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Background: The kala-azar elimination programme has resulted in a significant reduction in visceral leishmaniasis (VL) cases across the Indian Subcontinent. To detect any resurgence of transmission, a sensitive cost-effective surveillance system is required. Molecular xenomonitoring (MX), detection of pathogen DNA/RNA in vectors, provides a proxy of human infection in the lymphatic filariasis elimination programme. To determine whether MX can be used for VL surveillance in a low transmission setting, large numbers of the sand fly vector Phlebotomus argentipes are required. This study will determine the best method for capturing P. argentipes females for MX. Methodology/Principal findings: The field study was performed in two programmatic and two non-programmatic villages in Bihar, India. A total of 48 households (12/village) were recruited. Centers for Disease Control and Prevention light traps (CDC-LTs) were compared with Improved Prokopack (PKP) and mechanical vacuum aspirators (MVA) using standardised methods. Four 12x12 Latin squares, 576 collections, were attempted (12/house, 144/village,192/method). Molecular analyses of collections were conducted to confirm identification of P. argentipes and to detect human and Leishmania DNA. Operational factors, such as time burden, acceptance to householders and RNA preservation, were also considered. A total of 562 collections (97.7%) were completed with 6,809 sand flies captured. Females comprised 49.0% of captures, of which 1,934 (57.9%) were identified as P. argentipes. CDC-LTs collected 4.04 times more P. argentipes females than MVA and 3.62 times more than PKP (p<0.0001 for each). Of 21,735 mosquitoes in the same collections, no significant differences between collection methods were observed. CDC-LTs took less time to install and collect than to perform aspirations and their greater yield compensated for increased sorting time. No significant differences in Leishmania RNA detection and quantitation between methods were observed in experimentally infected sand flies maintained in conditions simulating field conditions. CDC-LTs were favoured by householders. Conclusions/Significance: CDC-LTs are the most useful collection tool of those tested for MX surveillance since they collected higher numbers of P. argentipes females without compromising mosquito captures or the preservation of RNA. However, capture rates are still low. Author summary: Molecular xenomonitoring, screening insects for pathogen DNA/RNA, may be used for surveillance of diseases transmitted by insects. Since the proportion of insects infected with pathogens is very low in areas targeted for disease elimination, large numbers of females need to be screened. We compared three different methods for collecting Phlebotomus argentipes sand fly females, the vector of parasites causing the disease visceral leishmaniasis in the Indian subcontinent, to determine which collected the largest number of females. Other factors that may also influence selection of a particular method of collection by a disease control programme, such as the time it takes to collect and sort samples, the acceptance of householders for a particular collection method and whether RNA degradation in insect samples differed between collection methods, were also considered. Centers for Disease Control and Prevention light traps (CDC-LTs) proved to be more useful than two types of aspiration methods for collecting higher numbers of sand fly females and RNA preservation was retained. Furthermore, they took less time to install than to perform aspirations and were favoured by householders. Therefore, CDC-LTs were considered to be the most suitable collection method for molecular xenomonitoring of sand flies in India. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL) is a vector-borne protozoan disease, which can be fatal if left untreated. Synthetic chemical insecticides are very effective tools for controlling of insect vectors, including the sand fly Phlebotomus argentipes, the vector of VL in the Indian subcontinent. However, repeated use of the same insecticide with increasing doses potentially can create high selection pressure and lead to tolerance and resistance development. The objective of this study was to determine the lethal concentrations and assess levels of susceptibility, diagnostic doses and times to death of laboratory-reared P. argentipes to five insecticides that are used worldwide to control vectors. Using the Center for Disease Control and Prevention (CDC) bottle bioassay, 