Vidya Nand Ravi Das

Efficacy and Safety of Amphotericin B Emulsion versus Liposomal Formulation in Indian Patients with Visceral Leishmaniasis: A Randomized, Open-Label Study

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 visceral leishmaniasis in Bihar India: comparison of the rK39 rapid diagnostic test on whole blood versus serum.

Background Antibody-detecting rapid diagnostic tests (RDTs) against rK39 are available to aid in the diagnosis of visceral leishmaniasis (VL). Although these rK39 RDTs have been developed, validated and approved for use with serum, they are universally performed using whole blood. It was therefore necessary to determine whether this RDT is as sensitive on whole blood as on serum. Method and Principal Findings In this study we compared the rK39 RDT on serum and blood samples from 624 individuals with symptoms of VL attending the outpatient clinic at the Rajendra Memorial Research Institute of Medical Sciences, Patna, India. A total of 251 cases (40%) were both serum and blood-positive and 26 cases (4%) were identified as blood-negative and serum-positive. These 26 individuals in general had low titer antibodies against rK39 as determined by ELISA and follow-up on most of these individuals revealed none had persistent VL symptoms. The Cohen kappa index comparing blood and serum was 0.88 indicating excellent concordance. Conclusion Although the concordance was excellent, it is possible to miss rK39 positive individuals when using blood and the titer of anti-rK39 antibodies is low. We recommend that when an individual from an endemic area has obvious clinical symptoms of VL and the whole blood rK39 RDT is negative, that the test should be redone 2–3 weeks later if the symptoms persist.

Induction of IL-10 and TGFβ from CD4+CD25+FoxP3+ T Cells Correlates with Parasite Load in Indian Kala-azar Patients Infected with Leishmania donovani.

Background Visceral leishmaniasis (VL) is distinguished by a complex interplay of immune response and parasite multiplication inside host cells. However, the direct association between different immunological correlates and parasite numbers remains largely unknown. Methodology/Principal Findings We examined the plasma levels of different disease promoting/protective as well as Th17 cytokines and found IL-10, TGFβ and IL-17 to be significantly correlated with parasite load in VL patients (r = 0.52, 0.53 and 0.51 for IL-10, TGFβ and IL-17, respectively). We then extended our investigation to a more antigen-specific response and found leishmanial antigen stimulated levels of both IL-10 and TGFβ to be significantly associated with parasite load (r = 0.71 and 0.72 for IL-10 and TGFβ respectively). In addition to cytokines we also looked for different cellular subtypes that could contribute to cytokine secretion and parasite persistence. Our observations manifested an association between different Treg cell markers and disease progression as absolute numbers of CD4+CD25+ (r = 0.55), CD4+CD25hi (r = 0.61) as well as percentages of CD4+CD25+FoxP3+ T cells (r = 0.68) all correlated with parasite load. Encouraged by these results, we investigated a link between these immunological components and interestingly found both CD4+CD25+ and CD4+CD25+FoxP3+ Treg cells to secrete significantly (p<0.05) higher amounts of not only IL-10 but also TGFβ in comparison to corresponding CD25- T cells. Conclusions/Significance Our findings shed some light on source(s) of TGFβ and suggest an association between these disease promoting cytokines and Treg cells with parasite load during active disease. Moreover, the direct evidence of CD4+CD25+FoxP3+ Treg cells as a source of IL-10 and TGFβ during active VL could open new avenues for immunotherapy towards cure of this potentially fatal disease.

Longitudinal study of transmission in households with visceral leishmaniasis, asymptomatic infections and PKDL in highly endemic villages in Bihar, India

Background Visceral Leishmaniasis (VL) is a neglected tropical disease that afflicts some of the poorest populations in the world including people living in the Bihar state of India. Due to efforts from local governments, NGOs and international organizations, the number of VL cases has declined in recent years. Despite this progress, the reservoir for transmission remains to be clearly defined since it is unknown what role post kala-azar dermal leishmaniasis (PKDL) and asymptomatic infections play in transmission. This information is vital to establish effective surveillance and monitoring to sustainably eliminate VL. Methodology/Principal Findings We performed a longitudinal study over a 24-month period to examine VL transmission and seroconversion in households with VL, PKDL and asymptomatic infections in the Saran and Muzaffarpur districts of Bihar. During the initial screening of 5,144 people in 16 highly endemic villages, 195 cases of recently treated VL, 116 healthy rK39 positive cases and 31 PKDL cases were identified. Approximately half of the rK39-positive healthy cases identified during the initial 6-month screening period were from households (HHs) where a VL case had been identified. During the 18-month follow-up period, seroconversion of family members in the HHs with VL cases, PKDL cases, and rK39-positive individuals was similar to control HHs. Therefore, seroconversion was highest in HHs closest to the time of VL disease of a household member and there was no evidence of higher transmission in households with PKDL or healthy rK39-positive HHs. Moreover, within the PKDL HHs, (the initial 31 PKDL cases plus an additional 66 PKDL cases), there were no cases of VL identified during the initial screen or the 18-month follow-up. Notably, 23% of the PKDL cases had no prior history of VL suggesting that infection resulting directly in PKDL is more common than previously estimated. Conclusions/Significance These observations argue that acute VL cases represent the major reservoir for transmission in these villages and early identification and treatment of VL cases should remain a priority for VL elimination. We were unable to obtain evidence that transmission occurs in HHs with a PKDL case.

