedema [12, 13], including clinical stages, different age groups and gender in all three health districts. Data were analysed using Statistical Package for Social Sciences

(SPSS) version 24.

Ethical considerations

Our study protocol was approved by the ethical committee of the Faculty of Medicine and

Odonto Stomatology and the Faculty of Pharmacy (2016/ISO/CE/FMPOS). Participation in this study was entirely voluntary. Before undertaking any research activity, written informed consent (and/or assent) was obtained from every study participant.

135

4.3. Results

Characteristics of people with lymphedema

Three hundred thirty-nine people with lymphedema were identified, 175 (51.62%) in

Kolondieba, 116 (34.22%) in Bougouni, and 48 (14.16%) in Kolokani (Table 4-2). Overall,

286 (84.36%) lymphedema cases were women (Table 4-1). The median age of all cases was

56 years (range 2-90). Lymphedema was reported in 266 (78.47%) people aged 41 years and above compared to 73 (21.53%) people below the age of 41 years (Chi2 =17.28, df=5, p=0.004). All stages of lymphedema were observed except stage VII. Stage III lymphedema was observed in 131 (38.64%), stage II in 108 (31.86%), stage IV in 46 (13.57%), stage I in 23

(6.78%), stage V in 21 (6.19%) and stage VI in ten (2.95%) (Table 4-1).

136

Table 4-1 Distribution of the lymphedema cases according to gender, age and body localization in three health districts of Mali

Kolondieba Bougouni Kolokani Total (N=175) (N=116) (N=48) Number (%) Number (%) Number (%) Number (%) p values Sex Female 155 (88.57%) 97 (83.63%) 34 (70.83%) 286 (84.36%) 0.01 Male 20 (11.43%) 19 (16.37%) 14 (29.17%) 53 (15.64%) Age in years Median age (range) 54 (13-88) 56.5 (2-90) 56 (18-84) 56 (2-90) 0.40 Age group 0 -20 2 (1.14%) 2 (1.72%) 2 (2.08%) 5 (1.47%) 21-40 39 (22.29%) 19 (16.38%) 10 (20.83%) 68 (20.06) 0.004 41-60 80 (45.71%) 55 (47.41%) 23 (47.92%) 158 (46.61%) >=61 54 (30.86%) 40 (34.48%) 14 (29.17%) 108 (31.86%) Anatomic location Legs 140 (80.00%) 104 (89.65%) 37 (77.08%) 281 (82.89%) Arms 29 (16.57%) 8 (6.90%) 5 (10.42%) 42 (12.39%) 0.008 Legs and Arms 6 (3.43%) 4 (3.45%) 6 (12.50%) 16 (4.72%) Stage I 13 (7.43%) 5 (4.31%) 5 (10.42%) 23 (6.78%) II 53 (30.29%) 32 (27.59%) 23 (47.92%) 108 (31.86%) III 71 (40.57%) 44 (37.93%) 16 (33.33%) 131 (38.64%) 0.02 IV 27 (15.43%) 17 (14.66%) 2 (4.17%) 46 (13.57%) V 8 (4.57%) 11 (9.48%) 2 (4.17%) 21 (6.19%) VI 3 (1.71%) 7 (6.03%) 0 (0.00%) 10 (2.95%)

137

Staging and body localization of lymphedema in the three health districts

Lymphedema affected the legs in 281(82.89%), the arms in 42 (12.39%) and both the arms and legs in 16 (4.72%) (Chi2=13.63, p=0.008) (Table 4-1). Stage III was slightly more common in

Kolondieba 71 (40.57%) and in Bougouni 44 (37.93%), stage II was most common in Kolokani

23 (47.92%) and Bougouni had the highest prevalence of stage VI 7 (6.03%). All stages were more frequent in the older age groups (Table 4-1).

Case detection approaches

Of the 339 people with lymphedema, only 104 (30.68%) were identified through passive case identification and 235 (69.32%) by active case identification with no statistically difference observed (Chi2 =3.323, df=2 , p=0.18) (Table 4-2).

138

Table 4-2 Number and percentage of lymphedema cases recorded per health district and per gender according to the method of identification

Number surveyed Type of identification Gender Health districts Passive Active n (%) n (%) n (%) Total Male 20 (11.42%) 7 (35.00%) 13 (65.00%) 20 (100%) Kolondieba Female 155(88.58%) 45 (29.00%) 110 (70.00%) 155 (100%) Sub-total 1* 175 (51.62%) 52 (29.71%) 123 (70.29%) 175 (100%) Male 19 (16.38%) 3 (15.79%) 16 (84.21%) 19 (100%) Bougouni Female 97(83.62%) 29 (29.90%) 68 (70.10%) 97 (100%) Sub-total 2* 116 (34.22%) 32 (27.59%) 84 (72.41%) 116 (100%) Male 14 (29.17%) 8 (57.14%) 6 (42.86%) 14 (100%) Kolokani Female 34(70.83%) 12 (35.29%) 22 (64.71%) 34 (100%) Sub-total 3* 48 (14.16%) 20 (41.66%) 28 (58.34%) 48 (100%) Total 339 (100%) 104 (30.68%) 235 (69.32%) 339 (100%) *Subtotal proportions were on the total cases of lymphedema identified

*Per identification type difference of reported lymphedema cases in the three-health district (Chi2 =3.323, df=2, p=0.18)

139

Estimation of lymphedema prevalence

The estimated prevalence of lymphedema in Kolondieba was 65.60 per 100,000 people, 19.17 per 100,000 people in Bougouni and 15.66 per

100,000 people in Kolokani; overall in the three health districts it was 28.77 per 100,000 people (Chi2 =163.5, df=2, p<0.0001) (Table 4-3).

Table 4-3 Estimation of lymphedema prevalence per health district in three health districts of Mali

District Population size in Prevalence of LF during the Number of Estimated lymphedema 95% CI 2017 mapping in 2005* lymphedema prevalence for 100,000 persons carriers

Kolondieba 266,753 66% 175 65.60/100,000 [55.89 -75.32]

Bougouni 604,962 48% 116 19.17/100,000 [15.69 - 22.66]

Kolokani 306,445 34% 48 15.66/100,000 [11.23 - 20.09]

Total 1,178,160 49% 339 28.77/100,000 [25.71 - 31.84]

*Data are from the LF national report in 2005

140

Examples of clinical presentations of lymphedema in Mali

Case 1: A 40-year-old woman with stage VI lymphedema with swelling of the right leg from the foot to above the knee with mossy lesions (Figure 4-2A). She reported two to three episodes of ADL per year.

Case 2: A 70-year-old woman with lymphedema affecting both upper and lower limbs: right leg at stage III, left leg and right arm at stage II and the left arm at stage I (Figure 4-2Bi and 4-

2 Bii). She reported at least two episodes of ADL per year.

Case 3: A 25-year-old mother and her three-year-old daughter born with bilateral and symmetrical lymphedema at the lower limbs. The mother presented with stage VI lymphedema

(Figure 4-2D) and the daughter with stage III lymphedema (Figure 4- 2Ci and 4-2Cii).

Case 4: A 18-year-old man with lymphedema affecting the two legs. The two legs were at stage

V (Figure 4- 2E). He reported having multiple episodes of ADL.

Case 5: A 52-year-old woman with stage III lymphedema in two arms (Figure 4- 2F). She reported having multiple ADL episodes over the past 10 years.

Case 6: A 65-year-old woman had lymphedema in the right leg (Figure 4- 2Gi) and left arm

(Figure 4-2Gii) at stage II. She had no episodes of ADL for the last three years and had noticed a fairly dramatic decline in lymphedema size as she aged.

Case 7: A 63-year-old woman had a lymphedema of the left leg at stage V associated with dermal hypochromia (Figure 4-2H). She reported having an average of three ADL attacks per year.

Case 8: A 48-year-old woman with stage II lymphedema of the left arm (Figure 4-2I). She had reported three ADL per year.

Case 9: A 60-year-old woman with stage II lymphedema of the left leg and right arm and stage

I lymphedema of the right leg (Figure 4-2J). She had a chronic non-weeping ulceration at the

141 medial malleolus of her left leg. She had reported having ADL attacks on average twice a month.

Case 10: A 85-year-old woman with lymphedema of two arms and one left leg at stage III

(Figure 4- 2K). There is a history of lymphedema in her family. She had reported an average three ADL attacks per month.

