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This thesis focuses on the development of protozoan parasites from Leishmania donovani complex in their insect vectors and summarizes results of five parts of the project I participated in during my Ph.D. studies. Sand flies of genera Phlebotomus and Lutzomyia are the only proven vectors of leishmaniasis, however, the role of alternative vectors, like ticks, fleas and biting midges is frequently discussed in the literature. In this work, we showed that Eurasian species of biting midge Culicoides nubeculosus does not support late stage infections of L. major and L. infantum. We also demonstrated that microscopical observation of Leishmania promastigotes in the digestive tract of bloodfeeding arthropods remains a crucial method for any conclusion about the vector competence of the suspected insect. In the second part of our study were compared the life-cycle parameters and vector competence of two Ethiopian P. orientalis colonies for L. donovani. Marked differences between colonies were found in life-cycle parametes, however, molecular analyses did not reveal any genetic differences. Experimental infections showed that both P. orientalis colonies are very susceptible to L. donovani infection and even the lowest infective dose tested (2 × 103 promastigotes/ml; corresponding to 1-2 promastigotes) was...

Visceral leishmaniasis (VL) or kala-azar is a parasitic infectious disease that is fatal if left untreated. Two types of Leishmania species are causal agents of VL: Leishmania infantum and Leishmania donovani. VL caused by L.infantum is a zoonosis and is endemic in countries around the Mediterranean basin and in Latin-America. VL caused by L. donovani is assumed to be an anthroponosis and is endemic in East-Africa and the Indian subcontinent.<p><p>VL is considered as a major public health problem in the Indian subcontinent and the annual case load of VL in this focus is represents around 80% of the global burden. In Nepal, a quarter of the country’s population is estimated to be at risk of this disease. The disease in the ISC is caused by L. donovani, which is transmitted from man to man by the bite of the sandfly Phlebotomus argentipes. VL occurs predominantly among the poorest of the poor. Since 2005, the governments of Bangladesh, India and Nepal have been engaged in a collaborative effort to eliminate VL from the region. The strategies to control the disease include early diagnosis and treatment, along with vector control measures, effective disease surveillance, social mobilization and partnership building, and clinical and operational research. In recent years, considerable efforts were made within the elimination initiative. Still, important gaps remain in the understanding of the VL epidemiology, and impact as well as on the best approach to case management or vector control. These knowledge gaps may affect the success of the ongoing VL elimination initiative and make it difficult to meet the set target of bringing the incidence down to less than 1 case per 10,000 by 2015. With this background we focused on some of the knowledge gaps; we wanted to generate evidence and offer sound recommendations for policy makers to underpin the ongoing VL elimination initiative in the Indian subcontinent in general and in Nepal in particular. <p><p>We have - for the first time- described the epidemiology of L. donovani infection in high transmission areas in Nepal. The sero-prevalence of L. donovani infection was 9% in these communities, but there was wide variation between endemic villages (5-15%). The seroprevalence rates remain however substantially lower than those observed in a parallel study in the neighbouring districts in Bihar, India. In our study 39% of individuals who live together in a house with at least one recent VL case were serologically (DAT) positive compared to 9% in the overall study population in the same endemic region. This pattern suggests that untreated VL cases are the main source of transmission and sharing the same household is an important risk factor for L. donovani infection. Therefore, the VL elimination campaign recently initiated an active case detection strategy including the search of active cases of VL and post-kala-azar dermal leishmaniasis (PKDL).<p><p>Generally the risk factors for VL are linked to precarious housing conditions and an environment that provides excellent breeding sites for the sandfly vector.VL has thus been largely considered as a disease of the rural poor. However, with occasional cases being reported also from town e.g. Dharan in south-eastern Nepal, questions were raised about possible extension of transmission to urban areas.