Impact of ASHA Training on Active Case Detection of Visceral Leishmaniasis in Bihar, India

Background One of the major challenges for management of visceral leishmaniasis (VL) is early diagnosis of cases to improve treatment outcome and reduce transmission. We have therefore investigated active case detection of VL with the help of accredited social health activists (ASHA). ASHAs are women who live in the community and receive performance-based incentives for overseeing maternal and other health-related issues in their village. Methods and Principal Finding Through conducting interviews with 400 randomly selected ASHAs from four primary health care centers (PHCs), it was observed that their level of knowledge about visceral leishmaniasis (VL) regarding transmission, diagnosis, and treatment was limited. The baseline data indicated that less than 10% of VL cases seeking treatment at the PHCs were referred by ASHAs. To increase the knowledge and the referral rate of VL cases by ASHAs, training sessions were carried out during the monthly ASHA meetings at their respective PHCs. Following a single training session, the referral rate increased from less than 10% to over 27% and the overall knowledge about VL substantially improved. It was not possible, however, to demonstrate that ASHA training reduced the time that individuals had fever before treatment at the PHC. Conclusions Training ASHAs to identify VL cases in villages for early diagnosis and treatment at the local PHC is feasible and should be undertaken routinely to improve knowledge about VL.

Noninvasive Diagnosis of Visceral Leishmaniasis: Development and Evaluation of Two Urine-Based Immunoassays for Detection of Leishmania donovani Infection in India.

Background Visceral Leishmaniasis (VL), a severe parasitic disease, could be fatal if diagnosis and treatment is delayed. Post kala-azar dermal leishmaniasis (PKDL), a skin related outcome, is a potential reservoir for the spread of VL. Diagnostic tests available for VL such as tissue aspiration are invasive and painful although they are capable of evaluating the treatment response. Serological tests although less invasive than tissue aspiration are incompetent to assess cure. Parasitological examination of slit-skin smear along with the clinical symptoms is routinely used for diagnosis of PKDL. Therefore, a noninvasive test with acceptable sensitivity and competency, additionally, to decide cure would be an asset in disease management and control. Methodology/principal findings We describe here, the development of antibody-capture ELISA and field adaptable dipstick test as noninvasive diagnostic tools for VL and PKDL and as a test of cure in VL treatment. Sensitivity and specificity of urine-ELISA were 97.94% (95/97) and 100% (75/75) respectively, for VL. Importantly, dipstick test demonstrated 100% sensitivity (97/97) and specificity (75/75) in VL diagnosis. Degree of agreement of the two methods with tissue aspiration was 98.83% (κ = 0.97) and 100% (κ = 1), for ELISA and dipstick test, respectively. Both the tests had 100% positivity for PKDL (14/14) cases. ELISA and dipstick test illustrated treatment efficacy in about 90% (16/18) VL cases when eventually turned negative after six months of treatment. Conclusions/significance ELISA and dipstick test found immensely effective for diagnosis of VL and PKDL through urine samples thus, may substitute the existing invasive diagnostics. Utility of these tests as indirect methods of monitoring parasite clearance can define infected versus cured. Urine-based dipstick test is simple, sensitive and above all noninvasive method that may help not only in active VL case detection but also to ascertain treatment response. It can therefore, be deployed widely for interventions in disease management of VL particularly in poor resource outskirts.

Leishmania donovani resistant to Ambisome or Miltefosine exacerbates CD58 expression on NK cells and promotes trans-membrane migration in association with CD2

HighlightsHigher expression of CD58 but not of CD2 was observed on CD56+ cells during Visceral Leishmaniasis.Expression of CD58 on CD56+ cells were further exacerbated in Ambisome or Miltefosine relapsed VL.Ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells reduces after stimulation with Ld.Ambisome or Miltefosine resistance Ld significantly increase CD58 expression on CD56+ cells.Antagonist to CD58 or CD2, down‐regulates CD56+ cell recruitment at Ld infection site. Abstract Visceral leishmaniasis (VL) is a disease that is associated with compromised immunity and drug un‐responsiveness as well as with the emergence of drug resistance in Leishmania donovani (Ld). Ld down‐modulates cellular immunity by manipulating signaling agents, including a higher expression of the adhesion molecule CD58. The expression of CD58 and CD2 on natural killer (NK) cells facilitates intercellular adhesion and signaling. The influence of drug‐resistant Ld on the expression of CD58 and CD2 was addressed in this study. The mean florescence intensity (MFI) of CD58 but not of CD2 was twofold higher on CD56+ cells during VL, but was down‐regulated after treatment. In addition, MFI of CD58 on CD56+ cells was further exacerbated in VL subjects who had relapsed after Ambisome or Miltefosine treatment. The same pattern of CD58 expression was also obtained upon stimulation of healthy peripheral blood mononuclear cells with Miltefosine‐ or Ambisome‐resistant Ld. The ratio of CD56+CD58+IFN‐&ggr;+/CD56+CD58+IL‐10+ cells was reduced by 6.98‐fold after stimulation with Ld. Further, an antagonist to CD58 or its counter‐receptor CD2 down‐regulated CD56+ NK cell recruitment across a polycarbonate trans‐membrane at Ld infection sites. This study reports that factors associated with drug resistance in Ld probably promote higher expression of CD58 on CD56+ cells and their migration to the infection site in association with CD2.