Figure 4-2 Variability of clinical presentation in patients with lymphedema in Mali

Panels showing unilateral lower extremity lymphedema (2A), four limb lymphedema each at a different stage (2Bi/2Bii),familial lymphedema affected a children of 3 years old (2Ci, 2Cii) and her mother (2D), lymphedema of two legs in 18 years old young man (2E), lymphedema of two arms in 52 years old woman(2F), asymmetric lymphedema of left arm(2Gii) and right leg (2Gi), lymphedema of left leg with hypochromia

(2H), lymphedema of the left arm in 48 years old woman (2I), lymphedema of two legs and right arm at different stages in 60 years old woman

(2J) and lymphedema of two arms and left leg in 85 years old woman (2K).

142

4.4. Discussion

We identified 339 cases of lymphedema, mostly through active case finding, in three previously

LF hyper-endemic health districts (Kolondieba, Bougouni, and Kolokani) in Mali. Most people with lymphedema were identified in Kolondieba, the district previously reported with the highest pre-MDA LF prevalence (66%) [10]. Lymphedema was found predominantly among those in the older age group (median age 56 years); most cases occured in women (84%). The preponderance of lymphedema among women is in line with observations in other LF endemic countries [12, 14, 15]. The age and gender distribution is different from what has been reported in podoconiosis which is mainly observed in individuals between 10-30 years old without any gender predominance [4, 13, 16].

Lymphedemas were more frequently observed in those older than 41 years than in those in the lower age groups. We only observed 1 case of lymphedema (in the health district of Bougouni

) in the age group of below 6 years after the MDA stopped in 2015.This difference is most likely a consequence of ivermectin- and albendazole-based MDA whereby the younger age groups had reduced exposure to LF parasites [17].

Most late stages of lymphedema were identified in Bougouni and Kolondieba, very large districts characterized by difficult geographic accessibility; these remote areas are locations in which lymphedema is believed to be caused by evil or malediction [18] It is against this backdrop that our study population may have been difficult to mobilize, which in turn may have underestimated the true burden of lymphedema.

Our study highlights the importance of conducting active case detection which was more successful (approximatively 70% of cases detected) than passive case detection. This is explained by the fact that people with lymphedema usually consider their condition to be incurable and therefore may not consult health workers. Moreover, stigma concerning

143 lymphedema may also play a role in further reducing uptake of community health services [19,

20].

We observed several uncommon cases of lymphedema such as a case of congenital lymphedema also known as Milroy disease (case 3), an autosomal dominant disorder that causes lymphatic vessel dysfunction or absence of functional lymphatics [21]. We also observed a case of lymphedema affecting both upper and lower limbs at different stages (case

2) and a case of lymphedema affecting symmetrically two legs in a 18 year old young man without multiple hard skin nodules like podoconiosis (case 4) and those cases seem not related to LF [22].

In tropical countries, LF and podoconiosis are the most frequent causes of acquired lymphedema as opposed to European countries and in the United States where cancer-related treatment is the most frequent cause [23]. Medical record review of 511 patients with lymphedema attending a dermatological clinic in Tigray, Ethiopia between 2005-9, revealed that 9.2% of them were people with LF related lymphedema [24]. Integrated morbidity mapping of LF and podoconiosis in 20 co-endemic districts in Ethiopia in 2018 detected 26,123 cases of lymphedema, 89.3% with bilateral lymphedema [25]. The prevalence of men reporting hydrocele was low, 2.4 per 10,000 population. This large number of bilateral lymphedema cases and the low prevalence of hydrocoele suggest that most lymphedema cases in Ethiopia are caused by podoconiosis. Recently, eleven countries including Burundi, Rwanda,

Cameroon, Equatorial Guinea, Ethiopia, Kenya, Sao Tome & Principe, Uganda, Cape Verde,

Tanzania and India started to report lymphedema cases due to podoconiosis with a prevalence ranging from 0.1% (Uganda ) to 8.08% (Cameroon) [26].

In Mali, LF control programs, akin to elsewhere in Africa, are more focused on MDA to interrupt transmission than on lymphedema morbidity management [27]. In a study conducted in Togo in 2007, only 28.2 % of 188 lymphedema cases were reported to have benefited from

144 some sort of treatment. As lymphedema is a chronic, progressive condition, it is important to implement a morbidity management and disability prevention (MMDP) programme for LF as recommended by the WHO [27].

As limitations of this study, we need to mention that we did not perform a door-to-door survey and therefore we may have underestimated the prevalence of lymphedema. We also focused on case finding and did not assess co-morbidities such as scrotum swelling-hydrocele, treatment practices nor assessed the perception of communities about lymphedema.

4.5. Conclusion

Lymphedema remains a public health problem in previously LF endemic regions in Mali where transmission has been interrupted. Consequently, it is imperative that active lymphedema case identification be scaled up in all previously LF endemic regions in Mali and that a MMDP programme at peripheral health system level is implemented to meet LF elimination goal in near future.

145

4.6. References

1. Kerchner K, Fleischer A, Yosipovitch G. Lower extremity lymphedema update: pathophysiology, diagnosis, and treatment guidelines. J Am Acad Dermatol. 2008;59:324–31. doi:10.1016/j.jaad.2008.04.013.

2. Gaffney RM, Casley-Smith JR. Excess plasma proteins as a cause of chronic inflammation and lymphoedema: biochemical estimations. J Pathol. 1981;133:229–42. doi:10.1002/path.1711330306.

3. Filariose lymphatique. https://www.who.int/fr/news-room/fact-sheets/detail/lymphatic-filariasis. Accessed 14 Apr 2019.

4. Davey G, Newport M. Podoconiosis: the most neglected tropical disease? The Lancet. 2007;369:888– 9. doi:10.1016/S0140-6736(07)60425-5.

5. Price EW. The association of endemic elephantiasis of the lower legs in East Africa with soil derived from volcanic rocks. Trans R Soc Trop Med Hyg. 1976;70:288–95. doi:10.1016/0035-9203(76)90078- X.

6. Gassama M, Karabinta Y, Diarra M, Koné M, Tall K, Sanogo AM, et al. Aspects Épidémiologiques et Cliniques du Lymphœdème dans le Service de Dermatologie du CHU Gabriel Touré (Bamako). Health sciences and diseases. 2018;29(1)

7. Nutman TB. Insights into the pathogenesis of disease in human lymphatic filariasis. Lymphat Res Biol. 2013;11:144–8. doi:10.1089/lrb.2013.0021.

8. Coulibaly YI, Dembele B, Diallo AA, Kristensen S, Konate S, Dolo H, et al. Wuchereria bancrofti transmission pattern in southern Mali prior to and following the institution of mass drug administration. Parasit Vectors. 2013;6:247. doi:10.1186/1756-3305-6-247.

9. Dolo H, Coulibaly YI, Dembele B, Guindo B, Coulibaly SY, Dicko I, et al. Integrated seroprevalence- based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test. PLoS Negl Trop Dis. 2019;13:e0007064. doi:10.1371/journal.pntd.0007064.

10. Dembélé M, Bamani S, Dembélé R, Traoré MO, Goita S, Traoré MN, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl Trop Dis. 2012;6:e1574. doi:10.1371/journal.pntd.0001574.

11. Dreyer G, Addiss D, Dreyer P, Norões J. Basic lymphoedema management: treatment and prevention of problems associated with lymphatic filariasis. Hollis, NY: Hollis Publishing Company; 2002:112.

12. Grada AA, Phillips TJ. Lymphedema: Pathophysiology and clinical manifestations. J Am Acad Dermatol. 2017;77:1009–20. doi:10.1016/j.jaad.2017.03.022.

13. Greene AK, Grant FD, Slavin SA. Lower-extremity lymphedema and elevated body-mass index. N Engl J Med. 2012;366:2136–7. doi:10.1056/NEJMc1201684.

14. Richard SA, Mathieu E, Addiss DG, Sodahlon YK. A survey of treatment practices and burden of lymphoedema in Togo. Trans R Soc Trop Med Hyg. 2007;101:391–7. doi:10.1016/j.trstmh.2006.08.011.

15. Walsh V, Little K, Wiegand R, Rout J, Fox LM. Evaluating the burden of lymphedema due to lymphatic filariasis in 2005 in khurda district, odisha state, india. PLoS Negl Trop Dis. 146

2016;10:e0004917. doi:10.1371/journal.pntd.0004917.

