Failure of miltefosine in visceral leishmaniasis is associated with low drug exposure.

BACKGROUND: Recent reports indicated high miltefosine treatment failure rates for visceral leishmaniasis (VL) on the Indian subcontinent. To further explore the pharmacological factors associated with these treatment failures, a population pharmacokinetic-pharmacodynamic study was performed to examine the relationship between miltefosine drug exposure and treatment failure in a cohort of Nepalese patients with VL. METHODS: Miltefosine steady-state blood concentrations at the end of treatment were analyzed using liquid chromatography tandem mass spectrometry. A population pharmacokinetic-pharmacodynamic analysis was performed using nonlinear mixed-effects modeling and a logistic regression model. Individual estimates of miltefosine exposure were explored for their relationship with treatment failure. RESULTS: The overall probability of treatment failure was 21%. The time that the blood concentration was >10 times the half maximal effective concentration of miltefosine (median, 30.2 days) was significantly associated with treatment failure: each 1-day decrease in miltefosine exposure was associated with a 1.08-fold (95% confidence interval, 1.01-1.17) increased odds of treatment failure. CONCLUSIONS: Achieving a sufficient exposure to miltefosine is a significant and critical factor for VL treatment success, suggesting an urgent need to evaluate the recently proposed optimal allometric miltefosine dosing regimen. This study establishes the first evidence for a drug exposure-effect relationship for miltefosine in the treatment of VL.

Increasing failure of miltefosine in the treatment of Kala-azar in Nepal and the potential role of parasite drug resistance, reinfection, or noncompliance.

BACKGROUND: Miltefosine (MIL), the only oral drug for visceral leishmaniasis (VL), is currently the first-line therapy in the VL elimination program of the Indian subcontinent. Given the paucity of anti-VL drugs and the looming threat of resistance, there is an obvious need for close monitoring of clinical efficacy of MIL. METHODS: In a cohort study of 120 VL patients treated with MIL in Nepal, we monitored the clinical outcomes up to 12 months after completion of therapy and explored the potential role of drug compliance, parasite drug resistance, and reinfection. RESULTS: The initial cure rate was 95.8% (95% confidence interval [CI], 92.2-99.4) and the relapse rate at 6 and 12 months was 10.8% (95% CI, 5.2-16.4) and 20.0% (95% CI, 12.8-27.2) , respectively. No significant clinical risk factors of relapse apart from age <12 years were found. Parasite fingerprints of pretreatment and relapse bone marrow isolates within 8 patients were similar, suggesting that clinical relapses were not due to reinfection with a new strain. The mean promastigote MIL susceptibility (50% inhibitory concentration) of isolates from definite cures was similar to that of relapses. Although more tolerant strains were observed, parasite resistance, as currently measured, is thus not likely involved in MIL treatment failure. Moreover, MIL blood levels at the end of treatment were similar in cured and relapsed patients. CONCLUSIONS: Relapse in one-fifth of the MIL-treated patients observed in our study is an alarming signal for the VL elimination campaign, urging for further review and cohort monitoring.

Adherence to miltefosine treatment for visceral leishmaniasis under routine conditions in Nepal.

OBJECTIVE: To assess patient adherence to unsupervised single-drug miltefosine treatment for visceral leishmaniasis and to identify the factors influencing adherence. METHODS: This is a prospective cohort study of 171 patients with Visceral leishmaniasis (VL) in three healthcare settings in Nepal. Adherence was assessed through pill count, checking of treatment cards and adherence questionnaires, as well as miltefosine concentration measurements at the end of treatment. Poor adherence was defined as less than 90% of required capsules taken. RESULTS: Patient adherence to miltefosine was 83%. Predictors of adherence were being the male sex (OR = 2.60, 95% CI 1.02-6.67) and knowing the duration of treatment (OR = 3.05, 95% CI 1.16-8.04). Adherence was also better for patients who were literate and knew the side effects of treatment. Gastrointestinal side effects and negligence after the resolution of clinical symptoms of VL were the main reasons for poor adherence. Poor adherence was associated (though not statistically significant) with future relapse. CONCLUSION: Effective counselling during the treatment, a short take-home message on VL and on side effects and action of miltefosine, and follow-up visits are the best way to prevent poor adherence. Single end-of-treatment measurements of miltefosine concentrations as objective assessment of adherence would only be useful in addition to the subjective assessments when substantial doses of miltefosine have been missed.

Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India.

OBJECTIVES: Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection. METHODS: Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA. RESULTS: PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity. CONCLUSION: A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers.

Retrospective Quarterly Cohort Monitoring for patients with Visceral Leishmaniasis in the Indian subcontinent: outcomes of a pilot project.

