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Background: Visceral leishmaniasis (VL) in patients with human immunodeficiency virus (HIV) presents an increasingly important patient cohort in areas where both infections are endemic. Evidence for treatment is sparce, with no high-quality studies from the Indian subcontinent., Methods: This is a randomized, open-label, parallel-arm, phase 3 trial conducted within a single hospital in Patna, India. One hundred and fifty patients aged ≥18 years with serologically confirmed HIV and parasitologically confirmed VL were randomly allocated to 1 of 2 treatment arms, either a total 40 mg/kg intravenous liposomal amphotericin B (AmBisome; Gilead Pharmaceuticals) administered in 8 equal doses over 24 days or a total 30 mg/kg intravenous AmBisome administered in 6 equal doses given concomitantly with a total 1.4 g oral miltefosine administered through 2 daily doses of 50 mg over 14 days. The primary outcome was intention-to-treat relapse-free survival at day 210, defined as absence of signs and symptoms of VL or, if symptomatic, negative parasitological investigations., Results: Among 243 patients assessed for eligibility, 150 were recruited between 2 January 2017 and 5 April 2018, with no loss to follow-up. Relapse-free survival at day 210 was 85% (64/75; 95% CI, 77-100%) in the monotherapy arm, and 96%, (72/75; 90-100%) in the combination arm. Nineteen percent (28/150) were infected with concurrent tuberculosis, divided equally between arms. Excluding those with concurrent tuberculosis, relapse-free survival at day 210 was 90% (55/61; 82-100%) in the monotherapy and 97% (59/61; 91-100%) in the combination therapy arm. Serious adverse events were uncommon and similar in each arm., Conclusions: Combination therapy appears to be safe, well tolerated, and effective, and halves treatment duration of current recommendations., Clinical Trials Registration: Clinical Trial Registry India (CTRI/2015/05/005807; the protocol is available online at https://osf.io/avz7r)., Competing Interests: Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: Visceral leishmaniasis (VL), also known as kala-azar (KA), is a neglected vector-borne disease, targeted for elimination, but several affected blocks of Bihar are posing challenges with the high incidence of cases, and moreover, the disease is spreading in newer areas. High-quality kala-azar surveillance in India, always pose great concern. The complete and accurate patient level data is critical for the current kala-azar management information system (KMIS). On the other side, no accurate data on the burden of post kala-azar dermal leishmaniasis (PKDL) and co-infections are available under the current surveillance system, which might emerge as a serious concern. Additionally, in low case scenario, sentinel surveillance may be useful in addressing post-elimination activities and sustaining kala-azar (KA) elimination. Health facility-based sentinel site surveillance system has been proposed, first time to do a proper accounting of KA, PKDL and co-infection morbidity, mortality, diagnosis, case management, hotspot identification and monitoring the impact of elimination interventions., Methodology/principal Findings: Kala-azar sentinel site surveillance was established and activated in thirteen health facilities of Bihar, India, using stratified sampling technique during 2011 to 2014. Data were collected through specially designed performa from all patients attending the outpatient departments of sentinel sites. Among 20968 symptomatic cases attended sentinel sites, 2996 cases of KA and 53 cases of PKDL were registered from 889 endemic villages. Symptomatic cases meant a person with fever of more than 15 days, weight loss, fatigue, anemia, and substantial swelling of the liver and spleen (enlargement of spleen and liver).The proportion of new and old cases was 86.1% and 13.9% respectively. A statistically significant difference was observed for reduction in KA incidence from 4.13/10000 in 2011 to 1.75/10000 in 2014 (p<0.001). There were significant increase (0.08, 0.10 per 10 000 population) in the incidences of PKDL and co-infection respectively in the year 2014 as compared to that of 2011 (0.03, 0.06 per 10 000 population). The proportion of HIV-VL co-infection was significantly higher (1.6%; p<0.05) as compared to other co-infections. Proportions of male in all age groups were higher and found statistically significant (Chi-square test = 7.6; P = 0.026). Utilization of laboratory services was greatly improved. Friedman test showed statistically significant difference between response of different anti kala-azar drugs (F = 25.0, P = 0.004).The initial and final cure rate of AmBisome was found excellent (100%). The results of the signed rank sum test showed significant symmetry of unresponsiveness rate (P = 0.03). Similarly, relapse rate of sodium antimony gluconate (SAG) was also found significantly higher as compared to other drugs (95%CI 0.2165 to 19.7035; P = 0.03). A statistically significant difference was found (p<0.001) between villages having 1-2 cases (74%) and villages with 3-5 cases (15%). Significantly higher proportion (95%) of cases were captured by existing Govt. surveillance system (KMIS) (p<0.001), as compared to private providers (5%)., Conclusions/significance: Establishment of a sentinel site based kala-azar surveillance system in Bihar, India effectively detected the rising trend of PKDL and co-infections and captured complete and accurate patient level data. Further, this system may provide a model for improving laboratory services, KA, PKDL and co-infection case management in other health facilities of Bihar without further referral. Program managers may use these results for evaluating program's effectiveness. It may provide an example for changing the practices of health care workers in Bihar and set a benchmark of high quality surveillance data in a resource limited setting. However, the generalizability of this sentinel surveillance finding to other context remains a major limitation of this study. The justifications for this; the sentinel sites were made in the traditionally high endemic PHC's. The other conditions were Program commitment for diagnostic (rk-39) and the first line anti kala-azar drug i.e. miltefosine throughout the study period in the sentinel sites. In addition, there were clause of fulfillment of readiness criteria at each sentinel site (already described in the line no 171 to 180 at page no-8, 181-189 at page no-9 and 192-212 at page no-10). Rigorous efforts were taken to improve all the sentinel sites to meet the readiness criteria and research activities started only after meeting readiness criteria at the site. Therefore sentinel site surveillance described under the present study cannot be integrated into other set up (medium and low endemic areas). However, it can be integrated into highly endemic areas with program commitment and fulfillment of readiness criteria., Competing Interests: The authors have declared that no competing interests exist.