16. Schook CC, Mulliken JB, Fishman SJ, Grant FD, Zurakowski D, Greene AK. Primary lymphedema: clinical features and management in 138 pediatric patients. Plast Reconstr Surg. 2011;127:2419–31. doi:10.1097/PRS.0b013e318213a218.

17. Ramaiah KD, Ottesen EA. Progress and impact of 13 years of the global programme to eliminate lymphatic filariasis on reducing the burden of filarial disease. PLoS Negl Trop Dis. 2014;8:e3319. doi:10.1371/journal.pntd.0003319.

18. Abdulmalik J, Nwefoh E, Obindo J, Dakwak S, Ayobola M, Umaru J, et al. Emotional Difficulties and Experiences of Stigma among Persons with Lymphatic Filariasis in Plateau State, Nigeria. Health Hum Rights. 2018;20:27–40.

19. Cassidy T, Worrell CM, Little K, Prakash A, Patra I, Rout J, et al. Experiences of a Community- Based Lymphedema Management Program for Lymphatic Filariasis in Odisha State, India: An Analysis of Focus Group Discussions with Patients, Families, Community Members and Program Volunteers. PLoS Negl Trop Dis. 2016;10:e0004424. doi:10.1371/journal.pntd.0004424.

20. Greene AK, Slavin SA, Brorson H, editors. Lymphedema: Presentation, Diagnosis, and Treatment. Cham: Springer International Publishing; 2015. doi:10.1007/978-3-319-14493-1.

21. Yimer M, Hailu T, Mulu W, Abera B. Epidemiology of elephantiasis with special emphasis on podoconiosis in Ethiopia: A literature review. J Vector Borne Dis. 2015;52:111–5.

22. Rockson SG, Rivera KK. Estimating the population burden of lymphedema. Ann N Y Acad Sci. 2008;1131:147–54. doi:10.1196/annals.1413.014.

23. Padovese V, Marrone R, Dassoni F, Vignally P, Barnabas GA, Morrone A. The diagnostic challenge of mapping elephantiasis in the Tigray region of northern Ethiopia. Int J Dermatol. 2016;55:563–70. doi:10.1111/ijd.13120.

24. Kebede B, Martindale S, Mengistu B, Kebede B, Mengiste A, H/Kiros F, et al. Integrated morbidity mapping of lymphatic filariasis and podoconiosis cases in 20 co-endemic districts of Ethiopia. PLoS Negl Trop Dis. 2018;12:e0006491. doi:10.1371/journal.pntd.0006491.

25. Deribe K, Cano J, Trueba ML, Newport MJ, Davey G. Global epidemiology of podoconiosis: A systematic review. PLoS Negl Trop Dis. 2018;12:e0006324. doi:10.1371/journal.pntd.0006324.

26. Schulze H, Nacke M, Gutenbrunner C, Hadamitzky C. Worldwide assessment of healthcare personnel dealing with lymphoedema. Health Econ Rev. 2018;8:10. doi:10.1186/s13561-018-0194-6.

27. Managing morbidity and preventing disability in the Global Programme to Eliminate Lymphatic Filariasis: WHO position statement. Wkly Epidemiol Rec. 2011;86(51-52):581–5.

147

148

Chapter 5 Factors associated with Wuchereria bancrofti microfilaremia in an endemic area of Mali

Dolo H, Coulibaly YI, Kelly-Hope L, Konate S, Dembele B, Coulibaly SY, Sanogo D,

Soumaoro L, Coulibaly ME, Doumbia SS, Diallo AA, Traore SF, Colebunders R, Nutman TB,

Klion AD. Factors associated with Wuchereria bancrofti microfilaremia in an endemic area of Mali. Am J Trop Med Hyg. 2018 Jun;98(6):1782-1787.

149

Abstract

Background: Although Wuchereria bancrofti (W. bancrofti), the causative agent of lymphatic filariasis, is endemic throughout Mali, the prevalence of W. bancrofti microfilaremia (Mf) can vary widely between villages despite similar prevalence of infection as assessed by circulating antigen.

This study aimed to determine the effect of spatial clustering, co-incident infection with

Mansonella perstans (M. perstans), bednet use and occupation on W. bancrofti prevalence and Mf levels within a single transmission area.

Methods: To examine this variation, cross-sectional data obtained during screening prior to an interventional study in two neighboring villages in Mali were analyzed.

Results: The overall prevalence of W. bancrofti, as assessed by W. bancrofti Cag (circulating antigen), was 50.3% among 373 participants, aged 14-65. W. bancrofti Mf-positive and negative individuals appeared randomly distributed across the two villages (Moran’s I spatial statistic = -

0.01, Z score =0.1, P>0.05). Among the 187 subjects positive for W. bancrofti CAg, 117 (62.5%) had detectable Mansonella perstans microfilaremia (M. perstans Mf) and 64 (34.2%) had detectable W. bancrofti microfilaremia. The prevalence of M. perstans microfilaremia was 73.4% in the W. bancrofti Mf-positive group (as compared to 56.9% in the W. bancrofti Mf-negative group; p=0.01), and median W. bancrofti Mf load was increased in co-infected subjects (267Mf/ml vs 100 Mf/ml; p<0.001). In multivariate analysis, village of residence, M. perstans Mf positivity and gender were significantly associated with W. bancrofti Mf positivity. After controlling for age, gender and village of residence, the odds of being W. bancrofti Mf positive was 2.67 times higher in M. perstans positive individuals (95% CI [1.42-5.01]).

Conclusion: Given the geographical overlap between M. perstans and W. bancrofti in Africa, a better understanding of the distribution and prevalence of M. perstans could assist national LF control programs in predicting areas of high W. bancrofti mf prevalence that may require closer surveillance.

150

5.1. Background

Lymphatic filariasis (LF) is a chronic neglected tropical disease that is endemic in 72 countries worldwide, including Mali [1,2]. LF is associated with significant morbidity due primarily to lymphedema and hydrocele [1,3] and has been targeted by the World Health Organization

(WHO) and its partners for elimination by 2020 [1]. The strategies for elimination are based on two pillars, community-based mass drug administration (MDA) to interrupt transmission through clearance of microfilaremia over the lifespan of the adult worms (~ 6 years) and morbidity management (hydrocele and lymphedema) [1]. Known risk factors for Wuchereria bancrofti (W. bancrofti ) infection include environmental [2,4], sociocultural and demographic

(age, sex) factors [5,6]. For example, in Mali, the prevalence of Wuchereria bancrofti circulating antigen (W. bancrofti Cag), a marker of active infection, ranged from 18.6% in the

South to 1% in the North, the latter being desertous and thereby ecologically less suitable for

W. bancrofti transmission [7]. These factors affect endemicity and thus impact control programs.

Microfilaremia (Mf) is the main factor that drives the endemicity level in a given area and can be used as a surrogate for transmission assessment before and during MDA. By definition, if microfilariae (mf) are found in the peripheral blood, fertile adults must be viable, and drug distribution must continue if transmission is to be interrupted [6,8]. Factors other than drug coverage may have an impact on W. bancrofti Mf load and thereby alter the efficacy of MDA at the individual or community level. These factors include spatial clustering, co-incident infection with other organisms, bednet use and occupation [9–11]. To determine the effect of these factors on W. bancrofti prevalence and Mf levels within a single transmission area, data obtained prior to an interventional study [12] in two neighboring villages (Tieneguebougou and Bougoudiana), were compared.

151

5.2. Material and methods

Study sites

The study was conducted in the villages of Tieneguebougou and Bougoudiana, ~105 km northwest of Bamako, Mali (Figure 5-1). These villages are not endemic for onchocerciasis.

They are in a northern savannah area with an average annual rainfall of 589.4 mm. The total population at the time of the data collection was 1945 for Tieneguebougou and 991 for

Bougoudiana.

152

Figure 5-1 Map of Koulikoro district with the two study villages indicated by black dots The inset shows the location of the district (depicted by a black square) within the country of Mali. Ethical considerations

153

The study was approved by both the ethical review committee of the Faculty of Medicine,

Pharmacy, and Dentistry at the University of Bamako (Bamako, Mali) and the Institutional

Review Board of the National Institutes of Allergy and Infectious Diseases (Bethesda,

Maryland). Before the launch of the study, community permission was obtained from village elders after a series of explanatory meetings. Individual written informed consent was obtained from all subjects (or from legal guardians for subjects less than 18 years of age).

