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A Leishmania donovani species-specific monoclonal antibody (monoclonal antibody D2) was evaluated for its diagnostic and prognostic potential by a competitive enzyme-linked immunosorbent assay (C-ELISA) in sera from Indian patients with visceral leishmaniasis (VL) and seven patients with post-kala-azar dermal leish-maniasis (PKDL). These results were compared with those obtained by microscopy with Giemsa-stained tissue smears and a direct enzyme-linked immunosorbent assay (direct ELISA) with crude parasite antigen. Of 121 patients with clinically diagnosed VL examined, 103 (85.1%) were positive and 11 (9.1%) were negative by all three methods. An additional 7 (5.8%) who were negative by microscopy were positive by both C-ELISA and direct ELISA. Seven PKDL patients were also examined and were found to be positive by all three methods. Analysis of the chemotherapeutic response to sodium Antimony Gluconate of these 110 serologically positive VL patients showed that 57 (51.8%) were drug responsive and 53 (48.2%) were drug resistant. The C-ELISA wit

The overexpression of genes of thiol metabolism contribute to drug resistance in clinical isolates of visceral leishmaniasis (kala azar) in India

Background Visceral leishmaniasis (VL), also called Kala Azar (KA) or black fever in India, claims around 20,000 lives every year. Chemotherapy remains one of the most important tools in the control of VL. Current chemotherapy for Kala Azar in India relies on a rather limited arsenal of drugs including sodium Antimony Gluconate and amphotericin B in addition to the very expensive drug miltefosine. Pentavalent antimonials have been used for more than half a century in the therapy of leishmaniasis as it is relatively safe and inexpensive, however, the spread of resistance to this drug is forcing clinicians in India to abandon this treatment. Consequently, improvement of antimonial chemotherapy has become a major challenging area of study by leishmaniacs worldwide. The alarming emergence of resistance to the commonly used antleishmanial drug, sodium Antimony Gluconate, in India, has led us to elucidate the resistance mechanism(s) in clinical isolates. Studies on laboratory mutants have shown that resistance to antimonials is highly dependent on thiol levels. The parasite evades cytotoxic effects of antimonial therapy by enhanced efflux of drug upon conjugation with thiols, through overexpressed membrane proteins belonging to the superfamily of ABC transporters. Methods We have carried out functional studies to determine the activity of the efflux pumps in antimonial resistant clinical isolates collected from disease endemic areas in India and also carried out molecular characterization of thiol levels in these parasites. Results Overexpression of the gene coding for γ glutamylcysteine synthetase was observed in these resistant clinical isolates thereby establishing that thiols represent the key determinants of antimonial resistance. The SbIII/thiol conjugates can be sequestered by ABC transporter multidrug resistance protein A (MRPA) into intracellular organelles or can be directly pumped out by an uncharacterized transporter. Conclusions Our studies investigating antimonial resistance in different L. donovani clinical isolates suggest that over functioning of MRP plays a role in generation of Antimony resistance phenotype in some L. donovani clinical isolates.

elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donovani

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.

elucidation of cellular mechanisms involved in experimental paromomycin resistance in leishmania donovani

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.

increased parasite surface antigen 2 expression in clinical isolates of leishmania donovani augments Antimony resistance

Abstract Resistance to sodium Antimony Gluconate (SAG) is a major cause of therapeutic failure in a large proportion of visceral leishmaniasis (VL) cases. Determinants of SAG resistance have been widely studied; however, the mechanism operating in clinical isolates is poorly understood. In the present study, expression of parasite surface antigen-2 (PSA-2) gene was studied in clinical isolates of Leishmania donovani comprising of Antimony resistant (n = 10) and sensitive (n = 4) parasites. The expression of PSA-2 gene was found to be consistently high in SAG resistant clinical isolates (⩾1.5-fold) at both transcript and protein level. Further, over-expression of PSA-2 in L. donovani isolates (LdPSA-2++) resulted in conversion of SAG sensitive phenotype to resistant. The LdPSA-2++ parasites showed significantly decreased susceptibility towards SAG (>12-fold), amphotericin B (>4-fold) and miltefosine (>2.5-fold). Marked decrease in Antimony accumulation and enhanced tolerance towards complement mediated lysis was evident in LdPSA-2++ parasites. The study established the role of PSA-2 gene in SAG resistance and its potential as a biomarker to distinguish resistant and sensitive clinical isolates of L. donovani.

genetic typing reveals monomorphism between Antimony sensitive and resistant leishmania donovani isolates from visceral leishmaniasis or post kala azar dermal leishmaniasis cases in india