Background: Treatment of post-kala-azar dermal leishmaniasis cases is of paramount importance for kala-azar elimination; however, limited treatment regimens are available as of now., Aim: To compare the effectiveness of liposomal amphotericin B vs miltefosine in post-kala-azar dermal leishmaniasis patients., Methodology: This was a randomized, open-label, parallel-group study. A total of 100 patients of post kala azar dermal leishmaniasis, aged between 5 and 65 years were recruited, 50 patients in each group A (liposomal amphotericin B) and B (miltefosine). Patients were randomized to receive either liposomal amphotericin B (30 mg/kg), six doses each 5 mg/kg, biweekly for 3 weeks or miltefosine 2.5 mg/kg or 100 mg/day for 12 weeks. All the patients were followed at 3rd, 6th and 12th months after the end of the treatment., Results: In the liposomal amphotericin B group, two patients were lost to follow-up, whereas four patients were lost to follow-up in the miltefosine group. The initial cure rate by "intention to treat analysis" was 98% and 100% in liposomal amphotericin B and miltefosine group, respectively. The final cure rate by "per protocol analysis" was 74.5% and 86.9% in liposomal amphotericin B and miltefosine, respectively. Twelve patients (25.5%) in the liposomal amphotericin B group and six patients (13%) in the miltefosine group relapsed. None of the patients in either group developed any serious adverse events., Limitations: Quantitative polymerase chain reaction was not performed at all the follow-up visits and sample sizes., Conclusion: Efficacy of miltefosine was found to be better than liposomal amphotericin B, hence, the use of miltefosine as first-line therapy for post-kala-azar dermal leishmaniasis needs to be continued. However, liposomal amphotericin B could be considered as one of the treatment options for the elimination of kala-azar from the Indian subcontinent.

Post-kala-azar dermal leishmaniasis is a skin disorder occurring in 5-10% of visceral leishmaniasis patients after treatment with miltefosine,the first-line drug for this skin disorder. We reported a case of acute anterior uveitis,a rare adverse effect, experienced by a patient treated with miltefosine for post-kala-azar dermal leishmaniasis. This adverse effect developed after 15 days of miltefosine consumption, and the patient himself discontinued the treatment. The ophthalmic complication was completely resolved with antibiotics and steroid eye drops. After recovery from the ophthalmic complication, the patient was successfully treated with liposomal amphotericin B for the skin lesions.

Introduction: Presence of asymptomatic individuals in endemic areas is common. The possible biomarkers in asymptomatic individuals 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 rK39 sero-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 individuals identified at base-line. The level of ADA, IL-10 and IFN-γ remained consistently high in asymptomatic cases and amongst these, ADA and IL-10 but not IFN-γ remained higher at the development of clinical symptoms into active VL. 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 may 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., Competing Interests: No authors have competing interest.

Post-kala-azar dermal leishmaniasis (PKDL) is clinical outcome of visceral leishmaniasis (VL) and is thought to be the potential reservoir of parasite. Miltefosine (MF) is the only oral drug existing for treatment of post-kala-azar dermal leishmaniasis (PKDL). Increased miltefosine tolerance in clinical isolates of Leishmania donovani has been reported and is one of the major concerns in the treatment of PKDL. Here, we report a highly ulcerated PKDL case that was successfully cured after miltefosine treatment., Competing Interests: The authors have declared that no competing interests exist.

Background: Surveillance of post-kala-azar dermal leishmaniasis (PKDL) is critical to the elimination of visceral leishmaniasis (VL). In this study we assessed the feasibility of using trained field workers for detecting suspected PKDL cases., Methods: A cross-sectional study using a multistage sampling technique was conducted in the Araria district of Bihar. Trained field workers were utilized for identification of suspected PKDL case., Results: We investigated 57 099 individuals from 11 300 households. The trained field workers were useful in identifying 107 (18%) probable PKDL cases. The calculated PKDL prevalences were 18.7/10 000 and 9.7/10 000 for probable and confirmed PKDL cases, respectively. The median duration of onset of PKDL was 23 months (interquartile range 16.5-56.5). The younger age group developed PKDL significantly more often compared with the older age group (p=0.007). Of the 107 patients, 25 (55.5%) were positive by microscopy of slit skin smear and 42 (93.3%) by polymerase chain reaction. Of 45 patients, 33 (73%) PKDL cases were cured after full treatment. The risk of not being cured with incomplete treatment was three times higher than with complete treatment (relative risk 3.12 [95% confidence interval 1.23 to 8.67], p=0.004)., Conclusions: We conclude that the prevalence of PKDL is high and the use of trained field workers may be feasible to actively detect PKDL cases in VL-endemic areas of Bihar, India., (© The Author(s) 2019. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Liposomal amphotericin B is being used increasingly to reduce the burden of kala-azar from the Indian subcontinent. There are studies which have evaluated efficacy and safety of liposomal amphotericin B for visceral leishmaniasis in all age groups. However, the only study that specifically addressed treatment of childhood visceral leishmaniasis did not include all ages or document renal and liver function. We, therefore, felt it was important to reassess the efficacy and safety of single dose liposomal amphotericin B in children and adolescents. A total of 100 parasitologically confirmed visceral leishmaniasis patients aged < 15 years were included in this study. Participants consisted of 65 males and 35 females. All of them had come from the endemic region of Bihar. They were administered one dose intravenous infusion of liposomal amphptericin B at 10 mg/kg body weight. Efficacy was assessed as initial and final cure at 1 and 6 months, respectively, and safety of all participants who were recruited in the study. The initial and final cure rate by per protocol analysis was 100% and 97.9%, respectively. Chills and rigors were the most commonly occurring adverse events (AEs). All the AEs were mild in intensity, and none of the patients experienced any serious AEs. No patients developed nephrotoxicity. Our finding indicates that liposomal amphotericin B at 10 mg/kg body weight is safe and effective in children. Results of our study support the use of single dose liposomal amphotericin B in all age group populations for elimination of kala-azar from the Indian subcontinent.