Study design

A cross-sectional study design was used to obtain data as part of the screening for participation in an interventional study of MDA dosing regimens (NCT00339417) [12]. All study activities were conducted at the primary school in Tieneguebougou or in 2 rooms provided by the chief of the village in Bougoudiana. Consenting participants underwent an extensive history and physical examination and blood collection. Data collected included demographics, related signs and symptoms of W. bancrofti infection, previous participation in MDA campaigns and bednet utilization.

Parasitological testing

W. bancrofti infection was defined by the presence of W. bancrofti Mf on a calibrated thick smear of blood collected between 10 pm and 2 am [13] or a positive test for circulating W. bancrofti antigen (CAg) (TropBio, Townsville, Australia) by ELISA [14]. For each participant,

3 Giemsa-stained slides, each containing 20 µl of whole blood collected at night, were examined for the presence of Mf. Counting of W. bancrofti and M. perstans Mf was done microscopically at 40X magnification. Speciation was confirmed based on size and morphology at 100X magnification under oil-immersion. Each slide was read by two independent readers. A third reader was used to resolve discrepancies of ≥ 10%. The final parasite load reported for each participant was the average of the Mf counts obtained. W.

154 bancrofti CAg was detected in serum by ELISA (TropBIO, Townsville, Australia) based on the manufacturer’s protocol. A value of >32 Units/ml was considered positive.

Mapping and spatial analysis

The geographical coordinates (latitude and longitude) of each participant’s household were determined using a GPS device. To examine the magnitude and geographical distribution of

W. bancrofti Mf load throughout the two villages, the household coordinates and all related data were compiled in a database and mapped using the geographic information system (GIS) software ArcGIS (ESRI 9.2, Redlands, CA). Spatial analysis of W. bancrofti load was examined using ArcGIS Spatial Analyst and Statistics tools (ESRI, Redland, CA).

Statistical analysis

Unless stated otherwise, medians were used to describe central tendency. Differences between groups were assessed using the Mann Whitney U test. Bivariate analyses were performed to explore the associations between W. bancrofti infection and each of the other variables including M. perstans infection, prior participation in MDA, the use of bednets, age, sex and villages of residence. Logistic regression was then performed to control for confounding factors. In this logistic regression, W. bancrofti Mf infection was the outcome of measure and

M. perstans infection primary risk factor with age, gender and village of residence as secondary cofactors. Spearman correlation was used to measure the relationship between two continuous variables. The Moran’s I statistic was used to test for spatial autocorrelation patterns i.e. clustered, dispersed, random.

155

5.3.Results

Of the 372 individuals included in the analyses, a total of 187 (50.27%) were W. bancrofti - positive based on W. bancrofti CAg testing. W. bancrofti Mf were detectable in blood from 64 of the 187 CAg positive subjects (34.22%) (Table 5-1). There were few differences between the CAg positive and negative groups, with the exception of age (45.5 years in the CAg positive group vs 29 years in the CAg negative group, p=0.007) (Table 5-1) and M. perstans infection prevalence (62.56% in the CAg positive group vs 56.9% in the CAg negative group, p=0.0002).

The prevalence of M. perstans microfilaremia was also increased in the W. bancrofti Mf+ group

(73.4%) compared to the W. bancrofti Mf- group (56.9%) (p=0.001, Fisher’s exact test) (Table

5-1). In contrast, gender distribution, median age, participation in either of the two previous

MDA campaigns in the study villages and bednet use were comparable between W. bancrofti

Mf+ and W. bancrofti Mf- individuals (Table 5-1).

156

Table 5-1 Characteristics of the study population

Wb-infected (CAg+) Wb-uninfected (CAg-) (n=187) (n=185)

Mf+ Mf- p values** CAg- p values† (n=64) (n=123) (n=185)

Median Age (range) 45.5 (14-64) 41 (14-65) 0.33 29 (14-77) 0.007 Male 47 (73.4%) 76 (61. 8%) 0.07 112 (60.5%) 0.17 Tieneguebougou 45 (70.3%) 62 (50.4%) 0.01 87 (47%) 0.06 Bednet owner 27 (42.2%) 44 (35.8%) 0.2 65 (35.1%) 0.32 Antifilarial drug use* 30 (46.9%) 69 (56.1%) 0.1 99 (53.5%) 0.49 M. perstans positive 47 (73.4%) 70 (56.9%) 0.01 79 (42.7%) <0.001

*Albendazole and ivermectin treatment during at least one of the 2 prior MDA

** Wb mf positive vs. mf negative

† Wb-infected (CAg+) vs. Wb-uninfected (CAg-) subjects

Mf: microfilariae

Wb CAg: Wuchereria bancrofti circulating antigen

157

In univariate and multivariate analysis, M. perstans status and village of residence were identified as significant risk factors for the presence of W. bancrofti Mf. Not only was W. bancrofti Mf load significantly increased in the W. bancrofti +M. perstans+ co-infected group as compared to the W. bancrofti +M. perstans- mono-infected group (p=0.0002, Mann Whitney

U test) (Figure 5-2), but there was a significant positive correlation between W. bancrofti and

M. perstans Mf levels in co-infected subjects (p<0.001, r=0.25, Spearman correlation) (Figure

5-3). The geometric mean W. bancrofti Mf level in Tieneguebougou (2.45 mf/mL; 95 % CI

[1.45; 3.77]) was increased compared to that in Bougoudiana (0.17 mf/mL; 95% CI [0.34-

1.15]) (p<0.001). In contrast, the geometric mean M. perstans levels were comparable in the two villages (11.11 mf/mL; 95 % CI [7.33; 16.59]) in Tieneguebougou and 12 mf/mL; 95 %

CI [7.91; 17.96] in Bougoudiana) (p=0.76). After controlling for age, sex and village of residence, the odds of being W. bancrofti Mf positive was ~3 times greater if M. perstans Mf positivity was also present (OR =2.67 95% CI [1.62-5.36]) (Table 5-2).

158

Figure 5-2 Wuchereria bancrofti load among Mansonella perstans negative and positive subjects

Gray dot: Wuchereria bancrofti microfilaremia for each M. perstans tested negative

Black dot: Wuchereria bancrofti microfilaremia for each M. perstans tested positive

Figure 5-3 Correlation between M. perstans and W. bancrofti microfilaremia loads within study subjects

159

Table 5-2 Risk factors associated with Wuchereria bancrofti microfilaremia in Tieneguebougou and Bougoudiana

Univariate Analysis Multivariate analysis Variables Category Crude OR [95 % CI] p values Adjusted OR [95 % CI] p values M. perstans negative Ref. Co- infection M. perstans positive 2.95 [1.62-5.36] < 0.001 2.67 [1.42-5.01] 0.0021 ≥38 years Ref. Age < 38 years 1.80 [1.03-3.13] 0.035 1.84 [0.99-3.42] 0.0522 Male Ref. Sex Female 1.76 [0.96-3.21] 0.061 0.47 [0.24-0.91] 0.0257 Tieneguebougou Ref. Village Bougoudiana 2.52 [1.41-4.51] 0.001 2.69 [1.48- 4.91] 0.0012 No bednet owner Ref. Bednet owner 1.33[0.76-2.30] 0.304 Protection measure No Antifilarial drug use* Ref. Antifilarial drug use* 0.73[0.42-1.26] 0.263

*Albendazole and ivermectin treatment during at least one of the 2 prior MDA

160

Despite comparable infection rates (as assessed by CAg positivity) in Tieneguebougou and

Bougoudiana 55.4% (107/195) and 44.9% (80/178), respectively; p=0.05 Fisher’s exact test), village of residence was also significantly associated with W. bancrofti Mf status (OR=2.52

[1.41-4.51]). This association was not due to differences in M. perstans microfilaremia, age, gender, or bednet use between the 2 study villages (Table 5-3). Moreover, there was a relatively even distribution of W. bancrofti Mf positivity across the 2 villages studied with 23.2 %

(45/149) versus 10.7% (19/159) W. bancrofti Mf positive respectively, and geospatial analyses failed to detect any systematic spatial association between Mf-positive and Mf-negative individuals (Moran’s I spatial statistic (MI= -0.01, Z score =0.1, p>0.05) (Figure 5-4).

