Browsing by Author "V. Ramesh"

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A simple and sensitive test for field diagnosis of Post Kala-azar Dermal Leishmaniasis
(Blackwell Publishing Ltd., 2001) P. Salotra; G. Sreenivas; V. Ramesh; S. Sundar
Background: Current methods for diagnosis of post kala-azar dermal leishmaniasis (PKDL) do not offer adequate sensitivity and specificity. Objectives To develop a simple and sensitive test for field diagnosis of PKDL. Methods: Immunochromatographic nitrocellulose strips precoated with recombinant k39 antigen were evaluated for the detection of circulating antibodies to leishmanial k39 in PKDL sera. A drop of serum applied to the strip followed by buffer led to the development of two visible bands indicating the presence of anti-k39 IgG. Results: The strip test was able to detect cases of PKDL with 91% sensitivity. The specificity of the test was evaluated using controls with other skin diseases, other common infections and healthy persons from endemic and non-endemic regions. Of 125 controls examined, all were negative on the strip test, indicating 100% specificity of the test. Conclusions: The immunochromatographic nitrocellulose strips provide a non-invasive, rapid and accurate method for diagnosing PKDL. © 2001 British Association of Dermatologists.
Drug susceptibility in Leishmania isolates following Miltefosine treatment in cases of Visceral Leishmaniasis and post Kala-Azar dermal Leishmaniasis
(2012) Vasundhra Bhandari; Arpita Kulshrestha; Deepak Kumar Deep; Olivia Stark; Vijay Kumar Prajapati; V. Ramesh; Shyam Sundar; Gabriele Schonian; Jean Claude Dujardin; Poonam Salotra
Background: With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program. Methodology and Principal Findings: We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC 50±SD = 2.43±1.44 μM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC 50 = 4.72±1.99 μM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC 50 = 1.86±0.75 μM). In PKDL, post-treatment isolates (n = 3, mean IC 50 = 16.13±2.64 μM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC 50 = 8.63±0.94 μM). Overall, PKDL isolates (n = 8, mean IC 50 = 11.45±4.19 μM) exhibited significantly higher tolerance (p
Increased miltefosine tolerance in clinical isolates of Leishmania donovani is associated with reduced drug accumulation, increased infectivity and resistance to oxidative stress
(Public Library of Science, 2017) Deepak Kumar Deep; Ruchi Singh; Vasundhra Bhandari; Aditya Verma; Vanila Sharma; Saima Wajid; Shyam Sundar; V. Ramesh; Jean Claude Dujardin; Poonam Salotra
Background: Miltefosine (MIL) is an oral antileishmanial drug used for treatment of visceral leishmaniasis (VL) in the Indian subcontinent. Recent reports indicate a significant decline in its efficacy with a high rate of relapse in VL as well as post kala-azar dermal leishmaniasis (PKDL). We investigated the parasitic factors apparently involved in miltefosine unresponsiveness in clinical isolates of Leishmania donovani. Methodology: L. donovani isolated from patients of VL and PKDL at pretreatment stage (LdPreTx, n = 9), patients that relapsed after MIL treatment (LdRelapse, n = 7) and parasites made experimentally resistant to MIL (LdM30) were included in this study. MIL uptake was estimated using liquid chromatography coupled mass spectrometry. Reactive oxygen species and intracellular thiol content were measured fluorometrically. Q-PCR was used to assess the differential expression of genes associated with MIL resistance. Results: LdRelapse parasites exhibited higher IC50both at promastigote level (7.92 ± 1.30 μM) and at intracellular amastigote level (11.35 ± 6.48 μM) when compared with LdPreTx parasites (3.27 ± 1.52 μM) and (3.85 ± 3.11 μM), respectively. The percent infectivity (72 hrs post infection) of LdRelapse parasites was significantly higher (80.71 ± 5.67%, P<0.001) in comparison to LdPreTx (60.44 ± 2.80%). MIL accumulation was significantly lower in LdRelapse parasites (1.7 fold, P<0.001) and in LdM30 parasites (2.4 fold, P<0.001) when compared with LdPreTx parasites. MIL induced ROS levels were significantly lower (p<0.05) in macrophages infected with LdRelapse while intracellular thiol content were significantly higher in LdRelapse compared to LdPreTx, indicating a better tolerance for oxidative stress in LdRelapse isolates. Genes associated with oxidative stress, metabolic processes and transporters showed modulated expression in LdRelapse and LdM30 parasites in comparison with LdPreTx parasites. Conclusion: The present study highlights the parasitic factors and pathways responsible for miltefosine unresponsiveness in VL and PKDL. © 2017 Deep et al.