Background: Post kala-azar dermal leishmaniasis (PKDL) is a dermatological disorder caused by protozoal parasite Leishmania donovani. PKDL cases are thought to be a reservoir of parasites and may increase cases of visceral leishmaniasis. The disease is not life threatening but cosmetic disfigurement associated with it may impair the patients' quality of life. This study aimed to assess the health related quality of life in patients with post kalaazar dermal leishmanasis for the first time., Methods: A total of 92 PKDL cases and 96 healthy participants filled out the questionnaires. The Dermatology Life Quality Index (DLQI) and SF 36 questionnaire were used to assess the quality of life. Data on socio-demographic and clinical features were also collected. The collected data were analyzed by using SPSS software (version 16), Student's t-test, analysis of variance (ANOVA) was applied for comparison of means., Results: PKDL patients experienced very large impact on their quality of life. The mean score of DLQI was 11.41. Highest impact was found in symptoms and feelings and lowest impact was observed for personal relationship domain. Patients below 20 years age group found to have lower quality of life. There was a significant difference in mean DLQI scores with regard to age and severity of lesions (P < 0.05). No significant difference was observed with respect to gender, duration and location of lesions (p > 0.05)., Conclusion: PKDL significantly impaired the patient's quality of life. Further studies to assess the impact of treatment on quality of life in these patients are recommended.

Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and Zinc Sulfate (ZnSO4). Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death. Therefore, cellular zinc homeostasis in Leishmania can be explored for new drug targets and chemotherapeutics to control Leishmanial growth and disease progression.

Background: Post kala-azar dermal leishmaniasis (PKDL) is a skin disorder that usually occurs among patients with a past history of visceral leishmaniasis (VL). Cases are also reported without a history of VL. There is no satisfactory treatment regimen available at present. We aimed to compare the efficacy and safety of amphotericin B in two different doses (0.5mg/kg vs 1mg/kg) in a prospective randomized trial in 50 PKDL patients., Methods: In this open label study 50 patients with PKDL, aged between 5-60 years were randomized in two groups. Group A received amphotericin B in the dose of 0.5 mg/kg in 5% dextrose, daily for 20 infusions for 3 courses at an interval of 15 days between each course and Group B received amphotericin B in the dose of 1mg/kg in 5% dextrose on alternate days, 20 infusions for 3 courses an interval of 15 days between each course and followed up for one year., Results: A total of 50 patients were enrolled, 25 in each of group A and group B. Two patients lost to follow up and three patients withdrew consent due to adverse events. The initial cure rate was 92% in group A and 88% in group B by intention to treat analysis and final cure rate by per protocol analysis was 95.65% and 95.45% in group A and group B respectively. Two patients each from either group relapsed. Nephrotoxicity was the most common adverse event occurring in both the groups., Conclusion: The lower dose appears to have fewer adverse events however, nephrotoxicity remains a problem in both regimens. The 0.5mg/kg regimen may be considered instead of the higher dosage however safer treatments remain critical for PKDL treatment.

In this work, we have described the expression of ecto-ATPDase on the external surface of Leishmania donovani. This enzyme has the ability to hydrolyze extracellular ATP. There is a low level of ATP hydrolysis in the absence of divalent cation 2.5 ± 0.51 nM Pi 10 7 cells/h which shows the divalent cation-dependent activity of this enzyme in the intact parasite. However, MgCl 2 stimulated the ATP hydrolysis to a greater extent compared with CaCl 2 and ZnCl 2 . This activity was also observed when replaced by MnCl 2 . The Mg-dependent ecto-ATPase activity was 46.58 ± 6.248 nM Pi 10 7 cells/h. The apparent K m for ATP was 5.76 mM. Since Leishmania also possesses acid phosphatase activity and to discard the possibility that the observed ATP hydrolysis was due to acid phosphatase, the effect of pH was examined. In the pH range 6.0-9.0, in which the cells were viable, the phosphatase activity decreased while ATPase activity increased. To show that the observed ATP hydrolysis was not due to phosphatase or nucleotidase activity, certain inhibitors for these enzymes were tested. Vandate and NaF inhibited the phosphatase activity; Ammonium molybdate inhibited 5'-nucleotidase activity, but these inhibitors did not inhibit the observed ATP hydrolysis. However, when ADP was used as a substrate, there was no inhibition of ATP hydrolysis showing the possibility of ATP diphosphohydrolase activity. To confirm that this Mg-dependent ATPase activity is an ecto-ATPase activity, we used an impermeable inhibitor, 4,4'-diisothiocyanostilbene 2,-2'-disulfonic acid, as well as suramin, an antagonist of P2-purinoceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg 2+ -dependent ATPase activity in a dose-dependent manner. The presence of L. donovani E-NTPDase activity was demonstrated using antibodies against NTPDase by Western blotting and flow cytometry. The presence of Mg 2+ -dependent ATP diphosphohydrolase activity on the surface of L. donovani modulates the nucleotide concentration and protects the parasite from the lytic effects of the nucleotides mainly ATP. Ecto-ATPDase from L. donovani may be further characterized as a good antigen and as a target for immunodiagnosis and drug development, respectively.