Although antifilarial drug use was not identified as a risk factor for W. bancrofti Mf positivity in the univariate or multivariate analyses, there was a small but statistically significantly higher frequency of antifilarial drug use in Bougoudiana (59.5% vs 47.4% in Tieneguebougou; p=0.02) (Table 5-3).

161

Figure 5-4 Geographic distribution of Wuchereria bancrofti antigen and Mansonella perstans

Panel 2A: Spatial distribution of Wuchereria bancrofti antigen with gray dot the positive and white dot the negative,

Panel 2 B: Spatial distribution of Wuchereria bancrofti microfilaremia with gray dot the positive and white dot the negative,

Panel 2 C: Spatial distribution of Mansonella perstans microfilaremia with gray dot the positive and white dot the negative

162 Table 5-3 Risk factors distribution by village

Tieneguebougou Bougoudiana p values (n=194) (n=178)

Median Age (range) 37 (14-65) 38.5 (14-65) 0.45†

Male 126 (64.9%) 109 (61.2%) 0.51††

Bednet owner 65 (33.5%) 71 (39.8%) 0.23††

Antifilarial drug use* 92 (47.4%) 106 (59.5%) 0.02††

M. perstans positive 101 (52.1%) 96 (53.9%) 0.75††

*Albendazole and ivermectin treatment during at least one of the 2 prior MDA

† Mann-Whitney U Test

† † Fishers exact test

163 5.4. Discussion

Understanding the factors that affect W. bancrofti microfilaremia at the individual and local levels can help identify regions within an endemic district that are likely to have a higher local transmission, which in turn may require closer surveillance. The present study examined some of these factors in two neighboring villages in an area endemic for W. bancrofti with an overall

CAg prevalence of >50%. Despite similar infection prevalences (rates of W. bancrofti CAg positivity) between the two villages, W. bancrofti microfilarial prevalences were significantly different, suggesting a role for factors other than ecology in W. bancrofti Mf prevalence in this setting. Whereas village of residence, M. perstans Mf positivity and age >38 years were significantly associated with W. bancrofti mf positivity in univariate analysis, only M. perstans microfilaremia and village of residence remained significant in a multivariate analysis. Gender was not a significant risk factor for W. bancrofti Mf positivity in an univariate analysis, but proved significant in the multivariate analysis, consistent with prior data demonstrating an increased prevalence of microfilaremia in males [15,16].

An association between W. bancrofti infection and M. perstans microfilaremia at the individual level was reported previously in a different region of Mali [17], but was not seen in a similar study in Uganda [4]. The reason for this discrepancy may be the presence of different strains of M. perstans in different parts of Africa, as was suggested in studies from Kenya [18,19]. In fact, the Uganda M. perstans strains appear to have lost the Wolbachia endosymbiont whereas in Mali, Gabon and Cameroon, M. perstans has been demonstrated to contain Wolbachia, both directly by PCR and indirectly as evidenced by treatment response to doxycycline [20,21].

M. perstans down-regulates the host's immune system as it is the case with Wuchereria bancrofti. Moreover, the two parasites are carrying the endosymbiont Wolbachia despite having different vectors [22]. Several biologic and climatic factors were shown to be significantly linked with M. perstans infection intensities as it is the case for W. bancrofti. In

164 Uganda, the level of co-infection with W. bancrofti was approximatively 0.3%, due to limited geographic overlap. However, where the two infections did overlap geographically but in Mali the overlap is common in all the region supported by the logic that the two vectors do co-exist due to ecological suitability and they all have preference in biting human in these areas [4].

In the present study, W. bancrofti Mf load was increased in subjects with concomitant M. perstans infection and correlated with M. perstans Mf load at the individual level. A positive association between M. perstans and W. bancrofti Mf densities was also reported in the

Ugandan study.4 Similar relationships have been seen in other filarial co-infections, including loiasis and onchocerciasis [23], suggesting common regulatory mechanisms controlling microfilarial numbers between species.

Since both M. perstans and W. bancrofti are spread by insect vectors that bite unprotected skin, inhabitants of endemic areas who engage in activities that put them at increased risk for vector bites are at high risk of acquiring both W. bancrofti and M. perstans infection [4]. This could lead to clustering of an infection, as has been described in LF [18]. Although Moran’s I spatial statistic failed to detect significant spatial correlation or clustering between M. perstans and W. bancrofti in this study, this could have been due to the relatively small sample size.

Surprisingly, neither W. bancrofti infection rates nor Mf density were significantly reduced by prior MDA-based antifilarial drug treatment or bednet use, which has been shown to synergize with MDA in eliminating LF [17]. Nevertheless, the significantly increased history of MDA participation in Bougoudiana as compared to Tieneguebougou is consistent with attributing a role for antifilarial drug therapy in reducing W. bancrofti Mf levels at the community level.

The lack of significance in the univariate and multivariate analysis may be due to the relatively small impact of two MDA rounds on LF microfilaremia, as observed in a previous study in a highly endemic area in Mali where as many as 6 MDA rounds were required to decrease LF microfilaremia to the recommended threshold levels for elimination [24].

165 5.5. Conclusion

In conclusion, in multivariate analysis, W. bancrofti microfilaremia was associated with the village of residence, the presence of M. perstans microfilaremia and gender in two neighboring rural villages in Mali. Given the geographical overlap between M. perstans and W. bancrofti in Africa [18], a better understanding of the distribution and prevalence of M. perstans could assist national LF control programs in predicting areas of high W. bancrofti mf prevalence that may require closer surveillance.

166 5.6. References

1. world Health organization. WHO | Lymphatic filariasis. WHO 2016;

2. Cano J, Rebollo MP, Golding N, et al. The global distribution and transmission limits of lymphatic filariasis: past and present. Parasit. Vectors 2014; 7:466.

3. Stanton MC, Best A, Cliffe M, et al. Situational analysis of lymphatic filariasis morbidity in Ahanta West District of Ghana. Trop. Med. Int. Health 2016; 21:236–244.

4. Stensgaard A-S, Vounatsou P, Onapa AW, et al. Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria. PLoS Negl. Trop. Dis. 2016; 10:e0004319.

5. Chesnais CB, Missamou F, Pion SD, et al. A case study of risk factors for lymphatic filariasis in the Republic of Congo. Parasit. Vectors 2014; 7:300.

6. Boyd A, Won KY, McClintock SK, et al. A community-based study of factors associated with continuing transmission of lymphatic filariasis in Leogane, Haiti. PLoS Negl. Trop. Dis. 2010; 4:e640.

7. Dembélé M, Bamani S, Dembélé R, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl. Trop. Dis. 2012; 6:e1574.

8. Richards FO, Eigege A, Miri ES, et al. Epidemiological and entomological evaluations after six years or more of mass drug administration for lymphatic filariasis elimination in Nigeria. PLoS Negl. Trop. Dis. 2011; 5:e1346.

9. Slater H, Michael E. Mapping, bayesian geostatistical analysis and spatial prediction of lymphatic filariasis prevalence in Africa. PLoS One 2013; 8:e71574.

10. Reimer LJ, Thomsen EK, Tisch DJ, et al. Insecticidal bed nets and filariasis transmission in Papua New Guinea. N. Engl. J. Med. 2013; 369:745–753.

11. Kelly-Hope LA, Molyneux DH, Bockarie MJ. Can malaria vector control accelerate the interruption of lymphatic filariasis transmission in Africa; capturing a window of opportunity? Parasit. Vectors 2013; 6:39.

12. Dembele B, Coulibaly YI, Dolo H, et al. Use of high-dose, twice-yearly albendazole and ivermectin to suppress Wuchereria bancrofti microfilarial levels. Clin. Infect. Dis. 2010; 51:1229–1235.

13. Onapa AW, Simonsen PE, Pedersen EM, Okello DO. Lymphatic filariasis in Uganda: baseline investigations in Lira, Soroti and Katakwi districts. Trans. R. Soc. Trop. Med. Hyg. 2001; 95:161–167.

14. Wattal S, Dhariwal AC, Ralhan PK, et al. Evaluation of Og4C3 antigen ELISA as a tool for detection of bancroftian filariasis under lymphatic filariasis elimination programme. J Commun Dis 2007; 39:75–84.