20–30 sand flies were exposed in insecticide- coated 500-ml glass bottles. Flies were then observed for 24 hours and mortality was recorded. Dose-response survival curves were generated for each insecticide using QCal software and lethal concentrations causing 50%, 90% and 95% mortality were determined. A bioassay was also conducted to determine diagnostic doses and diagnostic times by exposing 20–30 flies in each bottle containing set concentrations of insecticide. Mortality was recorded at 10-minute intervals for 120 minutes to generate the survival curve. Phlebotomus argentipes are highly susceptible to alpha-cypermethrin, followed by deltamethrin, malathion, chlorpyrifos, and least susceptible to DDT. Also, the lowest diagnostic doses and diagnostic times were established for alpha-cypermethrin (3μg/ml for 40 minutes) to kill 100% of the flies. The susceptibility data, diagnostic doses and diagnostic times presented here will be useful as baseline reference points for future studies to assess insecticide susceptibility and resistance monitoring of field caught sand flies and to assist in surveillance as VL elimination is achieved in the region. Author summary: Synthetic chemical insecticides are mainly used for controlling sand flies via indoor residual spraying (IRS) and insecticide treated nets. Sand flies had developed resistance against DDT in endemic areas of Bihar, India, and was replaced by synthetic pyrethroids (alpha-cypermethrin) in the second phase of IRS in 2015. The substantial increase in the use of synthetic pyrethroids will potentially lead to increased resistance because DDT and pyrethroids both have the same target site. To monitor insecticide resistance in field caught sand flies, susceptibility status and diagnostic doses and times are warranted, as no diagnostic dose for resistance detection has been established for Indian sand flies. A laboratory reared P. argentipes was used to quantify insecticide susceptibility and determination of diagnostic doses and times. The CDC bottle bioassay was used with five insecticides viz., alpha-cypermethrin, deltamethrin, malathion, chlorpyrifos and DDT. Results indicate that the P. argentipes were highly susceptible to alpha-cypermethrin followed by deltamethrin, malathion, chlorpyrifos and least susceptible to DDT. This study provides critical base line reference data for further studies to assess insecticide susceptibility and resistance monitoring in field populations, as well as determining diagnostic doses and times for other insecticide susceptible sand fly populations undergoing surveillance to sustain VL elimination. [ABSTRACT FROM AUTHOR]

Leishmaniasis on the Indian subcontinent is thought to have an anthroponotic transmission cycle. There is no direct evidence that a mammalian host other than humans can be infected with Leishmania donovani and transmit infection to the sand fly vector. The aim of the present study was to evaluate the impact of sand fly feeding on other domestic species and provide clinical evidence regarding possible non-human reservoirs through experimental sand fly feeding on cows, water buffalo goats and rodents. We performed xenodiagnosis using colonized Phlebotomus argentipes sand flies to feed on animals residing in villages with active Leishmania transmission based on current human cases. Xenodiagnoses on mammals within the endemic area were performed and blood-fed flies were analyzed for the presence of Leishmania via qPCR 48hrs after feeding. Blood samples were also collected from these mammals for qPCR and serology. Although we found evidence of Leishmania infection within some domestic mammals, they were not infectious to vector sand flies. Monitoring infection in sand flies and non-human blood meal sources in endemic villages leads to scientific proof of exposure and parasitemia in resident mammals. Lack of infectiousness of these domestic mammals to vector sand flies indicates that they likely play no role, or a very limited role in Leishmania donovani transmission to people in Bihar. Therefore, a surveillance system in the peri-/post-elimination phase of visceral leishmaniasis (VL) must monitor absence of transmission. Continued surveillance of domestic mammals in outbreak villages is necessary to ensure that a non-human reservoir is not established, including domestic mammals not present in this study, specifically dogs. Author summary: Leishmania donovani, the causative agent of visceral leishmaniasis (VL), has unique enzootic, zoonotic and anthroponotic life cycles dependent on geographic region. Most Leishmania