Background: Visceral leishmaniasis (VL), with the squeal of Post-kala-azar dermal leishmaniasis (PKDL), is a global threat for health. Studies have shown sodium stibogluconate (SSG) resistance in VL patients with chronic arsenic exposure. Here, we assessed the association between arsenic exposure and risk of developing PKDL in treated VL patients., Methods: In this retrospective study, PKDL patients (n = 139), earlier treated with SSG or any other drug during VL, were selected from the study cohort. Trained physicians, unaware of arsenic exposure, interviewed them and collected relevant data in a questionnaire format. All probable water sources were identified around the patient's house and water was collected for evaluation of arsenic concentration. A GIS-based village-level digital database of PKDL cases and arsenic concentration in groundwater was developed and individual point location of PKDL cases were overlaid on an integrated GIS map. We used multivariate logistic regression analysis to assess odds ratios (ORs) for association between arsenic exposure and PKDL development., Results: Out of the 429 water samples tested, 403 had arsenic content of over 10 μg/L, with highest level of 432 μg/L among the seven study villages. Multivariate adjusted ORs for risk of PKDL development in comparison of arsenic concentrations of 10.1-200 μg/L and 200.1-432.0 μg/L were 1.85 (1.13-3.03) and 2.31 (1.39-3.8) respectively. Interestingly, similar results were found for daily dose of arsenic and total arsenic concentration in urine sample of the individual. The multivariate-adjusted OR for comparison of high baseline arsenic exposure to low baseline arsenic exposure of the individuals in the study cohort was 1.66 (95% CI 1.02-2.7; p = 0.04)., Conclusion: Our findings indicate the need to consider environmental factors, like long time arsenic exposure, as an additional influence on treated VL patients towards risk of PKDL development in Bihar., Competing Interests: The authors have declared that no competing interests exist.

Background: Visceral Leishmaniasis, commonly known as kala-azar, is widely prevalent in Bihar. The National Kala-azar Control Program has applied house-to-house survey approach several times for estimating Kala-azar incidence in the past. However, this approach includes huge logistics and operational cost, as occurrence of kala-azar is clustered in nature. The present study aims to compare efficiency, cost and feasibility of snowball sampling approach to house-to-house survey approach in capturing kala-azar cases in two endemic districts of Bihar, India., Methodology/principal Findings: A community based cross-sectional study was conducted in two highly endemic Primary Health Centre (PHC) areas, each from two endemic districts of Bihar, India. Snowball technique (used to locate potential subjects with help of key informants where subjects are hard to locate) and house-to-house survey technique were applied to detect all the new cases of Kala-azar during a defined reference period of one year i.e. June, 2010 to May, 2011. The study covered a total of 105,035 households with 537,153 populations. Out of total 561 cases and 17 deaths probably due to kala-azar, identified by the study, snowball sampling approach captured only 221 cases and 13 deaths, whereas 489 cases and 17 deaths were detected by house-to-house survey approach. Higher value of McNemar's χ² statistics (64; p<0.0001) for house-to-house survey approach than snowball sampling and relative difference (>1) indicates that most of the kala-azar cases missed by snowball sampling were captured by house-to-house approach with 13% of omission., Conclusion/significance: Snowball sampling was not found sensitive enough as it captured only about 50% of VL cases. However, it captured about 77% of the deaths probably due to kala-azar and was found more cost-effective than house-to-house approach. Standardization of snowball approach with improved procedure, training and logistics may enhance the sensitivity of snowball sampling and its application in national Kala-azar elimination programme as cost-effective approach for estimation of kala-azar burden., Competing Interests: The authors declare that they have no conflict of interests.

Purpose: Visceral leishmaniasis (VL), a protozoan disease, is 100% fatal if left untreated. Anemia is common in VL which plays a role in expression of clinically overt VL disease. Laboratory clues are scarce for strengthening clinical suspicion for severity in VL. Hypertriglyceridemia has emerged as a new concept for the diagnosis and prognosis in VL. The present study is aimed at correlating the magnitude of hypertriglyceridemia with the severity in VL., Materials and Methods: A retrospective case-control study was conducted between January 2012 to December 2013 among 124 patients coming for treatment from VL endemic areas, who had fever of more than 15 days and did not respond to antimalarials and antibiotics. The parasitologically confirmed VL cases (n = 87) were categorized as mild/moderate (n = 60) and severe (n = 27) groups according to WHO classification for anemia and parasite burden. Serum triglycerides were assayed in VL groups along with controls (n = 37)., Results: Serum triglyceride level was significantly higher in VL than controls [mean values were 173.50 ± 47.67 versus 127.1 ± 53.79 mg/dl, respectively (p < 0.0001)]. Triglyceride level was significantly higher in severe than in mild/moderate group of VL [211.3 ± 50.2 mg/dl versus 134 ± 45.09 mg/dl, respectively (p < 0.0001)]. Hypertriglyceridemia (>161.7 mg/dl) was noted in all severe VL patients, compared to 31.66% of mild or moderate group (p < 0.0001). There was no significant difference between mild/moderate VL and controls., Conclusions: It is hypothesized that hypertriglyceridemia could be of additional diagnostic benefit to assess the probability and severity of VL in endemic areas.

