PhD defence Kristien Cloots

Supervisors

• Prof. dr. Epco Hasker (ITM)

• Prof. dr. Sake J. de Vlas (Erasmus University, Rotterdam)

• Prof. dr. Marleen Boelaert (†, ITM)

• Dr. Epke A. Le Rutte (Erasmus University, Rotterdam)

Summary

Visceral leishmaniasis (VL) is a parasitic infectious disease transmitted by sand flies and fatal if not treated timely. It is targeted for elimination as a public health problem in the Indian subcontinent (ISC). The overarching aim of this thesis is to explore how the surveillance system for VL should be optimized towards the post-elimination phase in the ISC.

Chapter 1 provides a general overview of leishmaniasis, before redirecting the focus to VL in the Indian subcontinent. We describe the clinical picture of both VL and its cutaneous sequel called post-kala-azar dermal leishmaniasis (PKDL), the diagnostic and treatment modalities, and current disease control and surveillance activities. We discuss how in the early 2000s, India, Nepal and Bangladesh together accounted for 70% of all global cases, with an estimated 160 to 320 thousand new VL cases each year in the ISC, and how this resulted in a regional initiative to eliminate VL as a public health problem, defined as an annual incidence < 1/10,000 population in all endemic (sub-)districts, with a deadline originally set at 2015 but currently revised to 2026. We summarize the progress made to date, and how Bangladesh was the first country to be certified for elimination of VL as a public health problem in 2023. In addition, we illustrate how close India and Nepal are to reaching this goal as well, and describe which key challenges still threaten a sustainable elimination in the long run. These include incomplete coverage of the surveillance system, limitations of the available tools, and waning of political commitment in a postelimination phase as the most important ones.

The research in this thesis addresses the following three research questions:

1) How has the epidemiology of VL changed since the start of the elimination initiative in the Indian subcontinent?

2) To what extent do specific infectious subgroups require an additional focus for VL surveillance?

3) What indicators should be monitored in the post-elimination phase?

In Chapter 2, we assess to what extent the observed decrease in reported VL cases since the start of the VL elimination initiative is mirrored by a decrease in transmission as well. We carried out a repeat serological survey using the Direct Agglutination Test (DAT) as a proxy for asymptomatically infected individuals, and compared age-group specific seroprevalence data from 2006 with those collected from the same Nepalese villages in 2016. Our results show that the risk of being infected has indeed decreased across all age groups and villages since the start of the elimination initiative, and that children born in the last 10 years have barely been exposed to Leishmania at all.

In Chapter 3, we challenge the official extent of endemicity in Nepal in response to the observed geographical expansion of reported VL cases in the country in recent years. We carried out a transmission assessment in seven presumably non-endemic districts in West-Nepal. Combining epidemiological, serological and entomological data, we conclude that local transmission in these districts is present, and that a thorough revision of the extent of VL endemicity in Nepal is crucial in order to achieve and sustain elimination.

In Chapter 4, we aimed to verify to what extent post-kala azar dermal leishmaniasis (PKDL) and VL-HIV patients contribute to transmission. By preforming a multivariate analysis on routine data from India, we found that both presence of PKDL and VL-HIV patients was associated with a twofold increase in VL incidence at the village level. These findings highlight the importance of both infectious subgroups in Leishmania transmission, and emphasize that both groups should receive a more targeted focus within the VL elimination initiative.

Chapter 5 reports on the results of a pilot study of a novel microbiopsy device that could potentially provide an alternative tool for assessing infectiousness of asymptomatically infected individuals, and facilitate large-scale studies to assess their role in transmission – if any. We sampled the skin of a total of 201 participants, including VL and PKDL patients, as well as asymptomatically infected individuals, endemic controls, and non-endemic controls. We discuss how skin parasite load measured with qPCR on the microbiopsy was clearly correlated with disease status, while also reporting on four positive skin results from asymptomatically infected individuals. We conclude that the microbiopsy device seems promising to help address the question of infectiousness of asymptomatically infected individuals once validated by xenodiagnosis studies. In addition, we highlight that it could be a less invasive tool to collect skin samples for diagnosis of PKDL.

Chapter 6 explores whether female VL patients require additional focus to reach and detect, as they consistently comprise a minority of reported VL cases. By comparing hospital-based data and population-based data from India and Nepal, we conclude that the male preponderance in reported data is not explained by differences in health-care seeking behavior. While differences in exposure between both sexes cannot be excluded, we describe how the increased risk of progression from infection to disease seems to put mainly post-pubertal males at a disadvantage, suggesting a physiological difference between both sexes, with sex hormones likely playing a role. We describe how the sex-specific difference in treatment outcomes should also be considered when evaluating new drug regimens in the future.

In Chapter 7, we explore whether the existing algorithm used to diagnose VL in the ISC is still valid in the current low-endemic setting, as we can expect the proportion of false-positive results to increase with decreasing disease prevalence. We describe how we were able to confirm 95% (70/74) of VL diagnoses by molecular confirmation, concluding that at present the diagnostic algorithm is still valid. In parallel, we describe how our findings can be partly explained by the very high specificity of the diagnostic test, which opens possibilities to increase testing among chronic fever patients before the development of VL-specific signs such as splenomegaly. With incidence further declining, even a very specific test may start to yield unacceptably high proportions of false positive results. We conclude that continued monitoring of the accuracy of the diagnostic algorithm will be required in the post-elimination phase.

In Chapter 8, we explore to what extent serological surveys are able to distinguish between areas with and without ongoing Leishmania transmission. We describe how we analyzed venous blood samples from participants from seven villages in India with three different serological tests in order to identify the best marker for transmission. We describe how the rK39 ELISA seems best fit for this purpose, though further validation is required. We discuss the need to translate these findings into practical policy recommendations, while also highlighting the need to explore possibilities for integration with surveillance of other diseases in order to improve sustainability of serological surveys in the long run.

Finally, in Chapter 9, we provide and discuss the answers to the three research questions. In addition, we explore cutaneous leishmaniasis (CL) as an emerging threat to the VL elimination initiative in Nepal, before formulating our conclusions and recommendations:

Conclusions

1. Although transmission has decreased since the start of the VL elimination initiative in the Indian subcontinent in 2005, VL seems to have spread geographically towards new areas.

2. Both PKDL and VL-HIV co-infected patients require a strengthened focus in order to reach and sustain VL elimination as a public health problem in the Indian subcontinent.

3. The low prevalence setting of the post-elimination phase in the Indian subcontinent would specifically benefit from monitoring the accuracy of the diagnostic algorithm as well as monitoring of transmission.

4. Cutaneous leishmaniasis (CL) should be considered as a potential threat to the VL elimination initiative in the ISC.

Recommendations for policy

1. Endemicity should be seen as a dynamic phenomenon, requiring availability of diagnostic tests and geographical coverage of the surveillance system beyond the known endemic areas.

2. Stronger collaborations with national programs for other diseases than VL are required, to improve VL-HIV detection and reporting, and explore integrated disease control activities.

3. Strong collaborations need to be established with dermatologists in order to detect and report PKDL and CL cases, and the disease reporting system needs to be adapted to allow for this.

4. All reported VL patients should be molecularly confirmed to monitor the accuracy of the diagnostic algorithm.

Recommendations for scientific research

1. Implementation research is needed to identify strategies to actively follow-up ex-VL patients to detect PKDL in a feasible way within the existing health infrastructure.

2. Pragmatic methodologies for the use of integrated serosurveillance should be established, and opportunities for integration with existing surveys should be explored.

3. The question whether the same L. donovani strain can cause both VL and CL should be answered.