spp. are zoonotic, transmitted between humans and non-human mammals. Leishmaniasis is endemic in over 98 countries and the estimated population at risk on the Indian sub-continent (ISC) is around 0.2–0.4 million people. Herein, we assess knowledge gaps in disease transmission that challenge dogma regarding the reservoir(s) of visceral leishmaniasis and control efforts on the Indian sub-continent. Better understanding of L. donovani infection in domestic animals and its transmission to sand flies will provide answers to fundamental questions in VL epidemiology and ecology. Blood samples were collected from livestock and rodents in endemic villages and colonized sand flies were fed on these animals and analyzed post feeding to see if they could pick up parasites via bloodmeal. We found that livestock and to a smaller extent rodents could be infected with or exposed to Leishmania infection. However, these mammals were not infectious to Phlebotomus argentipes, the predominant vector species incriminated as transmitting L. donovani in India. The inability of these domestic mammals to transmit L. donovani to sand flies in Bihar suggests that they play a limited role in the spread of infection. A key component in reducing and preventing the re-emergence of VL in the context of the elimination program is to understand the role of vector feeding on non-human animals in the transmission cycle. Better estimation of the proportion of the livestock and rodent population exposed to sand flies during different seasons and ensuring that there are not reservoir species that contribute to transmission will help implement appropriate control strategies for sustainable elimination. [ABSTRACT FROM AUTHOR]

Background: Few prospective data exist on incidence of post kala-azar dermal leishmaniasis (PKDL) and visceral leishmaniasis (VL) relapse after different treatment regimens. Methodology/Principal findings: A Phase IV trial included 1761 VL patients treated between 2012–2014 with single dose AmBisome (SDA; N = 891), miltefosine-paromomycin (Milt-PM; n = 512), or AmBisome-miltefosine (AmB-Milt; n = 358). Follow-up for PKDL and VL relapse was scheduled for 6, 12 and 24 months after treatment, lasting until 2017. Patients with lesions consistent with PKDL were tested by rK39 rapid test, and if positive, underwent skin-snip sampling, smear microscopy and PCR. Probable PKDL was defined by consistent lesions and positive rK39; confirmed PKDL required additional positive microscopy or PCR. PKDL and relapse incidence density were calculated by VL treatment and risk factors evaluated in Cox proportional hazards models. Among 1,750 patients who completed treatment, 79 had relapse and 104 PKDL. Relapse incidence density was 1.58, 2.08 and 0.40 per 1000 person-months for SDA, AmB-Milt and Milt-PM, respectively. PKDL incidence density was 1.29, 1.45 and 2.65 per 1000 person-months for SDA, AmB-Milt and Milt-PM. In multivariable models, patients treated with Milt-PM had lower relapse but higher PKDL incidence than those treated with SDA; AmB-Milt rates were not significantly different from those for SDA. Children <12 years were at higher risk for both outcomes; females had a higher risk of PKDL but not relapse. Conclusions/Significance: Active surveillance for PKDL and relapse, followed by timely treatment, is essential to sustain the achievements of VL elimination programs in the Indian sub-continent. Author summary: Efforts to eliminate visceral leishmaniasis (VL) in the Indian subcontinent have had an unprecedented impact on the number of cases over the past decade, however, the disease is known to be cyclical and previous periods of low incidence have been followed by a major resurgence. The disease is thought to be maintained between epidemics as post-kala-azar dermal leishmaniasis (PKDL), a skin disease affecting 5 to 15% of apparently cured VL patients in the Indian subcontinent. PKDL is difficult to cure and since PKDL patients are not systemically ill, they seldom seek treatment, meaning that PKDL is likely to be a major obstacle to elimination. A key question is how different VL treatment regimens affect the risk of subsequent PKDL. We followed-up a cohort of more than 1700 patients treated with three different drug regimens in an effectiveness trial in Bihar and found that patients treated with miltefosine-paromomycin had a lower risk of relapse but higher PKDL incidence, while AmBisome treated patients had the reverse pattern. In order to sustain the gains already made, active surveillance for PKDL and relapse is essential, along with timely treatment, and new VL treatment regimens that avoid future PKDL are needed. [ABSTRACT FROM AUTHOR]

