The clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconatenonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD 50 ) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts of S-adenosyl-L-methionine:C-24-⌬-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3␤-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani. L eishmaniasis, or kala azar, is a group of diseases caused by a protozoan parasite of the genus Leishmania. Kala azar is a symptomatic infection of liver, spleen, and bone marrow. The global estimates for the incidence and prevalence of kala azar cases per year are 0.5 and 2.5 million, respectively (57). In India, visceral leishmaniasis (VL) has been reported in parts of West Bengal, Uttar Pradesh, and Bihar. It poses a major health problem in the state of Bihar, which accounts for nearly 90% of the total cases in India (51). Chemotherapy has proven to be the only effective way of controlling infections and is highly dependent upon antimonycontaining drugs, such as sodium stibogluconate (Pentostam). Amphotericin B (AmB) is used as the drug of choice when acquired drug resistance emerges for traditional antimony therapy, and nearly 64% of cases in regions of Bihar where VL is hyperendemic are now resistant to antimonials (28). Hexadecylphosphocholine (miltefosine) is the first orally administered drug for VL and the latest to enter the market. The dosing scheme of hexadecylphosphocholine is 100 mg/kg of body weight/day for 28 days in adults weighing Ն50 kg, 50 mg/kg/day in adults weighing Ͻ50 kg, and 2.5 mg/kg/day in children (maximum dose, 100 mg/day). Major concerns abou...

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A cohort of 91 asymptomatic individuals with visceral leishmaniasis (VL) were identified during base line screening using recombinant 39-aminoacid antigen (rk-39) and polymerase chain reaction (PCR) conducted from December 2005 to June 2006 involving 997 individuals of two highly endemic villages of Vaishali district, Bihar. The point prevalence of asymptomatic infection was 98 per 1000 persons at baseline. There was no statistically significant difference between rk-39 and PCR positivity rate (P>0.05), even though PCR positivity alone was found significantly higher (4.2%) than rk-39 positivity alone (2.6%). The monthly follow-up of the asymptomatic cohort revealed a disease conversion rate of 23.1 per 100 persons within a year. There was a statistically significant difference in conversion of disease when individuals were positive by both tests as compared to single tests by rk-39 and PCR (P<0.01). Disease conversion rate in the subjects residing in households with a history of VL (62%, 13/21) was higher than those residing in the households without a history of VL (38%, 8/21). Most of the identified asymptomatic individuals were from low socio-economic strata similar to that of VL cases in general. Apart from rk-39, PCR may be considered for screening of asymptomatic Leishmania donovani infection in large-scale epidemiological studies. Screening of asymptomatic cases and their close follow-up to ascertain early detection and treatment of VL may be considered in addition to the existing VL control strategies.

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Sir2 plays a critical role in AmB resistance by regulating MDR1, ROS concentration and apoptosis-like phenomena and may be a new resistance marker for visceral leishmaniasis.

BackgroundThe World Health Organization has targeted elimination of visceral leishmaniasis (VL) in the Indian subcontinent (ISC) by 2020. Despite distinctive decline seen in the number of VL cases in ISC, there is still a quest for development of a diagnostic test which has the utility for detection of active infection and relapse cases and as a test of cure. The present study validated the sensitivity and specificity of SYBR Green I based closed tube LAMP assay reported by us for diagnosis of VL.MethodologyThe validation study was carried out at two endemic sites in India, located at Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna and Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi. Standard operating protocols were provided at the two sites for applying LAMP assay on confirmed VL cases. The diagnostic accuracy of LAMP assay was evaluated by Receiver operator curve (ROC) analysis. Furthermore, a simplified LAMP assay based on direct blood lysis, DBL-LAMP, was developed and verified for its diagnostic accuracy.Principal findingsA total of 267 eligible participants were included in the study which comprised of 179 VL cases and 88 controls. Sensitivity and specificity of the LAMP assay were 98.32% (95% C.I– 95.2–99.7%) and 96.59% (95% C.I.-90.4–99.3%), respectively. ROC curve analysis depicted no significant difference between area under curve (AUCROC) for LAMP assay and rK39 RDT, indicative of LAMP as an excellent diagnostic test. DBL-LAMP assay, performed on 67 VL and 100 control samples, yielded a sensitivity of 93.05% (95% C.I- 84.75–97%) and specificity of 100% (95% C.I.- 96.30–100%).Conclusions/SignificanceThe validated closed tube LAMP for diagnosis of VL will provide impetus to the ongoing VL elimination programme in ISC. The assay based on direct blood lysis promotes its scope for application in field settings by further reducing time and cost.

Abstract. 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 dermatosis caused by Leishmania donovani parasites in the skin after apparently successful treatment of patients with visceral leishmaniasis (VL) or those without a history of VL. It is characterized by macular, maculopapular, and nodular lesions in patients recovering from VL who are otherwise healthy.1 It occurs in nearly 10-20% of patients cured of VL within 1-5 years in India, and in approximately 50% of patients in Sudan within six months. [1][2][3][4] In the Indian subcontinent, kala-azar appears to occur at an interval of 10-15 years.5 Patients with PKDL have immense public health importance because they act as reservoirs of infection. 6Because there is no animal reservoir of L. donovani, in this sub-continent, it is important to detect patients with PKDL and treat them properly to prevent future outbreaks of VL. 7It has been reported that a large proportion of VL patients treated with sodium antimony gluconate (SAG) have a high probability of development of PKDL.8 It is not known whether other anti-leishmanial drugs have a similar posttreatment effect in development of PKDL. Clinical and epidemiologic aspects of PKDL are important for understanding transmission dynamics and defining VL control strategies. Because there is no mechanism currently available to detect PKDL cases at the community level, control strategies currently operational in disease-endemic areas would fail to achieve their goal of elimination. Therefore, we studied a cohort of PKDL cases diagnosed during 2007-2010 to identify clinical and epidemiologic features that are important to public health.The study was conducted at the Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Patna, India during June 2007-December 2010. A total of 102 confirmed patients with PKDL were enrolled in the study after providing informed consent. A semi-structured questionnaire was administered to each person to obtain demographic characteristics, area of residence, history of VL, relapse of VL, family history of VL or...