167

15. Grove DI, Valeza FS, Cabrera BD. Bancroftian filariasis in a Philippine village: clinical, parasitological, immunological, and social aspects. Bull. World Health Organ. 1978; 56:975–984.

16. Pani SP, Balakrishnan N, Srividya A, Bundy DA, Grenfell BT. Clinical epidemiology of bancroftian filariasis: effect of age and gender. Trans. R. Soc. Trop. Med. Hyg. 1991; 85:260–264.

17. Keiser PB, Coulibaly YI, Keita F, et al. Clinical characteristics of post-treatment reactions to ivermectin/albendazole for Wuchereria bancrofti in a region co-endemic for Mansonella perstans. Am. J. Trop. Med. Hyg. 2003; 69:331–335.

18. Simonsen PE, Onapa AW, Asio SM. Mansonella perstans filariasis in Africa. Acta Trop. 2011; 120 Suppl 1:S109-20.

19. Uttah E, Ibeh DC. Multiple filarial species microfilaraemia: a comparative study of areas with endemic and sporadic onchocerciasis. J. Vector Borne Dis. 2011; 48:197–204.

20. Coulibaly YI, Dembele B, Diallo AA, et al. A randomized trial of doxycycline for Mansonella perstans infection. N. Engl. J. Med. 2009; 361:1448–1458.

21. Keiser PB, Coulibaly Y, Kubofcik J, et al. Molecular identification of Wolbachia from the filarial nematode Mansonella perstans. Mol. Biochem. Parasitol. 2008; 160:123–128.

22. Wanji S, Tayong DB, Layland LE, et al. Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin. Parasit. Vectors 2016; 9:311.

23. Pion SDS, Clarke P, Filipe JAN, et al. Co-infection with Onchocerca volvulus and Loa loa microfilariae in central Cameroon: are these two species interacting? Parasitology 2006; 132:843–854.

24. Coulibaly YI, Dembele B, Diallo AA, et al. The Impact of Six Annual Rounds of Mass Drug Administration on Wuchereria bancrofti Infections in Humans and in Mosquitoes in Mali. Am. J. Trop. Med. Hyg. 2015; 93:356–360.

168 Chapter 6 General discussion

169 6.1. General discussion

Given the progress made towards the elimination of onchocerciasis and LF in Africa, there is great interest in stopping mass drug administration (MDA) and entering the elimination and surveillance phase [1,2]. However, as the prevalences and intensities of onchocerciasis and LF have decreased due to MDA, the diagnostic tools used in the mapping, monitoring and evaluation phase of these diseases have become obsolete [3,4]. In the WHO roadmap and the

London declaration on NTD, particular emphasis is placed on the importance of serological tests in deciding whether to discontinue MDA for the elimination of onchocerciasis and LF

[5,6]. The WHO recommends the Ov16 ELISA for onchocerciasis serology, but different

ELISA protocols vary in their performance characteristics [7,8]. Therefore, it is essential to standardize the current available ELISA protocols to measure the levels of exposure to infection with O. volvulus and W. bancrofti.

Sero-diagnostic tools for onchocerciasis and LF elimination

The availability of the Biplex SD Bioline Onchocerciasis/ LF IGg4 Ov16/Wb123 (Biplex) test, which can antibodies against O. volvulus and W. bancrofti antigens simultaneously, has opened new prospects [9,10]. The SD Bioline Biplex test has a (manufacturer)-reported sensitivity of

92– 98% for onchocerciasis and 81–95% for LF. Specificity estimates are 97– 100% and 96–

99%, respectively. [11]. In our study using the SD Bioline Biplex test, all Ov16 antibody- positive subjects were identified in pre-control onchocerciasis meso- and hyperendemic districts [12], and all Ov16 IgG4 antibody-positive children aged ≤ ten years of age in three pre-control onchocerciasis hyperendemic villages in Bakoye and one in Falémé were from previously meso- or hyperendemic areas. The variation in seropositivity status between villages underscores the importance of random selection of villages during onchocerciasis and LF transmission elimination surveys. For LF, there is an online transmission assessment survey tool known as sample size builder (SSB)) for the random selection of school or community

170 cluster in "evaluation units" (one or more health districts depending on the population size)

[13]. For onchocerciasis, there are protocols available to evaluate the transmission of onchocerciasis in hypo-endemic areas where ivermectin was never distributed. These protocols could be used as a basis for the development of a standard protocol using rapid serological tests to investigate whether, in previously meso-and hyperendemic zones, ivermectin distribution can be stopped.

Antibodies against Wb123 appear earlier than circulating filarial antigen (CFA) [14]. In our study using the SD Bioline Biplex test, we observed excellent concordance with the prevalence determined with the CFA antigen test. Therefore, the measurement of IgG4 to Wb123 might be preferable in decisions to stop MDA or during TAS following cessation of MDA, as it allows the measurement of more recent exposure. Within the context of onchocerciasis elimination, new data are needed to re-categorize O. volvulus transmission potentials in sub-regions of countries such as in Mali, where many areas have undergone more than 24 years of CDTi with no or few epidemiological assessments [15]. Where co-endemicity is present, the SD Bioline

Biplex test can be used for elimination mapping, as it can be performed in the field without additional lab processing, in contrast to the Ov16 ELISA [9,10].

Age specific serology profile evaluation of onchocerciasis an LF

Examination of serological profiles in the total population of the previously hyperendemic villages of Kantila, Nioumala and Galé provides information about historical exposure trends to onchocerciasis in Bakoye. Ov16 seroprevalence was very low in ≤14-year olds, with upper

95% confidence limits of less than 2% in 3–6, 7–10 and 11–14-year olds, suggesting pronounced transmission suppression. Four of the 5 seropositive children aged ≤10 years were from the historically highly hyperendemic village of Galé. Seroprevalence increases slowly with age from 15–19, reaching 16% in those aged ≥40 years. These individuals would have been ≥15–20 years old when ivermectin distribution began and, under intense pre-treatment

171 transmission, their age-specific mf prevalence would have been >60% at that time [16]. These results are consistent with those of Paulin in Tanzania [17], indicating that Ov16 seropositivity likely declines over time after prolonged treatment. Our Ov16 serological findings among children aged <10 years, showing that all seven seropositive individuals were found in the

River Gambia and Fálémé, previously onchocerciasis endemic foci [18], indicate that not all

Ov16 seropositive results should be dismissed as false positives. The strength of evidence for the serological threshold of <0.1% in children aged <10 years being indicative of onchocerciasis elimination is low [5]. If 9 or fewer positives are negative by skin snip PCR, then the entire district passes the elimination. Recent modelling work has suggested that a threshold below 2% may be safe for stopping MDA, and that children aged 5–14 years may be most informative [19].

The all-age seroprevalence profiles for Wb123 also increased somewhat with age, reaching 3% in individuals ≥40 years of age but remaining below 2% in those aged <40 years of age. A recent study in Mali found a strong correlation between ICT and Wb123 seropositivity in children aged 6–7 years, suggesting that Wb123 IgG4 prevalence might be preferable in guiding stop-MDA decisions or in TAS following MDA cessation, as it is a better measure of recent exposure than antigen testing [20]. These findings on age-specific seroprevalence suggest that programmes should consider the age group of £ 14 years as their sentinel age of follow up and that rapid serologic tests are useful in measuring recent exposure to the parasites causing onchocerciasis and LF.

Integrated evaluation of transmission elimination of onchocerciasis and LF

The WHO's roadmap for combating NTDs recommends the integration of control activities, including MDA and evaluation, to reduce costs, since these NTDs overlap in many countries and areas as it is the case in Mali [3]. Using the SD Bioline Onchocerciasis/LF IgG4 Rapid

Test, we have shown that it is possible to conduct an integrated transmission assessment and

172 surveillance for onchocerciasis and LF. Although there may be programmatic barriers to the integration of the assessment of LF and onchocerciasis, because LF programs use districts as the treatment unit and combine one or several districts as evaluation unit while onchocerciasis programs are based on foci of transmission and the distance between villages and streams [21], we have shown that the integrated simultaneous evaluation of both onchocerciasis and LF using the SD Bioline Onchocerciasis/LF IgG4 Rapid Test is practical and will reduce overall cost.