Repeated training of accredited social health activists (ASHAs) for improved detection of visceral leishmaniasis cases in Bihar, India

Accredited Social Health Activists (ASHAs) are incentive-based, female health workers responsible for a village of 1000 population and living in the same community and render valuable services towards maternal and child health care, polio elimination program and other health care-related activities including visceral leishmaniasis (VL). One of the major health concerns is that cases remain in the endemic villages for weeks without treatment causing increased likelihood to treatment failure and disease transmission in the community. To address this problem, we have begun a training program for ASHAs to enhance early detection of potential VL cases and referring them to their local Primary Health Centers (PHCs) for diagnosis and treatment. The result of this training showed increased referral rate to PHCs for diagnosis and treatment. Encouraged with the results from a single training session, we determined in the present study whether repeated training of ASHAs resulted in an a further increase in VL case referral to the local PHCs. After two training sessions, VL referrals by ASHAs increased to 46% as compared to 28% after a single training session in this cohort and a baseline of 7% before training. ASHA training is an effective way to conduct active case detection of VL cases and should be repeated once a year.

Immunoproteomic Identification and Characterization of Leishmania Membrane Proteins as Non-Invasive Diagnostic Candidates for Clinical Visceral Leishmaniasis

Visceral leishmaniasis (VL), a potentially fatal disease is an outcome of infection caused by the parasite Leishmania donovani. The clinical diagnostic tests for this disease are still related to invasive tissue aspiration or serological immunochromatography. Advancements in immunoproteomics such as two-dimensional gel electrophoresis, mass spectrometry, B cell epitope prediction, and peptide synthesis have enabled researchers to discover newer biomarkers for disease diagnosis. In this study, we have screened several urine-reactive leishmanial membrane proteins as potential biomarker candidates. In the immunoblot assay, three proteins 51, 55 and 63 kDa showed 100% reactivity to the urine of 47 VL patients and nonreactive to 18 healthy and other diseases. Mass spectrometry revealed the identity of 51, 55 and 63 kDa proteins as elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63, respectively. B cell reactive epitopes of these proteins were mapped through bioinformatic tools and one epitope from each protein that had the highest score were synthesized. All the three native electroeluted proteins and their corresponding synthetic peptides were tested through ELISA for reactivity with VL and control urine samples. While all three demonstrated good reactivity, the diagnostic performance of EF1-α was the best. Our findings illustrate the use of urine-based proteomic approach for biomarker discovery in non-invasive clinical diagnosis of VL.

CD8 dim but not CD8 bright cells positive to CD56 dominantly express KIR and are cytotoxic during visceral leishmaniasis

This study reports a structural and functional heterogeneity of CD8+CD56+NKT cells, which usually decrease quantitatively during visceral leishmaniasis. Based on fluorescence intensity of CD8 receptors on CD56+NKT cells, two populations of CD8+CD56+NKT cells have been identified. These cells were recognized as CD8dimCD56+NKT and CD8brightCD56+NKT cells. We further analyzed the functional nature of CD8dim and CD8bright positive CD56+NKT cells. In comparison to CD8brightCD56+NKT cells, a significantly higher percentage of CD8dimCD56+NKT cells expressed KIR during VL. The percentage of CD8dimCD56+NKT cells expressing KIR was found 4 fold higher in VL as compared to healthy subjects. But, the difference was insignificant in case of CD8brightCD56+NKT cells. CD8+CD56+NKT cells release granzyme B to kill the infected cells. A categorical difference was also observed in the function of CD8dimCD56+NKT and CD8brightCD56+NKT cells during visceral leishmaniasis. The percentage of granzyme B expressing CD8dimCD56+NKT cells was 2.83 fold higher in VL compared to healthy subjects. But, there was no significant difference in granzyme B expressing CD8brightCD56+NKT cells in samples from healthy and VL subjects. However, within VL subject, the percentage of granzyme B expressing CD8dimCD56+NKT cells was 5.7 fold higher in comparison to CD8brightCD56+NKT cells. This study concludes that CD8dimCD56+NKT cells are more cytotoxic than CD8brightCD56+NKT cells during VL.