OBJECTIVE: To evaluate a new tool for the monitoring of Visceral Leishmaniasis (VL) treatment outcomes in primary healthcare (PHC) settings, adapted from the standardised Retrospective Quarterly Cohort Monitoring done in tuberculosis control. METHODS: We developed standard case definitions for early and late VL treatment outcomes, a single register allowing for one-line entry per patient as registration tool, and quarterly reporting formats for the clinical outcomes. We pilot-tested these tools in three Indian Primary Health Centres and two Nepalese district hospitals, as well as in a charity VL treatment centre and a university hospital. RESULTS: Data collection for early treatment outcome was easily implemented but information on late treatment outcome was hard to obtain. Effectiveness of Miltefosine under routine care conditions was about 87% at end of treatment, and 76% at 6 months post-treatment related to the high number of patients lost to follow up at the latter end point. CONCLUSION: A retrospective cohort monitoring methodology is conceptually a good framework for monitoring clinical outcomes for chronic conditions as VL. The monitoring of early outcomes of VL treatment is perfectly feasible in Primary Care settings. The completeness of information on late outcomes can be improved by a number of strategies that remain to be field tested. Generally, clinical outcome monitoring should be strengthened in the VL control programmes.

Genetic markers for SSG resistance in Leishmania donovani and SSG treatment failure in visceral leishmaniasis patients of the Indian subcontinent.

The current standard to assess pentavalent antimonial (SSG) susceptibility of Leishmania is a laborious in vitro assay of which the result has little clinical value because SSG-resistant parasites are also found in SSG-cured patients. Candidate genetic markers for clinically relevant SSG-resistant parasites identified by full genome sequencing were here validated on a larger set of clinical strains. We show that 3 genomic locations suffice to specifically detect the SSG-resistant parasites found only in patients experiencing SSG treatment failure. This finding allows the development of rapid assays to monitor the emergence and spread of clinically relevant SSG-resistant Leishmania parasites.

Efficacy of miltefosine in the treatment of visceral leishmaniasis in India after a decade of use.

BACKGROUND: Miltefosine is the only oral drug available for treatment of Indian visceral leishmaniasis (VL), which was shown to have an efficacy of 94% in a phase III trial in the Indian subcontinent. Its unrestricted use has raised concern about its continued effectiveness. This study evaluates the efficacy and safety of miltefosine for the treatment of VL after a decade of use in India. METHODS: An open-label, noncomparative study was performed in which 567 patients received oral miltefosine (50 mg for patients weighing <25 kg, 100 mg in divided doses for those weighing ≥25 kg, and 2.5 mg per kg for those aged <12 years, daily for 28 days) in a directly observed manner. Patients were followed up for 6 months to see the response to therapy. RESULTS: At the end of treatment the initial cure rate was 97.5% (intention to treat), and 6 months after the end of treatment the final cure rate was 90.3%. The overall death rate was 0.9% (5 of 567), and 2 deaths were related to drug toxicity. Gastrointestinal intolerance was frequent (64.5%). The drug was interrupted in 9 patients (1.5%) because of drug-associated adverse events. CONCLUSIONS: As compared to the phase III trial that led to registration of the drug a decade ago, there is a substantial increase in the failure rate of oral miltefosine for treatment of VL in India.

Post-kala-azar dermal leishmaniasis (PKDL) in visceral leishmaniasis-endemic communities in Bihar, India.

OBJECTIVE: To assess the prevalence of Post-kala-azar dermal leishmaniasis (PKDL) in 16 visceral leishmaniasis-endemic communities in Bihar, India. METHODS: Three-stage house-to-house survey of 2020 households to identify and confirm PKDL cases. RESULTS: The prevalence of confirmed PKDL cases was 4.4 per 10 000 individuals and 7.8 if probable cases were also considered. The clinical history and treatment of the PKDL cases are discussed in detail. CONCLUSION: PKDL can develop in visceral leishmaniasis patients treated with different anti-leishmanial drugs. Migration of PKDL cases to other villages may expand visceral leishmaniasis-affected areas.

Post-kala-azar dermal leishmaniasis in visceral leishmaniasis-endemic communities in Bihar, India.

We assessed the prevalence of post-kala-azar dermal leishmaniasis (PKDL), a late cutaneous manifestation of visceral leishmaniasis (VL), in 16 VL-endemic communities in Bihar, India. The prevalence of confirmed PKDL cases was 4.4 per 10 000 individuals and 7.8 if probable cases were also considered. The clinical history and treatment of the post-kala-azar dermal leishmaniasis cases are discussed.

Domestic animals and epidemiology of visceral leishmaniasis, Nepal.