Lymphedema epidemiology in post-MDA period

Morbidity management and disability prevention have long been ignored as an essential component of LF programs. Patients suffering from the overwhelming consequences of LF morbidity should have access to sustainable care. Despite the successful implementation of

MDA to eliminate LF as public health problem in many countries, including Mali, morbidity management remains a challenge. There is a need for more active lymphedema case finding to ascertain the true burden of this morbidity. Socio- anthropological studies need to be conducted to break down the socio-cultural barriers of lymphedema declaration and diagnosis. In addition, lymphedema management programmes need to be implemented for each country to meet the goal of LF elimination. The current distribution of ivermectin and albendazole is intended to eliminate transmission but has little effect on clinical morbidities in those already affected [22].

Improvement of the management of lymphedema using doxycycline in the early stages of lymphedema needs to be investigated. We also suggest that the presence of podoconiosis should be investigated in Mali, despite the fact that, in contrast with Rwanda and Ethiopia the geography is not suitable for podoconiosis [23,24].

Mansonella perstans infection in the context of onchocerciasis and LF elimination

Infection with the filarial parasite, Mansonella perstans, is neglected among the neglected tropical diseases, despite being widespread with prevalence of up to 100% in some tropical regions [25–29]. It is also neglected because its pathogenicity is unknown or controversial

173 [25,30]. Studies have shown that M. perstans infection can cause cross-reactions during serological tests for LF [28,31–33]. Moreover, our study in two villages in Mali showed that

M. perstans infection increased the risk of being infected with W. bancrofti by approximatively

3 times after controlling for age, gender and village of residence. We also observed that co- infection was more common than mono-infection. A similar study in Uganda reported a widespread distribution of M. perstans infection and identified important potential risk drivers of this poorly known filarial parasite and its Culicoides vector species, including diurnal temperature range, normalized difference in vegetation index, and cattle densities [34].

However, in the Ugandan study, a very low co-infection prevalence (0.3%) between M. perstans and W. bancrofti was found, compared to the very high co-infection rates ( >70%) in

Mali [34]. Due to the overlapping clinical signs (eosinophilia, subcutaneous swellings, aches, pains and skin rashes in a considerable proportion of patients) of LF and M. perstans and the widespread distribution of the two infections in Mali, more attention should be directed toward

M. perstans infection. To prepare for LF elimination, further assessment of M. perstans prevalence needs to be investigated because of the close similarity of the two parasites and the possible cross-reactions during FTS testing.

174 6.2. Conclusion

In different epidemiological settings, we have demonstrated that it is possible to conduct an integrated transmission assessment of onchocerciasis and LF in co-endemic settings using the

SD Bioline Onchocerciasis/LF IgG4 Rapid Test. Only a few Ov16-positive children were detected in zones known to be meso- and hyperendemic for onchocerciasis before MDA implementation. Ov16 and Wb123 seroprevalence results were consistent with elimination of onchocerciasis transmission (EOT) and the elimination of LF as a Public Health Problem

(EPHP) since MDA was stopped in 2016. The few Ov16-seropositive children should be tested for O.volvulus infection using skin snips and followed.

Lymphedema remains a public health problem in previously LF endemic regions in Mali where transmission has been interrupted. It is imperative that active lymphedema case identification be scaled up in all previously LF endemic regions in Mali and that a MMDP programme at peripheral health system level be implemented to meet the LF elimination goal.

Given the geographical overlap between M. perstans and W. bancrofti in Africa, a better understanding of the distribution and prevalence of M. perstans is important for national onchocerciasis and LF elimination programs, due to the potential impact on the interpretation of serologic testing.

6.3. Research priorities

During the preparatory phase for onchocerciasis and lymphatic filariasis elimination and with regard to our finding and observation we suggest the following research priorities:

- The development and manufacture of standardized and quality-assured ELISA kits with

sensitivity/specificity compatible with measuring (revised) serological thresholds,

- Modelling studies to corroborate these empiric findings in field as well as the determination

of useful threshold for decision making when using Wb123 test for LF.

175 - Study the technical threshold for elimination which is confounded by limited understanding

of O. volvulus transmission dynamics and population biology at low transmission levels

- Develop of guidance on how onchocerciasis elimination surveys should be implemented

and the appropriate spatial unit,

- Develop standard protocols for the surveillance of onchocerciasis an LF by serology to

predict the risk of re-emergence,

- Conduct more operational studies on the possibilities to combine the assessment of

onchocerciasis and LF in co-endemic setting using available evaluation tools

176 6.4. References

1. Tekle AH, Zouré HGM, Noma M, et al. Progress towards onchocerciasis elimination in the participating countries of the African Programme for Onchocerciasis Control: epidemiological evaluation results. Infect Dis Poverty 2016; 5:66.

2. WHO | Progress report on the elimination of human onchocerciasis, 2017–2018. 2018. Available at: http://www.who.int/onchocerciasis/resources/who_wer9347/en/. Accessed 3 December 2018.

3. World Health Organization. Accelerating Work to Overcome the Impact of Neglected Tropical Diseases: A Roadmap for Implementation. 2012. Available at: https://www.who.int/neglected_diseases/NTD_RoadMap_2012_Fullversion.pdf. Accessed 4 October 2019.

4. Dickinson B, KGaA M, Sano M. London declaration on neglected tropical diseases. Sustaining the drive to overcome the global impact of neglected tropical diseases, 5 2013; 5.

5. World Health Organization/Department of Control of Neglected Tropical Diseases. Guidelines for stopping mass drug administration and verifying elimination of human onchocerciasis. Criteria and procedures (Ukety T, Ed.), 44 pp. 2016. Available at: https://apps.who.int/iris/bitstream/handle/10665/204180/9789241510011_eng.pdf;jsessi onid=85ADE0FAAE6368A0802984B4A3810DCF?sequence=1. Accessed 4 October 2019.

6. World Health Organization/Department of Control of Neglected Tropical Diseases. Lymphatic filariasis: monitoring and epidemiological assessment of mass drug administration. A manual for national elimination programmes (Ichimori K, Ed.), 79pp. 2011. Available at: https://www.who.int/lymphatic_filariasis/resources/9789241501484/en/. Accessed 4 October 2019.

7. World Health Organization. Report of the 1st Meeting of the WHO Onchocerciasis Technical Advisory Subgroup. 2017. Available at: https://apps.who.int/iris/bitstream/handle/10665/273705/WHO-CDS-NTD-PCT- 2018.05-eng.pdf. Accessed 4 October 2019.

8. Shott J, Ducker C, Unnasch TR, Mackenzie CD. Establishing quality assured (QA) laboratory support for onchocerciasis elimination in Africa. Int. Health 2018; 10:i33–i39.

9. Steel C, Golden A, Stevens E, et al. Rapid Point-of-Contact Tool for Mapping and Integrated Surveillance of Wuchereria bancrofti and Onchocerca volvulus Infection. Clin. Vaccine Immunol. 2015; 22:896–901.

10. Golden A, Stevens EJ, Yokobe L, et al. A Recombinant Positive Control for Serology Diagnostic Tests Supporting Elimination of Onchocerca volvulus. PLoS Negl. Trop. Dis. 2016; 10:e0004292.

11. SD BIOLINE Onchocerciasis/LF IgG4 Rapid Test - Alere is now Abbott. Available at:

177 https://www.alere.com/en/home/product-details/sd-bioline-onchocerciasis-LF-igg4- rapid-test.html. Accessed 14 August 2019.

12. Traore MO, Sarr MD, Badji A, et al. Proof-of-principle of onchocerciasis elimination with ivermectin treatment in endemic foci in Africa: final results of a study in Mali and Senegal. PLoS Negl. Trop. Dis. 2012; 6:e1825.

13. Health OW. Lymphatic filariasis: monitoring and epidemiological assessment of mass drug administration: A manual for national elimination programmes. World Health Organization: Geneva, Switzerland 2011;

14. Hamlin KL, Moss DM, Priest JW, et al. Longitudinal monitoring of the development of antifilarial antibodies and acquisition of Wuchereria bancrofti in a highly endemic area of Haiti. PLoS Negl. Trop. Dis. 2012; 6:e1941.

15. Dembélé M, Bamani S, Dembélé R, et al. Implementing preventive chemotherapy through an integrated National Neglected Tropical Disease Control Program in Mali. PLoS Negl. Trop. Dis. 2012; 6:e1574.

