Browsing by Author "Poonam Salotra"

Now showing 1 - 11 of 11

Control of the leishmaniases
(2008) Richard W. Ashford; Caryn Bern; Marleen Boelaert; Anthony Bryceson; François Chappuis; Simon Croft; Jean-Pierre Dedet; Philippe Desjeux; Luigi Gradoni; Robert R. Killick-Kendrick; Elmer Alejandro Llanos-Cuentas; Rogelio López-Vélez; Farrokh Modabber; Suman Rijal; Afif Ben Salah; Poonam Salotra; Nancy Gore Saravia; Jeffrey Jon Shaw; Shyam Sundar; Chandreshwar P. Thakur; Dinesh Mondal; Guilherme L. Werneck
[No abstract available]
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
Elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donoVani
(American Society for Microbiology, 2014) Vasundhra Bhandari; Shyam Sundar; Jean Claude Dujardin; Poonam Salotra
Leishmania donoVani is the causative agent of the potentially fatal disease visceral leishmaniasis (VL). Chemotherapeutic options available to treat VL are limited and often face parasite resistance, inconsistent efficacy, and toxic side effects. Paromomycin (PMM) was recently introduced to treat VL as a monotherapy and in combination therapy. It is vital to unDerstand the mechanisms of PMM resistance to safeguard the drug. In the present study, we utilized experimentally generated PMM-resistant L. donoVani to elucidate the mechanisms of resistance and parasite biology. We found increased membrane fluidity accompanied by decreased intracellular drug accumulation in the PMM-resistant parasites. There were marked increases in gene expression of ATP-binding cassette (ABC) transporters (MDR1 and MRPA) and protein phosphatase 2A that evince increased drug efflux. Further, evaluation of parasite tolerance toward host leishmanicidal mechanisms revealed PMM-resistant parasites as being more tolerant to nitrosative stress at the promastigote and amastigote stages. The PMM-resistant parasites also predicted a better survival capacity, as indicated by resistance to complement-mediated lysis and increased stimulation of host interleukin-10 (IL-10) expression. The susceptibilities of PMM-resistant isolates to other antileishmanial agents (sodium antimony gluconate and miltefosine) remained unchanged. The data implicated the roles of altered membrane fluidity, decreased drug accumulation, increased expression of ABC transporters, and greater tolerance of parasites to host defense mechanisms in conferring PMM resistance in Leishmania. Copyright © 2014, American Society for Microbiology.
Experimental induction of paromomycin resistance in antimony-resistant strains of L. donovani: Outcome dependent on in vitro selection protocol
(2012) Sarah Hendrickx; Raquel Andrea Inocêncio da Luz; Vasundhra Bhandari; Kristel Kuypers; Craig D. Shaw; Julien Lonchamp; Poonam Salotra; Katharine Carter; Shyam Sundar; Suman Rijal; Jean-Claude Dujardin; Paul Cos; Louis Maes
Paromomycin (PMM) has recently been introduced for treatment of visceral leishmaniasis in India. Although no clinical resistance has yet been reported, proactive vigilance should be warranted. The present in vitro study compared the outcome and stability of experimental PMM-resistance induction on promastigotes and intracellular amastigotes. Cloned antimony-resistant L. donovani field isolates from India and Nepal were exposed to stepwise increasing concentrations of PMM (up to 500 μM), either as promastigotes or intracellular amastigotes. One resulting resistant strain was cloned and checked for stability of resistance by drug-free in vitro passage as promastigotes for 20 weeks or a single in vivo passage in the golden hamster. Resistance selection in promastigotes took about 25 weeks to reach the maximal 97 μM inclusion level that did not affect normal growth. Comparison of the IC50 values between the parent and the selected strains revealed a 9 to 11-fold resistance for the Indian and 3 to 5-fold for the Nepalese strains whereby the resistant phenotype was also maintained at the level of the amastigote. Applying PMM pressure to intracellular amastigotes produced resistance after just two selection cycles (IC50 = 199 μM) compared to the parent strain (IC50 = 45 μM). In the amastigote-induced strains/clones, lower PMM susceptibilities were seen only in amastigotes and not at all in promastigotes. This resistance phenotype remained stable after serial in vitro passage as promastigote for 20 weeks and after a single in vivo passage in the hamster. This study clearly demonstrates that a different PMM-resistance phenotype is obtained whether drug selection is applied to promastigotes or intracellular amastigotes. These findings may have important relevance to resistance mechanism investigations and the likelihood of resistance development and detection in the field. © 2012 Hendrickx et al.