Post-kala-azar dermal leishmaniasis (PKDL) is an important factor in kala-azar transmission; hence its early detection and assessment of effective treatment is very important for disease control. In present study on 60 PKDL cases presented with macular, mixed papulonodular, or erythematous lesions, Leishmania parasites were demonstrated microscopically in 91% of papulonodular and 40% of macular lesions. Cellular infiltrates in skin biopsy imprint smears from lesions were mononuclear cells, 25-300/OIF (oil immersion field), predominantly histiocytes with vacuolation, many lymphocytes, some plasma cells, and Leishmania amastigotes 0-20/OIF. Cases with no demonstrable parasites were diagnosed on the basis of past history of VL, lesion's distribution, cytopathological changes, and positive DAT (86.83%). Following antileishmanial treatment with SAG, papulonodular forms of PKDL lesions disappeared clinically but microscopically the mononuclear cells (20-200/OIF) persisted in the dermal lesions. Response observed in macular PKDL lesions was poor which persisted both clinically and cytopathologically. Follow-up of PKDL will assess the effectivity of treatment as either disappearance of lesions or any relapse. Studies on involvement of immunological factors, that is, certain cytokines (IL-10, TGF-β, etc.) and chemokines (macrophage inflammatory protein, MIP 1-α, etc.) in PKDL, may provide insight for any role in the treatment response.

Background: India is home to 60% of the total global visceral leishmaniasis (VL) population. Use of long-term oral (e.g. miltefosine) and parenteral drugs, considered the mainstay for treatment of VL, is now faced with increased resistance, decreased efficacy, low compliance and safety issues. The authors evaluated the efficacy and safety of an alternate treatment option, i.e. single infusion of preformed amphotericin B (AmB) lipid emulsion (ABLE) in comparison with that of liposomal formulation (LAmB)., Methods: In this multicentric, open-label study, 500 patients with VL were randomly assigned in a 3:1 ratio to receive 15 mg/kg single infusion of either ABLE (N = 376) or LAmB (N = 124). Initial cure (Day 30/45), clinical improvement (Day 30) and long term definitive cure (Day 180) were assessed., Findings: A total of 326 (86.7%) patients in the ABLE group and 122 (98.4%) patients in the LAmB group completed the study. Initial cure was achieved by 95.9% of patients in the ABLE group compared to 100% in the LAmB group (p = 0.028; 95% CI: -0.0663, -0.0150). Clinical improvement was comparable between treatments (ABLE: 98.9% vs. LAmB: 98.4%). Definitive cure was achieved in 85.9% with ABLE compared to 98.4% with LAmB. Infusion-related pyrexia (37.2% vs. 32.3%) and chills (18.4% vs. 18.5%) were comparable between ABLE and LAmB, respectively. Treatment-related serious adverse events were fewer in ABLE (0.3%) compared to LAmB (1.6%). Two deaths occurred in the ABLE group, of which one was probably related to the study drug. Nephrotoxicity and hepatotoxicity was not observed in either group., Conclusions: ABLE 15 mg/kg single infusion had favorable efficacy and was well tolerated. Considering the demographic profile of the population in this region, a single dose treatment offers advantages in terms of compliance, cost and applicability., Trial Registration: www.clinicaltrials.gov NCT00876824.

Diagnosis of post-kala-azar dermal leishmaniasis (PKDL), particularly the macular form, is difficult when based on microscopy. This study compared the results of nested PCR (91.9% positive samples) with imprint smear microscopy (70.9% positive samples) for 62 PKDL samples. We found that nested PCR, which indicated 87.5% positivity for the macular lesions, compared to 41.6% positivity by imprint smear microscopy, is an efficient method for early diagnosis of PKDL.

Project: Chronic visceral leishmaniasis (VL) is an increasingly common problem in disease endemic states of India. Identification of prognosis risk factor in patients with VL may lead to preventive actions, toward decreasing its mortality in chronic individuals. Though serum Zinc levels are decreased in patients of VL, limited information is available regarding trace elements status in acute and chronic VL patients. The present study was undertaken to compare serum trace elements concentrations in acute and chronic VL patients., Procedure: Acute (mean age=28.64 years), chronic (mean age=23.68 years) VL patients and healthy controls (mean age=23.05 years) who agreed to provide blood specimens for laboratory investigations participated in this study. Serum zinc (Zn), copper (Cu), iron (Fe), magnesium (Mg) and calcium (Ca) were measured spectrophotometrically using chemistry analyzer., Results: Serum Zn concentration was comparatively much decreased in chronic VL than to acute ones (p=0.007) while serum Mg was higher in chronic VL than acute (p=0.002) ones. There was no statistically significant difference between acute and chronic VL in serum concentrations of Cu, Fe and Ca., Conclusions: Serum Zn levels were much decreased and serum Mg were increased in chronic VL as compared to acute cases. The serum concentrations of Fe and Ca did not show any difference between two groups. The serum Cu was increased in both groups but more in chronic ones. Serum Zn and Mg could be a potential prognosis factor for chronic VL patients. We hypothesize zinc supplementation as a chemo preventive agent for chronic VL cases, particularly in endemic areas., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Visceral leishmaniasis, caused by Leishmania donovani, is a serious form of leishmaniasis and fatal if untreated. Nearly half of the VL cases are children. There are very few studies of renal function in pediatric visceral leishmaniasis. The aim of this study was to evaluate renal dysfunction by studying glomerular filtration rate (GFR), microalbuminuria, and microscopic examination of urine. Laboratory analysis was performed on blood and urine samples of 40 parasitologically confirmed pediatric VL cases. Laboratory data of urine examination showed albuminuria in 10% (4/40), white blood cells in 20% (8/40), hematuria in 10% (4/40), microalbuminuria in 37.5% (15/40), and decreased GFR in 27.5% (11/40). Renal involvement was manifested in most of the pediatric VL cases. These findings may help clinicians in decision making for safe and suitable antileishmanial treatment particularly in childhood VL.