Introduction: Presence of asymptomatic individualsin endemic areas is common. The possible biomarkers inasymptomatic individualspatients once they get exposed to infection as well as following conversion to symptomatic disease are yet to be identified.We identified asymptomatic Visceral leishmaniasis (VL) infection amongst rK39+sorted direct agglutination test positive (DAT+) endemic healthy population and confirmed it by quantitative PCR(qPCR).The immunological determinants such as Adenosine deaminase (ADA), Interferon gamma (IFN-γ), Tumour Necrosis Factor alpha (TNF-α) and Interleukin 10 (IL-10)were examined to predict probable biomarkers for conversion to symptomatic VL. Methods: Sample size was 5794 healthy individuals from VL endemic region. Antibody tests(DAT &rK39) were performed and later a qPCR assay was employed using kDNA specific primers and probes. Immunological biomarkers examined were ADA level by ADA–MTP kit and quantitative cytokines(IFN-γ, IL-10 and TNF-α) by ELISA. Results: 120 asymptomatic individuals of 308 rK39sero-positives were DAT positive comprising of 56 with previous history and 64 with no history of VL. RT-PCR confirmed asymptomatic VL in 42 sero-positives. These were followed up through repeated qPCR and evaluation of immunological determinants. We observed10 symptomatic cases converted from a total of 42 asymptomatic individualssubjects identified at base-line. The level of ADA, IL-10 and IFN-γ remained consistently high in asymptomatic individuals and amongst these, ADA and IL-10 but not IFN-γ remained higher at the development of clinical symptoms into active VL suspects. On the contrary, there was no significant change in the mean concentration of TNF-α at both stages of the disease. Discussion: We surmise from our data that considerable proportion of asymptomatic cases can be a reservoir and play a crucial role in transmission of visceral leishmaniasis in endemic areas. The data also suggests that ADA and IL-10 can serve as a potential biomarker during the conversion of asymptomatic into symptomatic VL. Author summary: The most threatening form of leishmaniasis is human visceral leishmaniasis, which is caused by L. donovani in Indian subcontinent. The disease accounts for huge annual burden of infectious disease in India. Efforts towards disease elimination programme are far beyond satisfaction and there is need to re-strengthen health monitoring, surveillance programme in endemic areas. There is a greater need to identify asymptomatic individuals amongst endemic healthy control by active surveillance programme. Further, except a few, many asymptomatic subjects become sero-negative without developing VL due to strong inherent cellular immunity possessed by them. This study enumerates use of rK39, DAT and quantitative PCR(qPCR) in asymptomatic detection and then evaluate immunological biomarkers(Adenosine deaminase, IFN-γ, TNF-α and IL-10) which help in disease conversion to symptomatic VL, at 6 month follow- up. Follow–up analysis revealed that antibody testing with rK39 (3.24%), DAT (8.33%) had poor agreement with disease conversion. By qPCR, we could identify the level of parasitemia, and collectively with serology, the test detected 23.80% of asymptomatic cases converted into disease. The level of ADA and IL-10 remained consistently high during conversion. IFN-γ became high in asymptomatic infection but became low on conversion, whereas, no significant change in level of TNF-α was observed at both stages of disease. Thus by use of qPCR, concomitantly with DAT over rK39+ sorted endemic healthy control may be employed for early case detection. Adenosine is produced by ecto-nucleotide during L.donovaniinfection, has been previously reported and in parallel to this study we conclude that determination of adenosine deaminase level (ADA) might help identify early cases with more tendency to convert into disease. This will help to boost health monitoring programme to eradicate Kala-azar in Bihar, India. [ABSTRACT FROM AUTHOR]