In vitro susceptibility of leishmania donovani to miltefosine in Indian visceral leishmaniasis
(2013) Vijay Kumar Prajapati; Smriti Sharma; Madhukar Rai; Bart Ostyn; Poonam Salotra; Manu Vanaerschot; Jean-Claude Dujardin; Shyam Sundar
Promastigote miltefosine (MIL) susceptibility was performed on Leishmania donovani isolates from Indian patients with visceral leishmaniasis treated with MIL. Isolates that were obtained before the onset of MIL treatment, after completion of treatment (29th day), or at the time of treatment failure, were screened using in vitro promastigote assay. The MIL susceptibility of the pre-treatment isolates (N = 24, mean IC50 ± SEM= 3.74 ± 0.38 mM) was significantly higher than that of the post-treatment group (N = 26, mean IC50 ± SEM = 6.15 ± 0.52 mM; P = 0.0006) but was similar in the cured patients (N = 22, mean IC50 ± SEM= 5.58 ± 0.56 mM) and those who failed treatment (N = 28, mean IC50 ± SEM= 4.53 ± 0.47 mM). The pre/post-treatment results thus showed a 2-fold difference, whereas isolated from cured versus failed patients showed a similar susceptibility, suggesting that this higher tolerance is not responsible forMIL-treatment failure. Our work highlights the need for careful monitoring of MIL susceptibility for implementation in national VL elimination programs. Copyright © 2013 by The American Society of Tropical Medicine and Hygiene.
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.
Multilocus microsatellite typing (MLMT) reveals genetic homogeneity of Leishmania donovani strains in the Indian subcontinent
(2009) Mohammad Zahangir Alam; Katrin Kuhls; Carola Schweynoch; Shyam Sundar; Suman Rijal; Abul Khair M. Shamsuzzaman; Balaraju Venkata Subba Raju; Poonam Salotra; Jean-Claude Dujardin; Gabriele Schönian
In this population genetic study of Leishmania donovani parasites in the Indian subcontinent, 132 isolates obtained from patients in Bangladesh, India, Nepal and Sri Lanka suffering from Kala-azar (100), post-Kala-azar dermal leishmaniasis (PKDL) (25) and cutaneous leishmaniasis (CL) (2), and from 5 patients whose clinical patterns were not defined, were analysed by using 15 hyper-variable microsatellite loci. Multilocus microsatellite typing (MLMT) data were analysed by using a Bayesian model-based clustering algorithm and constructing phylogenic tree based on genetic distances. In total, 125 strains from Bangladesh, Bihar (India) and Nepal formed a very homogeneous population regardless of geographical origin, clinical manifestation, and whether they presented in vitro or in vivo susceptibility to antimonial drugs. Identical multilocus microsatellite profiles were found for 108 strains, other strains differed in only one marker. Considerably different microsatellite profiles were identified for three Indian strains most closely related to L. donovani from Kenya, and for four strains from Indian and Sri Lankan CL cases. The circulation of a single homogeneous population of L. donovani in Bihar (India), Bangladesh and Nepal is, most probably, related to the epidemic spread of visceral leishmaniasis in this area. © 2008 Elsevier B.V. All rights reserved.
Transcriptome profiling identifies genes/pathways associated with experimental resistance to paromomycin in Leishmania donovani
(Elsevier Ltd, 2017) Aditya Verma; Vasundhra Bhandari; Deepak Kumar Deep; Shyam Sundar; Jean Claude Dujardin; Ruchi Singh; Poonam Salotra
Widespread resistance towards antimony and reports of relapses following miltefosine treatment has severely affected the management of visceral leishmaniasis (VL) in the Indian subcontinent. Paromomycin (PMM), an aminoglycoside antibiotic, has been licensed for VL treatment in India in 2007. Although its use is still restricted in the field, unraveling the molecular mechanism of resistance towards PMM is the key to preserve the drug. In this study, PMM resistant lines were selected up to 100 μM of PMM in three distinct field isolates of Leishmania donovani at promastigote stage. The resistance induced at promastigote level was also evident in amastigotes which showed 6 fold decreases in PMM susceptibility. Comparative transcriptome profiling of PMM resistant (PMM-R) and the corresponding PMM sensitive (PMM-S) parasites revealed modulated expression of 500 genes (1.5 fold cut off) in PMM-R parasites. Selected genes were validated for their modulated expression by quantitative real-time PCR. Functional classification and pathway analysis of modulated genes indicated probable adaptations in drug resistant lines which included a) reduced oxidative phosphorylation; b) increased glycosomal succinate fermentation and substrate level phosphorylation; c) dependency on lipids and amino acids for energy generation; d) reduced DNA synthesis and increased DNA damage repair and e) decreased protein synthesis and degradation. Interestingly, PMM-R parasites showed a marked increase in PMM susceptibility in presence of verapamil and amlodipine, antagonists of Ca2+ channel that are also modulators of ABC transporters. Moreover, infection of macrophages by PMM-R parasites led to modulated nitric oxide (NO) levels while reactive oxygen species (ROS) level remained unaltered. The present study highlights the putative mechanisms of PMM resistance in Leishmania. © 2017
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.
Validation of SYBR green I based closed tube loop mediated isothermal amplification (LAMP) assay and simplified direct-blood-lysis (DBL)-LAMP assay for diagnosis of visceral leishmaniasis (VL)
(Public Library of Science, 2018) Keerti Kaumudee Dixit; Sandeep Verma; Om Prakash Singh; Dharmendra Singh; Akhil Pratap Singh; Ratan Gupta; Narendra Singh Negi; Pradeep Das; Shyam Sundar; Ruchi Singh; Poonam Salotra
Background: The 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. Methodology: The 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 findings: A 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 (AUC ROC ) 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/Significance: The 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. © 2018 Dixit et al. http://creativecommons.org/licenses/by/4.0/.