On the Indian subcontinent, visceral leishmaniasis (VL) is considered an anthroponosis. To determine possible reasons for its persistence during interepidemic periods, we mapped Leishmania infections among healthy persons and animals in an area of active VL transmission in Nepal. During 4 months (September 2007-February 2008), blood was collected from persons, goats, cows, and buffaloes in 1 village. Leishmania infections were determined by using PCR. We found infections among persons (6.1%), cows (5%), buffaloes (4%), and goats (16%). Data were georeferenced and entered into a geographic information system. The bivariate K-function results indicated spatial clustering of Leishmania spp.-positive persons and domestic animals. Classification tree analysis determined that among several possible risk factors for Leishmania infection among persons, proximity of Leishmania spp.-positive goats ranked first. Although our data do not necessarily mean that goats constitute a reservoir host of L. donovani, these observations indicate the need for further investigation of goats' possible role in VL transmission.

Serological markers for leishmania donovani infection in Nepal: Agreement between direct agglutination test and rK39 ELISA.

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49­0.57). More research is needed to develop validated markers for Leishmania infection.

The epidemiology of Leishmania donovani infection in high transmission foci in India.

OBJECTIVE: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission dynamics are not well understood. We report data from a sero-survey in Bihar. METHODS: Demographic and immunological surveys were carried out in July and November 2006, respectively in 16 highly VL endemic foci in Muzaffarpur district in Bihar. Household and individual information was gathered and capillary blood samples were collected on filter papers. Direct agglutination test (DAT) was used to determine infected individuals (cut-off titre 1:1600). DAT results were tabulated against individual and household variables. A multivariate generalized estimating equation (GEE) model was used to study the prevalence of serologically positive individuals taking into account the clustering at household and cluster levels. RESULTS: Of study subjects 18% were DAT positive, and this proportion increased with age. Women had a significantly lower prevalence than men >14 years old. Owning domestic animals (cows, buffaloes or goats) was associated with a higher risk of being DAT positive [OR 1.16 (95% CI 1.01-1.32)], but socio-economic status was not. CONCLUSIONS: Prevalence of leishmanial antibodies was high in these communities, but variable. Demographic factors (i.e. marriage) may explain the lower DAT positivity in women >14 years of age. Within these homogeneously poor communities, socio-economic status was not linked to L. donovani infection risk at the individual level, but ownership of domestic animals was.

Epidemiology of Leishmania donovani infection in high-transmission foci in Nepal.

OBJECTIVE: Nepal reports a visceral leishmaniasis (VL) incidence of 5 per 10 000 per year on the basis of notification by health facilities, but little community-based epidemiological information exists. We report data on prevalence rates of Leishmania donovani infection in ten communities in East Nepal. METHODS: Ten clusters with highest VL incidence rates were purposefully selected in Nepal. All households were mapped and socio-demographic data and data on past VL incidence were collected. An exhaustive serological survey was performed of individuals aged >2 years, by collecting finger prick blood on filter paper in November-December 2006. The samples were tested by direct agglutination, and a titre >or=1:1600 was taken as marker of infection. A generalized estimating equation (GEE) model was used to assess risk factors for Direct Agglutination Test (DAT) positivity taking into account the clustering at household and village level. RESULTS: The sero-survey (n = 5397) showed an infection prevalence rate of 9%, (range 5-15% per cluster) with higher prevalence in men (9.9%) than in women (8.3%) (P = 0.049). Male gender, increasing age and poverty were significant risk factors in the final GEE model. CONCLUSION: Leishmania infection rate in high-transmission areas in Nepal is associated with gender, age and socio-economic status.

Spatial analysis of Leishmania donovani exposure in humans and domestic animals in a recent kala azar focus in Nepal.

SUMMARY: Visceral leishmaniasis (VL) is a major public health problem in the Indian subcontinent where the Leishmania donovani transmission cycle is described as anthroponotic. However, the role of animals (in particular domestic animals) in the persistence and expansion of VL is still a matter of debate. We combined Direct Agglutination Test (DAT) results in humans and domestic animals with Geographic Information System technology (i.e. extraction maps and scan statistic) to evaluate the exposure to L. donovani on these 2 populations in a recent VL focus in Nepal. A Poisson regression model was used to assess the risk of infection in humans associated with, among other factors, the proportion of DAT-positive animals in the proximities of the household. The serological results showed that both humans and domestic animals were exposed to L. donovani. DAT-positive animals and humans were spatially clustered. The presence of serologically positive goats (IRR=9.71), past VL cases (IRR=2.62) and the proximity to a forest island dividing the study area (IRR=3.67) increased the risk of being DAT-positive in humans. Even if they are not a reservoir, domestic animals, and specially goats, may play a role in the distribution of L. donovani, in particular in this new VL focus.