<p><p>We conducted an outbreak investigation including a case-control study among the residents of Dharan town. We documented several clusters of VL cases in the more peripheral wards of the town. These are wards with new settlements where the poorest migrants install themselves. They are typically a rural-urban interface with most residents dependent on daily wages as agricultural labourers. However, several factors pointed to urban transmission: firstly, we found a strong association between VL and certain housing factors. Secondly, the clustering of VL cases in space and the intra-household clustering makes urban transmission more likely than infection due to migration. Finally, the entomological data also provide further evidence in support of local transmission of VL inside the town. The vector P. argentipes was captured repeatedly inside the town, and some of them were infested with L. donovani.<p><p>We studied the health seeking behavior and documented the households cost of VL care in a miltefosine-based programme after the intensified implementation of VL control efforts in Nepal. We enrolled 168 patients that had been treated for VL within twelve months prior to the survey in five districts in south-eastern Nepal. We observed a median delay of 25 days to present to the appropriate level of the primary healthcare system. Most patients first visited unqualified local practitioners or traditional faith healers for VL care. With a median total cost of US$ 165 per episode of VL treatment, the economic burden of VL across all households was 11% of annual household income or 57% of median annual per capita income. About half of the households exceeded the catastrophic expenditure threshold of 10% of annual household income. Our findings seem to suggest that, compared to previous studies, the economic burden of VL (as a % of household income) has indeed decreased. However, despite the provision of free diagnostics and drugs by the government, households still incurred substantial medical out-of-pocket expenditure, especially at private providers. The government should consider specific policies to reduce VL care costs such as a conditional cash programme for travel and food, and a better health insurance scheme. <p><p>We monitored clinical outcomes of VL treatment with miltefosine up to 12 months after the completion of therapy and explored the potential role of patient compliance, drug resistance, and reinfection. The initial cure rate was 95.8% and cure rate at 6 months after treatment was 82.5%, which further dropped to 73.3% at 12 months after miltefosine treatment. The relapse rate at 6 months was 10.8% and 20.0% at 12 months i.e. relapse is observed in one-fifth of miltefosine treated VL patients in Nepal. The decreased effectiveness of miltefosine observed in our study is an alarming signal for the ongoing VL elimination initiative and implicates the need for reviewing the drug policy in the Indian subcontinent. Relapse was most common among children (<12 years of age) and continued to occur beyond the commonly used 6-month follow-up period. No significant clinical risk factors or predictors of relapse apart from age <12 years were found. Parasite fingerprints of pre-treatment and relapse bone marrow isolates were similar within 8 tested patients, suggesting that clinical relapses were not due to re-infection with a new strain, but due to true recrudescences. MIL blood levels at the end of treatment were similar for cured and relapsed patients.The MIL-susceptibility of 131 VL isolates was also analysed in vitro with a promastigote assay and the mean promastigote MIL-susceptibility (IC50) of isolates from definite cures was similar to that of relapses.<p><p>We also assessed patient adherence to miltefosine treatment for VL given on an unsupervised ambulatory basis, prescribed under routine conditions (i.e. little or no time for treatment counselling) in government primary healthcare facilities. Our findings showed that adherence is a problem and the target of 90% of capsules taken is not reached in 15% of the enrolled patients. The gastrointestinal related side-effects and treatment-negligence after the resolution of clinical symptoms of VL were the main reasons for poor adherence. Effective counselling during the treatment, a short take-home message on the action and side effects of miltefosine, and on the importance of adherence are the best way to prevent poor adherence.<p><p>Post-kala-azar dermal leishmaniasis is more commonly seen in inadequately treated cases which is considered as a reservoir of infection maintaining disease transmission. The occurrence of PKDL in Nepal is relatively low compared to neighbouring countries involved in the elimination initiative. Supervised and adequate treatment of VL seems essential to reduce the risk of PKDL development. Policy makers should include surveillance and case management of PKDL in the VL elimination programme.<p> / Doctorat en Santé Publique / info:eu-repo/semantics/nonPublished