Post-kala-azar dermal leishmaniasis (PKDL) has important public health implications for transmission of visceral leishmaniasis (VL). Clinical and epidemiologic profiles of 102 PKDL patients showed that median age of males and females at the time of diagnosis was significantly different (P = 0.013). A significant association was observed between family history of VL and sex of PKDL patients (χ(2) = 5.72, P < 0.01). Nearly 33% of the patients showed development of PKDL within one year of VL treatment. The observed time (median = 12 months) between appearance of lesions and diagnosis is an important factor in VL transmission. A significant association was observed between type of lesions and duration of appearance after VL treatment (χ(2) = 6.59, P = 0.001). Because PKDL was observed during treatment with all currently used anti-leishmanial drugs, new drug regimens having high cure rates and potential to lower the PKDL incidence need to be investigated.

Post kala-azar dermal leishmaniasis (PKDL) is a skin manifestation that usually develops after treatment of visceral leishmaniasis (VL), a major public health problem in India. The diagnosis and management of PKDL is complex. This is the first case report from India in which PKDL occurred after paromomycin treatment for VL in an Indian patient.

Case: We report a 32-year old relapse case of Visceral leishmaniasis, treated with Paromomycin who belonged from a endemic zone of Bihar state, India. After confirmation, he was treated with Amphotericin B, followed by Liposomal Amphotericin B in full course and even in higher dose. But after each therapy, the patient either did not responded or relapsed after treatment. Ultimately, the patient was successfully treated with combination therapy of Liposomal amphotericin B and Miltefosine without any relapse., Conclusion: The multi-drug unresponsive Visceral leishmaniasis cases could pose a major threat to treatment strategy in the elimination program. In such situation, combination therapy seems to be a better approach that needs to be explored.

Background: Improved treatment approaches are needed for visceral leishmaniasis. We assessed the efficacy and safety of three potential short-course combination treatments compared with the standard monotherapy in India., Methods: Standard treatment (1 mg/kg amphotericin B infusion on alternate days for 30 days, total dose 15 mg/kg) was compared with three drug combinations (single injection of 5 mg/kg liposomal amphotericin B and 7-day 50 mg oral miltefosine or single 10-day 11 mg/kg intramuscular paromomycin; or 10 days each of miltefosine and paromomycin) in an open-label, parallel-group, non-inferiority, randomised controlled trial in two hospital sites in Bihar, India. Patients aged 5-60 years with parasitologically confirmed visceral leishmaniasis were randomly assigned one of the four treatments by the trial statistician by use of a computer-generated list. Clinical assessments were done at the end of treatment (15 days on combination treatment; 31 days for standard treatment) and after 45 days and 6 months. The primary endpoint was definitive cure (defined as no sign or symptom of visceral leishmaniasis and parasitologically cured to the last follow-up). Analyses were done both by intention to treat and per protocol. This trial is registered with ClinicalTrials.gov, number NCT00696969., Findings: Between June, 2008, and July, 2009, 634 patients were assigned amphotericin B (n=157), liposomal amphotericin B with miltefosine (n=160) or paromomycin (n=158), or miltefosine and paromomycin (n=159). 618 patients were in the per-protocol population. There were two relapses in each group. The numbers with definitive cure at 6 months for the intention-to-treat population were 146 (cure rate 93·0%; CI 87·5-96·3) for amphotericin B, 156 (97·5%; 93·3-99·2) for liposomal amphotericin B and miltefosine, 154 (97·5%; 93·24-99·2) for liposomal amphotericin B and paromomycin, and 157 (98·7%; 95·1-99·8) for miltefosine and paromomycin. All combinations were non-inferior to the standard treatment, in both the intention-to-treat and per-protocol populations. Patients in the combination groups had fewer adverse events than did those assigned standard treatment., Interpretation: Combination treatments for visceral leishmaniasis are efficacious and safe, and decrease the duration of therapy, thereby encouraging adherence and reducing emergence of drug-resistant parasites., Funding: Drugs for Neglected Diseases initiative and the Indian Council of Medical Research., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Post-kala-azar dermal leishmaniasis is usually a sequel to visceral leishmaniasis. A 25-year-old woman presented with hypopigmented maculopapular lesions all over the body for the past 4 years without any previous history of visceral leishmaniasis. She was on treatment for leprosy and pulmonary tuberculosis for the past 2 months, but did not show any improvement. Investigations confirmed that she had post-kala-azar dermal leishmaniasis associated with pulmonary tuberculosis and HIV-1 infection. She was started on treatment for the triad of diseases, and showed improvement.

The membrane fluidity of antigen-presenting cells (APCs) has a significant bearing on T-cell-stimulating ability and is dependent on the cholesterol content of the membrane. The relationship, if any, between membrane fluidity and defective cell-mediated immunity in visceral leishmaniasis has been investigated. Systemic administration of cholesterol by liposome delivery (cholesterol liposomes) in Leishmania donovani-infected hamsters was found to cure the infection. Splenic macrophages as a prototype of APCs in infected hamsters had decreased membrane cholesterol and an inability to drive T cells, which was corrected by cholesterol liposome treatment. The effect was cholesterol specific because liposomes made up of the analogue 4-cholesten-3-one provided almost no protection. Infection led to increases in interleukin-10 (IL-10), transforming growth factor beta, and IL-4 signals and concomitant decreases in gamma interferon (IFN-gamma), tumor necrosis factor alpha, and inducible NO synthase signals, which reverted upon cholesterol liposome treatment. The antileishmanial T-cell repertoire, whose expansion appeared to be associated with protection, was presumably type Th1, as shown by enhanced IFN-gamma signals and the predominance of the immunoglobulin G2 isotype. The protected group produced significantly more reactive oxygen species and NO than the infected groups, which culminated in killing of L. donovani parasites. Therefore, cholesterol liposome treatment may be yet another simple strategy to enhance the cell-mediated immune response to L. donovani infection. To our knowledge, this is the first report on the therapeutic effect of cholesterol liposomes in any form of the disease.