Impact of the visceral leishmaniasis elimination initiative on Leishmania donovani transmission in Nepal: a 10-year repeat survey.

BACKGROUND: Nepal launched a visceral leishmaniasis (also known as kala-azar) elimination initiative in 2005. We primarily aimed to assess whether transmission of Leishmania donovani had decreased since the launch of the initiative. We also assessed the validity of the direct agglutination test (DAT) as a marker of infection, in view of future surveillance systems. METHODS: We did a repeat survey in a population aged 2 years and older for whom baseline serological data were available from 2006. Data were from three districts in the eastern region of Nepal. The primary outcome of interest was prevalent infection with L donovani as measured with DAT (cutoff value ≥1:3200). We compared age group-specific and cluster-specific seroprevalences in 2016 with those in 2006, using χ2 tests, with a specific focus on the comparison of seroprevalences in children born between 1996 and 2005, and those born between 2006 and 2015. To estimate the overall adjusted risk ratio for being seropositive in 2016 compared with 2006, we fitted a Poisson model controlling for age, sex, and cluster. FINDINGS: Between Oct 17, 2016, and Dec 26, 2016, we assessed 6609 individuals. DAT prevalence in children younger than 10 years was 4·1% (95% CI 3·2-5·4) in 2006 versus 0·5% (0·1-1·7) in 2016 (p<0·0001). Seroprevalence was lower in 2016 than in 2006 in all age groups and in all repeated clusters. The overall adjusted risk ratio of being seropositive was 0·44 (95% CI 0·37-0·52) for 2016 compared with 2006, and 0·04 (0·01-0·16) in children younger than 10 years. INTERPRETATION: Our findings show that transmission of L donovani in Nepal has decreased significantly between 2006 and 2016, coinciding with the elimination programme. DAT seems useful for monitoring of L donovani transmission. FUNDING: The Directorate-General for Development Cooperation of Belgium.

PCR and direct agglutination as Leishmania infection markers among healthy Nepalese subjects living in areas endemic for Kala-Azar.

OBJECTIVE: To compare a PCR assay and direct agglutination test (DAT) for the detection of potential markers of Leishmania infection in 231 healthy subjects living in a kala-azar endemic focus of Nepal. METHODS: The sample was composed of 184 (80%) persons without any known history of KA and not living in the same house as known kala-azar cases (HNK), 24 (10%) Healthy Household Contacts (HHC) and 23 (10%) past kala-azar cases which had been successfully treated (HPK). RESULTS: PCR and DAT positivity scores were, respectively: HNK, 17.6% and 5.6%; HHC, 12.5% and 20.8%; HPK, 26.1% and 95.7%. The ratio PCR-positives/DAT-positives was significantly higher in HNK (ratio = 3.1) than in HHC (ratio = 0.6, P = 0.036) and in HPK (ratio = 0.2, P = 0.012). The ratio PCR-positives/DAT-positives did not significantly differ between HHC (ratio = 0.6) and HPK (ratio = 0.2, P = 0.473). The positive agreement index between PCR and DAT in HNK was 5%; in HHC, 0%; in HPK, 43%. CONCLUSIONS: Our study highlights the specific character of PCR and DAT for the exploration of Leishmania asymptomatic infections. PCR is probably more informative for very recent infections among HNK, while DAT provides more information among HHC and HPK, a feature likely related to the power of serology to track less recent infections.

Determinants for progression from asymptomatic infection to symptomatic visceral leishmaniasis: A cohort study.

BACKGROUND: Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL). METHODS: We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6-12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR. RESULTS: Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1-8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up. CONCLUSION: We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated.

Long-lasting insecticidal nets fail at household level to reduce abundance of sandfly vector Phlebotomus argentipes in treated houses in Bihar (India).

OBJECTIVE: To determine whether the use of long-lasting insecticidal nets (LLINS) at household level are effective in reducing the abundance of Phlebotomus argentipes, vector of anthroponotic visceral leishmaniasis in India, Nepal and Bangladesh. METHODS: The impact of two long-lasting nets (Olyset and PermaNet) on indoor sandfly abundance was evaluated in selected houses of three endemic hamlets in Bihar (India). It was assumed that most sandflies breed inside the houses and that LLINs would progressively reduce the indoor density during the reproduction season. A campaign of indoor spraying with dichloro-diphenyl-trichloroethane (DDT) interfered with the trial but did not affect the sandfly population. Results Only the density of males of P. argentipes was significantly reduced by both the LLINs but not the females. CONCLUSIONS: These findings suggest that most female sandflies are coming from outside and that LLINs do not reduce their entry rate.