16. Basáñez MG, Boussinesq M. Population biology of human onchocerciasis. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1999; 354:809–826.

17. Paulin HN, Nshala A, Kalinga A, et al. Evaluation of onchocerciasis transmission in tanzania: preliminary rapid field results in the tukuyu focus, 2015. Am. J. Trop. Med. Hyg. 2017; 97:673–676.

18. Wilson NO, Badara Ly A, Cama VA, et al. Evaluation of Lymphatic Filariasis and Onchocerciasis in Three Senegalese Districts Treated for Onchocerciasis with Ivermectin. PLoS Negl. Trop. Dis. 2016; 10:e0005198.

19. Coffeng LE, Stolk WA, Golden A, de Los Santos T, Domingo GJ, de Vlas SJ. Predictive value of ov16 antibody prevalence in different subpopulations for elimination of african onchocerciasis. Am. J. Epidemiol. 2019; 188:1723–1732.

20. Dolo H, Coulibaly YI, Dembele B, et al. Integrated seroprevalence-based assessment of Wuchereria bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with the SD Bioline Onchocerciasis/LF IgG4 Rapid Test. PLoS Negl. Trop. Dis. 2019; 13:e0007064.

21. WHO A. Conceptual and Operational Framework of Onchocerciasis Elimination with Ivermectin Treatmen. 2010. Available at: https://apps.who.int/iris/bitstream/handle/10665/275466/JAF16.6(II)-eng.pdf? Accessed 21 June 2019.

22. Mathew CG, Bettis AA, Chu BK, et al. The health and economic burden of lymphatic filariasis prior to mass drug administration programmes. Clin. Infect. Dis. 2019;

23. Deribe K, Mbituyumuremyi A, Cano J, et al. Geographical distribution and prevalence of podoconiosis in Rwanda: a cross-sectional country-wide survey. Lancet Glob. Health 2019; 7.

178

24. Sime H, Deribe K, Assefa A, et al. Integrated mapping of lymphatic filariasis and podoconiosis: lessons learnt from Ethiopia. Parasit. Vectors 2014; 7:397.

25. Simonsen PE, Onapa AW, Asio SM. Mansonella perstans filariasis in Africa. Acta Trop. 2011; 120 Suppl 1:S109-20.

26. Bassene H, Sambou M, Fenollar F, et al. High Prevalence of Mansonella perstans Filariasis in Rural Senegal. Am. J. Trop. Med. Hyg. 2015; 93:601–606.

27. Debrah LB, Nausch N, Opoku VS, et al. Epidemiology of Mansonella perstans in the middle belt of Ghana. Parasit. Vectors 2017; 10:15.

28. Wanji S, Tayong DB, Layland LE, et al. Update on the distribution of Mansonella perstans in the southern part of Cameroon: influence of ecological factors and mass drug administration with ivermectin. Parasit. Vectors 2016; 9:311.

29. Korbmacher F, Komlan K, Gantin RG, et al. Mansonella perstans, Onchocerca volvulus and Strongyloides stercoralis infections in rural populations in central and southern Togo. Parasite Epidemiol. Control 2018; 3:77–87.

30. Ritter M, Ndongmo WPC, Njouendou AJ, et al. Mansonella perstans microfilaremic individuals are characterized by enhanced type 2 helper T and regulatory T and B cell subsets and dampened systemic innate and adaptive immune responses. PLoS Negl. Trop. Dis. 2018; 12:e0006184.

31. Wanji S, Amvongo-Adjia N, Koudou B, et al. Cross-Reactivity of Filariais ICT Cards in Areas of Contrasting Endemicity of Loa loa and Mansonella perstans in Cameroon: Implications for Shrinking of the Lymphatic Filariasis Map in the Central African Region. PLoS Negl. Trop. Dis. 2015; 9:e0004184.

32. Bloch P, Simonsen PE, Weiss N, Nutman TB. The significance of guinea worm infection in the immunological diagnosis of onchocerciasis and bancroftian filariasis. Trans R Soc Trop Med Hyg 1998; 92:518–21.

33. Uttah E, Ibeh DC. Multiple filarial species microfilaraemia: a comparative study of areas with endemic and sporadic onchocerciasis. J. Vector Borne Dis. 2011; 48:197–204.

34. Stensgaard A-S, Vounatsou P, Onapa AW, et al. Ecological Drivers of Mansonella perstans Infection in Uganda and Patterns of Co-endemicity with Lymphatic Filariasis and Malaria. PLoS Negl. Trop. Dis. 2016; 10:e0004319.

179 Curriculum vitae

I , Housseini Dolo graduated as medical doctor from the Faculty of Medicine, Pharmacy and

Odonto Stomatology of Bamako, University of Bamako, Mali in 2007 and obtained a Master of Science in Public Health, Diseases Control in the option of tropical diseases from Institute of

Tropical Medicine in Antwerp, Belgium in 2014.

I worked for several years (2007 to 2013) as a clinician and junior scientist at the National

Institute of Health funded projects in collaboration with the University of Science Technique and Technologies of Bamako at the Faculty of Medicine and Odonto Stomatology of Bamako.

During this period, I was the clinical coordinator of several field studies in Mali.

In 2015, I obtained a scholarship from Islamic development Bank to register in a PhD

Programme in Medical Sciences at the University of Antwerp in Belgium and was appointed as Associate Professor in Epidemiology in the Faculty of Medicine and Odonto Stomatology.

In 2018, I won two awards, first from the Michel Dumas Scholarship to work on epilepsy by powering the community health workers on the identification and management of person with epilepsy in collaboration with the university of Limoges and the second was research fellowship grant from the international society of infectious diseases (ISID) to conduct modelling studies in collaboration with Imperial college London and the Royal Veterinary college London.

In 2019, I was appointed as epidemiologist in National Onchocerciasis control programme.

180 Publications used for this Thesis

Chapter 2

1. Dolo H, Coulibaly YI, Dembele B, Guindo B, Coulibaly SY, Dicko I, Doumbia SS, Dembele

M, Traore MO, Goita S, Dolo M, Soumaoro L, Coulibaly ME, Diallo AA, Diarra D, Zhang Y,

Colebunders R, Nutman TB. Integrated seroprevalence-based assessment of Wuchereria

bancrofti and Onchocerca volvulus in two lymphatic filariasis evaluation units of Mali with

the SD Bioline Onchocerciasis/LF IgG4 Rapid Test. PLoS Negl Trop Dis. 2019 Jan

30;13(1):e0007064. doi: 10.1371/journal.pntd.0007064. eCollection 2019 Jan. PubMed PMID:

30699120; PubMed Central PMCID: PMC6370230.

2. Chapter 3

Dolo H, Coulibaly YI, Sow M, Dembélé M, Doumbia SS, Coulibaly SY, Sangare MB, Dicko

I, Diallo AA, Soumaoro L, Coulibaly ME, Diarra D, Colebunders R, Nutman TB, Walker M,

Basáñez MG. Serological Evaluation of Onchocerciasis and Lymphatic Filariasis

Elimination in the Bakoye and Falémé foci, Mali. Clin Infect Dis. 2020 Mar 24. pii: ciaa318.

doi: 10.1093/cid/ciaa318. [Epub ahead of print] PubMed PMID: 32206773.

3. Chapter 4

Dolo H, Coulibaly YI, Konipo FN, Coulibaly SY, Doumbia SS, Sangare MS, Soumaoro L,

Coulibaly ME, Diallo AA, Diarra Y, Sangare M, Doumbia S, Colebunders R, Thomas B.

Nutman TB. Lymphedema in three previously Wuchereria bancrofti-endemic health

districts in Mali after cessation of mass drug administration. Under review

4. Chapter 5

Dolo H, Coulibaly YI, Kelly-Hope L, Konate S, Dembele B, Coulibaly SY, Sanogo D,

Soumaoro L, Coulibaly ME, Doumbia SS, Diallo AA, Traore SF, Colebunders R, Nutman TB,

Klion AD. Factors Associated with Wuchereria bancrofti Microfilaremia in an Endemic

Area of Mali. Am J Trop Med Hyg. 2018 Jun;98(6):1782-1787. doi: 10.4269/ajtmh.17-0902.

Epub 2018 Apr 26. PubMed PMID: 29714157; PubMed Central PMCID: PMC6086186.

181