We report two cases of post-kala-azar dermal leishmaniasis (PKDL), which had subsequently developed after successful treatment of visceral leishmaniasis with miltefosine. Both patients had maculo-nodular lesions all over the body, and they were diagnosed as PKDL by parasitologic examination for Leishmania donovani bodies in a skin snip of lesions. Patients were put on amphotericin B and responded very well for nodular lesions with one course of treatment. However, longer duration of the treatment is needed for total clearance of macular lesions from body surface in PKDL cases. This is the first case report of PKDL in India, which developed after successful treatment of visceral leishmaniasis with miltefosine.

The communication presents clinical response of cases of visceral leishmaniasis to treatment by two different brands of Amphotericin B. Fungizone was found to be slightly better than Amphotericin B, however, the difference is not statistically significant.

In the Indian state of Bihar, the sensitivities and specificities of direct agglutination tests (DAT) and rK39 test strips for the detection of Leishmania donovani infection in humans were explored and found to be generally good (92%-100%). When 172 asymptomatic individuals [16 'case-contacts' who lived in the same households as past or current, confirmed cases of visceral leishmaniasis (VL) and 156 other subjects from neighbouring households] were tested, the same 36 (21%) individuals, including all 16 'case-contacts', were found seropositive using each type of test. When followed-up after 3 months, 18 of the individuals who had been found seropositive in the baseline survey remained seropositive, and eight (44%) of these had developed symptomatic VL, with amastigotes in their splenic aspirates. Seven (44%) of the 16 'case-contacts' but only one (5%) of the other 20 subjects found seropositive at baseline went on to develop VL within 3 months. Although the strip test appeared slightly better than DAT for predicting the development of VL in the 172 subjects, either type of test may be very useful for the early detection of asymptomatic L. donovani infection and thus the identification of those at relatively high risk of developing VL.

Visceral leishmaniasis (VL), which is caused by the protozoa Leishmania donovani and transmitted by the bite of the female sand fly Phlebotomus argentipes, is common in Bihar, India. Wilson disease is an autosomal recessive disorder of copper metabolism in which copper is deposited in the brain and liver. We report a case of an extremely uncommon combination of these diseases in a patient. Treatment options for such a combination of diseases are limited and difficult.

Tyndalized milk of goat, cow, and buffalo was found to be a potential substitute for fetal bovine serum (FBS) in the medium for the cultivation of Leishmania donovani promastigotes. The numbers (means) of promastigotes reached 2.6 x 10(7), 2.3 x 10(7), and 2.1 x 10(7)/ml, respectively, in the medium supplemented with 10% milk of goat, cow, and buffalo, in comparison to 1.9 x 10(7)/ml in the control with 10% FBS. In primary isolation, the milk-supplemented medium showed that 22 out of 26 samples were positive for promastigotes (84.6%) and the cells were maintained successfully during the observed period of 6 months.

From a hospital-based surveillance carried out in Rajendra Memorial Research Institute of Medical Sciences, Patna, Bihar, India, the socio-economic, demographic and treatment response information of 737 patients admitted with visceral leishmaniasis (VL) during January 2001-December 2003, were analysed. The disease was two times higher in males than in females because of several factors including clothing pattern, sleeping habits and occupation. In Bihar, the second poorest state in India, poverty plays a major role in perpetuation of the disease, contributing to malnutrition, illiteracy (60%), and poor housing (82%). Further, presences of peri-domestic animal shelters around houses (63%) and vegetations (77%) facilitate breeding of sand fly vector. Clinical and laboratory characteristics were similar in the age groups <12 years and >12 years. The increasing unresponsiveness of VL patients to conventional anti-leishmanial drugs, e.g. sodium antimony gluconate (SAG) and pentamidine, has definitely posed a major therapeutic challenge in combating the disease. Amphotericin B, though costly, is highly effective. Miltefosine is a highly promising new oral drug for VL.

We describe here two cases, one male and one female, both age 40 years, with visceral leishmaniasis and HIV-1 co-infection. The female patient had features of Koch's abdomen. The male patient had features of tuberculous lymphadenitis and bilateral pleural effusion more marked on the right side. Both were treated with highly active antiretroviral therapy, antituberculous drugs, antibiotics, antifungal medicine (fluconazole) and miltefosine. Both patients showed marked improvement with therapy.