Utility of Blood as the Clinical Specimen for the Molecular Diagnosis of Post-Kala-Azar Dermal Leishmaniasis
(American Society for Microbiology, 2021) Keerti Kaumudee Dixit; V. Ramesh; Shreya Upadhyay; Abhishek Kumar Singh; Om Prakash Singh; Shyam Sundar; Ruchi Singh; Poonam Salotra
The countries in the Indian subcontinent have reported a dramatic decline in visceral leishmaniasis (VL) cases. However, the presence of the parasite reservoir in the form of post-kala-azar dermal leishmaniasis (PKDL), a dermal sequel of VL, is a hurdle in attaining VL elimination. Presently employed clinical specimens for the diagnosis of PKDL include skin biopsy specimens and slit skin smears. In this study, the use of blood as a clinical specimen was investigated in different manifestations of PKDL in India. This is a bicentric study (National Institute of Pathology, Indian Council of Medical Research [ICMR], New Delhi, and Institute of Medical Sciences [IMS], Banaras Hindu University, Varanasi), with 215 participants (120 PKDL patients and 95 controls). Highly sensitive quantitative real-time PCR (Q-PCR) and field-deployable loop-mediated isothermal amplification (LAMP) were employed using blood samples for diagnosis. Promising sensitivities of 77.50% (95% confidence interval [CI], 69.24 to 84.05%) for Q-PCR and 70.83% (95% CI, 62.16 to 78.22%) for LAMP were obtained for the diagnosis of PKDL. Further, enhanced sensitivities of 83.33% (95% CI, 71.28 to 90.98%) and 77.78% (95% CI, 65.06 to 86.80%) for Q-PCR and LAMP, respectively, were recorded for the detection of macular cases. The study revealed an inverse correlation between the parasite load estimated in slit and blood samples, thereby favoring the use of blood for the diagnosis of the macular variant, which may be missed due to scant parasite loads in the slit. This study is the first to propose the promising potential of blood as a clinical specimen for accurate diagnosis of PKDL, which would aid in fast-tracking VL elimination. © 2021 American Society for Microbiology.
Validation of a simple resazurin-based promastigote assay for the routine monitoring of miltefosine susceptibility in clinical isolates of Leishmania donovani
(2013) Arpita Kulshrestha; Vasundhra Bhandari; Rupkatha Mukhopadhyay; V. Ramesh; Shyam Sundar; Louis Maes; Jean Claude Dujardin; Syamal Roy; Poonam Salotra
Simple, cost-effective approach for routine surveillance of parasite susceptibility to antileishmanial drug miltefosine (MIL) is highly desirable for controlling emergence of drug resistance in visceral leishmaniasis (VL). We validated a simple resazurin-based fluorimetric assay using promastigotes to track natural MIL tolerance in Leishmania donovani parasites from VL cases (n = 17) against standard amastigote assay, in two different labs in India. The inter-stage MIL susceptibility correlated strongly (r = 0.70, p = 0.0018) using J774.A.1 macrophage cell line-based amastigote assay and fluorescence-based resazurin assay for promastigotes. Investigation of inter-stage MIL susceptibility for the same set of clinical isolates in another lab also showed a strong correlation (r = 0.72, p = 0.0012) using mouse peritoneal macrophages for amastigote assay and resazurin-based alamar blue assay for promastigotes. Additionally, parasites from post-kala-azar dermal leishmaniasis (PKDL) lesions (n = 7, r = 0.78, p = 0.046) and MIL-induced parasites (r = 0.92, p = 0.0001; n = 3) also exhibited a strongly correlated inter-stage miltefosine susceptibility. Thus, our results support the utility of resazurin assay as a simplified biological tool for MIL susceptibility monitoring in clinical isolates from MIL-treated VL/PKDL patients. © 2012 Springer-Verlag Berlin Heidelberg.