Introduction: Studies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early Leishmania donovani infection and as a predictor of progression to symptomatic disease. Methods: The study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes. Results: A large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load. Discussion: Thus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission. Author Summary: Anthroponotic VL caused by Leishmania donovani in the Indian subcontinent accounts for 70% of the world burden of VL. Among the estimated 100,000 cases of VL acquired annually in India, 90% occur in the state of Bihar. Leishmania infection can result in either symptomatic or asymptomatic infection. L. donovani infection can also manifest as post-kala azar dermal leishmaniasis, a chronic cutaneous form thought to provide the reservoir for anthroponotic transmission of VL in regions endemic for this parasite species. We hypothesized that, in areas endemic for L. donovani, asymptomatic infections might also play a crucial role in disease transmission. This study describes use of quantitative PCR (qPCR) to determine the infection status in individuals living in an endemic region of India. We hypothesized that parasite load estimation by qPCR of peripheral blood cells among healthy individuals living in the endemic region might reveal the true frequency of infections through direct evidence of parasitemia. We reasoned this test would detect both asymptomatic non-progressors as well as asymptomatic individuals who will progress to fully symptomatic VL. Serologic testing by ELISA or DAT showed poor agreement with molecular detection of parasite DNA by qPCR, suggesting the tests differentiate between infection and immune response. Amongst ten healthy individuals who progressed to VL, only six were serologically positive whereas eight were initially qPCR positive, among whom five had high parasite loads in their blood. Thus, deployment of qPCR technique to estimate the presence and level of parasitemia in healthy individuals from Leishmania endemic regions may contribute to early case detection, thereby reducing morbidity and mortality. Consistent with the goals of the VL control and elimination program, this early intervention approach could help interrupt disease transmission. [ABSTRACT FROM AUTHOR]

Background: The skin disorder Post Kala-Azar Dermal Leishmaniasis (PKDL) occurs in up to 10% of patients treated for visceral leishmaniasis (VL) in India. The pathogenesis of PKDL is not yet fully understood. Cases have been reported in India following therapy with most available treatments, but rarely in those treated with liposomal amphotericin B (Ambisome). Between July 2007 and August 2012 with the support of the Rajendra Memorial Research Institute (RMRI), Médecins Sans Frontières (MSF) supported a VL treatment programme in Bihar, India—an area highly endemic for Leishmania donovani—in which 8749 patients received 20 mg/kg intravenous Ambisome as first-line treatment. This study describes the characteristics of patients who returned to the MSF supported treatment programme with PKDL. Methods and Principal Findings: Over a 5-year period, Ambisome was administered to 8749 patients with laboratory-confirmed VL (clinical signs, rK39 positive, with/without parasite confirmation) in four intravenous doses of 5 mg/kg to a total of 20 mg/kg, with a high initial-cure rate (99.3%) and low default rate (0.3%). All patients received health education highlighting the possibility and symptoms of developing PKDL, and advice to return to the MSF programme if these symptoms developed. This is an observational retrospective cohort study of the programme outcomes. Of the 8311 patients completing treatment for their first episode of VL, 24 (0.3%) returned passively to the programme complaining of symptoms subsequently confirmed as PKDL, diagnosed from clinical history, appearance consistent with PKDL, and slit-skin smear examination. Of the 24 patients, 89% had macular lesions, with a median time (interquartile range) to development of 1.2 (0.8–2.2) years following treatment. Comparison of the demographic and clinical characteristics of the VL patients treated with Ambisome who later developed PKDL, with those of the remaining cohort did not identify any significant risk factors for PKDL. However, the time to developing PKDL was significantly shorter with Ambisome than in a subset of patients presenting to the programme with PKDL following previous sodium stibogluconate treatment for VL. Conclusions: In this large cohort of patients with VL in Bihar who were treated with 20 mg/kg Ambisome, PKDL following treatment appears to be infrequent with no predictive risk factors. The shorter median time to developing symptoms of PKDL compared with that after conventional VL treatments should be taken into account when counseling patients treated with regimens including Ambisome. Author Summary: Visceral leishmaniasis (VL), also known as Kala-azar, is caused by the parasite L.donovani. Half of cases worldwide occur in