A case-control study was conducted to understand the risk factors associated with kala-azar in disease-endemic areas of Bihar, India. A total of 134 kala-azar cases treated at the Rajendra Memorial Research Institute of Medical Sciences in Patna and 406 healthy controls selected randomly from the neighborhoods of cases in their native villages were included in the study. Univariate analysis showed that education, a history of other diseases in the previous year, a history of kala-azar in the family, type of walls in houses, presence of a granary inside houses, presence of vegetation around houses, bamboo trees near houses, and irregular spraying around houses with DDT were risk factors. Multivariate analysis showed that a history of other diseases in the previous year (odds ratio [OR] = 3.6, P = 0.002), a history of kala-azar in the family (OR = 1.8, P = 0.03), mud-plastered walls in houses, (OR = 2.4, P = 0.0001], a granary inside houses (OR = 4.3, P = 0.0001), presence of bamboo trees around houses (OR = 2.3, P = 0.001), and houses not sprayed with DDT in the past six months (OR = 3.4, P = 0.0001) were significant risk factors for kala-azar. These results will be useful in developing kala-azar control programs for identifying intervention strategies such as better housing, regular and proper insecticide spraying, and promoting health awareness to the community residing in disease-endemic areas for reducing transmission and incidence of this disease.

A 37-year-old man was diagnosed as being infected with human immunodeficiency virus (HIV), tuberculosis (TB), tuberculoma of the brain, and visceral leishmaniasis (VL) at the Rajendra Memorial Institute of Medical Sciences in Bihar, India. He had taken anti-tuberculosis therapy (ATT) for two and a half months and had episodes of convulsions with loss of consciousness, tongue bites, and incontinence of urine. The results of a neurologic examination were normal except for a left plantar extensor. He was positive for both HIV-I (confirmed by Western blot) and VL (confirmed by splenic aspirate). Treatment was initiated with amphotericin B lipid complex, a four-drug regimen (rifampicin, isoniazid, ethambutol, and pyrazinamide) of ATT, highly active antiretroviral therapy, anti-convulsants, and other supportive therapies. A repeat computed tomography scan of the brain showed the disappearance of the lesion followed by gliosis. After six months, he was also cured of VL. The triad of infections (HIV, VL, and TB) is a real threat in Bihar as an emerging combination of diseases of public health importance. Keeping these facts in mind, efforts to develop simple and cost effective diagnostic techniques coupled with affordable therapeutic facilities are urgently needed in developing countries.

In a bid to characterize the antigens and immunization mechanisms which may be used to produce a protective response against L. donovani, role of lipid associated polysaccharide (LPS) antigen and whole antigen was evaluated. BALB/C mice were immunized with whole or LPS antigen in combination with one of three putative adjuvents (anti CD-2 antibody/FIA/0.85% Saline). LPS antigen emulsified in anti CD-2 antibody was found to induce significant antibodies in mice on day 28 against challenge with lethal dose of L. donovani. Immunoprophylactic properties of LPS and whole antigen was investigated on day 40 through cytokine elicitation (IL-2), MIF) in culture supernatants of spleen cells, but before that MHC-II expressed on macrophage was studied. The LPS antigen in combination with anti CD-2 antibody was found to be most immuno-reactive inducing higher MHC-II expression on macrophages which was associated with substantial rise in the level of MIF and IL-2. It coincided with decline in antibody titre in 100% mice immunized with LPS antigen while Leishmania injected as whole antigen failed to induce specific macrophage and T-cell response with all the above formulations. We surmise from our data that lipid associated polysaccharide antigen linked to anti CD-2 antibody has potential for eliciting protective immunity against Leishmania.

Objectives: A randomized clinical trial of low dosage combination of pentamidine and allopurinol was carried out with objectives to assess the efficacy and toxicity as compared to full dosage of pentamidine in antimony unresponsive visceral leishmaniasis (VL) patients., Methods: Using a randomized control clinical trial, a total of 158 antimony unresponsive patients of VL were randomly allocated into two treatment groups. Patients in one group (n=80) received half the dosage of pentamidine i.e. 2 mg/kg body weight by IM route on alternate day and allopurinol in dose of 15 mg/kg body weight in three divided dosages for 30 days; patients in the second group (n=78) received pentamidine in dose of 4 mg/kg body weight by IM route on alternate day for 15 injections in 30 days. The efficacy and safety of the two regimens were compared., Results: Apparent cure i.e. clinical and pathological cure at the end of therapy, in 78 (97.5%) and 67 (86%), and ultimate cure i.e. clinical and parasitological cure at the end of follow-up of six months, in 73 (91.25%) and 58 (74.35%) patients was observed in the combination regimen and single regimen group respectively. The difference of the ultimate cure between two groups of the patients was statistically significant (p < 0.01). In single regimen group, 11 (14%) patients showed primary unresponsiveness (with no response during treatment) and nine (13%) relapse (after six months of follow-up) respectively, where as in combination regimen group, two (2.5%) patients showed primary unresponsiveness and five (6.4%) relapse respectively. By the end of the treatment, the incidence of injection-related toxicity, such as rigor and fever, was same in both groups. No hyperglycemia was observed in combination therapy probably due to reduced dose of pentamidine and three patients in single regimen developed hyperglycemia and one of them developed irreversible hyperglycemia., Conclusions: The study showed that the combination of pentamidine (half dose) and allopurinol is more effective in achieving ultimate cure with an added advantage of reduced toxicity in unresponsive cases as compared to full pentamidine dose.

After presenting processed glycoprotein of Leishmania donovani to T-cell, macrophage seeks the help of a panel of T-cells lymphokines to transform from a state that sustains intra cellular replication of parasite to an effector state for destructing parasites. But esterase and trypsin of macrophage membrane prevent T-cells to release MIF. Role of soya-bean trypsin inhibitor (STI) has been exposed in the present study with a view to alter esterase functional behaviour of macrophage for control of T-cell activation and also, if T-cells once made responsive to antigen by STI do alter macrophage response to T-cells or not. Results establish STI as potent effector molecule, which can serve as an adjuvant to candidate T-cell epitope and synthetic peptide for development of anti-Kala-azar vaccine protocol in future.