India, with up to 90% of these in Bihar state. Post Kala-azar dermal leishmaniasis (PKDL) is a difficult to treat skin condition that develops in up to 10% of VL cases following treatment in the Indian subcontinent. Although often mild, PKDL can be severe and disfiguring. Patients are otherwise healthy. PKDL is considered a reservoir of L.donovani and requires treating to support disease elimination. Between 2007–2012, 8311 patients diagnosed with a first episode of VL completed treatment with 20 mg/kg intravenous liposomal amphotericin B (Ambisome) in a Médecins Sans Frontières (MSF) programme supported by the Rajendra Memorial Research Institute (RMRI) in Bihar. Ambisome is a safe and effective treatment that is recommended by the WHO for first-line use in the Indian subcontinent; although not yet included in the Indian guidelines. PKDL has been described following all VL treatments, but so far in only two patients treated with Ambisome. Here, we describe 24 patients treated with Ambisome who returned to the MSF treatment programme with confirmed PKDL. We found no risk factors for PKDL following treatment; however, the average time to development of PKDL was much shorter than that seen following older treatments. [ABSTRACT FROM AUTHOR]

Background: Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades, but increasing failure rates under antimonial treatment have challenged further use of these drugs in the Indian subcontinent. Experimental evidence has suggested that parasites which are resistant against antimonials have superior survival skills than sensitive ones even in the absence of antimonial treatment. Methods and Findings: We use simulation studies based on a mathematical L. donovani transmission model to identify parameters which can explain why treatment failure rates under antimonial treatment increased up to 65% in Bihar between 1980 and 1997. Model analyses suggest that resistance to treatment alone cannot explain the observed treatment failure rates. We explore two hypotheses referring to an increased fitness of antimony-resistant parasites: the additional fitness is (i) disease-related, by causing more clinical cases (higher pathogenicity) or more severe disease (higher virulence), or (ii) is transmission-related, by increasing the transmissibility from sand flies to humans or vice versa. Conclusions: Both hypotheses can potentially explain the Bihar observations. However, increased transmissibility as an explanation appears more plausible because it can occur in the background of asymptomatically transmitted infection whereas disease-related factors would most probably be observable. Irrespective of the cause of fitness, parasites with a higher fitness will finally replace sensitive parasites, even if antimonials are replaced by another drug. Author Summary: The protozoan flagellate Leishmania donovani causes the neglected, life-threatening disease visceral leishmaniasis. Parasites are transmitted from man to man by the bite of the sand fly Phlebotomus argentipes, the vector of the disease. Pentavalent antimonials have been the mainstay of antileishmanial therapy for decades but rapidly increasing failure rates up to 65% observed between 1980 and 1997 in the state of Bihar, India, have challenged further use of these drugs. Comparative in vitro and in vivo experiments indicate that antimony-resistant parasites have a higher fitness than antimony-sensitive ones even in the absence of antimonial treatment. Simulation studies based on a previously published mathematical L. donovani transmission model suggest that resistance to antimonial treatment alone cannot explain the Bihar observations but that resistance together with higher fitness offers the potential to explain the data. After an antimony-resistant parasite with higher fitness has emerged, it will finally replace the antimony-sensitive ones, even in complete absence of antimonial treatment. [ABSTRACT FROM AUTHOR]

This article discusses the decline in the effectiveness of antimonial compounds in treating visceral leishmaniasis in Bihar, India. The decrease in efficacy has been attributed to the misuse of antimonials and resistance in parasites. The article proposes an additional hypothesis that chronic exposure to arsenic, which is present in the groundwater in Bihar, may contribute to the decreased efficacy of antimony. The article explores the evidence for this hypothesis and suggests that further research is needed to fully understand the relationship between arsenic contamination and antimonial treatment failure. [Extracted from the article]