Resistance to pentavalent antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also known as kala-azar (KA), caused by Leishmania donovani. In India, over 60 % of KA patients are unresponsive to the first-line drug sodium Antimony Gluconate (SAG). Resistance determinants in laboratory strains are partly known; however, the mechanism operating in field isolates is not well understood. In this study, we attempted to analyze the genetic polymorphism between SAG sensitive and resistant parasites using a total of 52 L. donovani isolates obtained either from bone marrow of VL patients or from skin lesions of post kala-azar dermal leishmaniasis (PKDL) patients that constitute an important reservoir of parasite. The clinical isolates were analyzed in comparison with L. donovani parasites from reference strains belonging to distinct geographical locations, at internal transcribed spacer 1 region; coding region of gp63 and nine microsatellite repeat regions. Our results demonstrated that both SAG resistant (n = 26) and sensitive (n = 19) Indian isolates, whether causing VL or PKDL, were monomorphic at all the genetic loci tested, unlike the L. donovani in East African or Leishmania infantum in Mediterranean countries where intraspecies variations exist at these loci. Further, the Indian isolates were found closest to the Kenyan isolates of L. donovani on the basis of fragment analysis of microsatellite markers.

Over-expression of 60s ribosomal L23a is associated with cellular proliferation in SAG resistant clinical isolates of Leishmania donovani

Background: Sodium Antimony Gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism. Methodology and principal findings: The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode. Conclusion/Significance: This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosoma

proteome mapping of overexpressed membrane enriched and cytosolic proteins in sodium Antimony Gluconate sag resistant clinical isolate of leishmania donovani

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT

Over 60% of patients with visceral leishmaniasis (VL) in India and Sudan have become unresponsive to treatment with pentavalent antimonials, the first line of drugs for over 60 years. The drug resistance mechanism, studied so far in in vitro selected laboratory strains, has been attributed to various biochemical parameters. The resistance to Sb (V) in Leishmania field isolates is still unexplored.

WHAT THIS STUDY ADDS

In order to elucidate for the first time the mechanism of drug resistance in field isolates, this study was done in those clinically relevant field isolates which were either responsive or non responsive to SAG. A comparison of proteome profiles of membrane-enriched as well as cytosolic protein fractions of these isolates has pinpointed the multiple overexpressed proteins in resistant isolates. This study has indicated their possible essential role in Antimony resistance of the parasite and provides a vast field to be exploited to find much needed novel treatment strategies against VL.

AIMS

This study aimed to identify differentially overexpressed membrane-enriched as well as cytosolic proteins in SAG sensitive and resistant clinical strains of L. donovani isolated from VL patients which are involved in the drug resistance mechanism.

METHODS

The proteins in the membrane-enriched as well as cytosolic fractions of drug-sensitive as well as drug-resistant clinical isolates were separated using two-dimensional gel electrophoresis and overexpressed identified protein spots of interest were excised and analysed using MALDI-TOF/TOF.

RESULTS

Six out of 12 overexpressed proteins were identified in the membrane-enriched fraction of the SAG resistant strain of L. donovani whereas 14 out of 18 spots were identified in the cytosolic fraction as compared with the SAG sensitive strain. The major proteins in the membrane-enriched fraction were ABC transporter, HSP-83, GPI protein transamidase, cysteine–leucine rich protein and 60S ribosomal protein L23a whereas in the cytosolic fraction proliferative cell nuclear antigen (PCNA), proteasome alpha 5 subunit, carboxypeptidase, HSP-70, enolase, fructose-1,6-bisphosphate aldolase, tubulin-beta chain have been identified. Most of these proteins have been reported as potential drug targets, except 60S ribosomal protein L23a and PCNA which have not been reported to date for their possible involvement in drug resistance against VL.

CONCLUSION

This study for the first time provided a cumulative proteomic analysis of proteins overexpressed in drug resistant clinical isolates of L. donovani indicating their possible role in Antimony resistance of the parasite. Identified proteins provide a vast field to be exploited for novel treatment strategies against VL such as cloning and overexpression of these targets to produce recombinant therapeutic/prophylactic proteins.

identification of genetic markers in sodium Antimony Gluconate sag sensitive and resistant indian clinical isolates of leishmania donovani through amplified fragment length polymorphism aflp

Sodium Antimony Gluconate (SAG) is currently used worldwide as the first-line drugs for the treatment of visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL) since 1940s. Unfortunately, the resistance of Leishmania parasite to this drug is increasing in several parts of the world. The mechanism of drug resistance in clinical isolates is still not very clear. Earlier, we have established a differentiation between six clinical isolates as sensitive and resistant on the basis of their sensitivity to SAG in vitro and in vivo as well as expression of proteophosphoglycan contents. In this preliminary study, we have further analyzed these isolates on the basis of their genetic diversity, molecular variance and phylogenetic structure using for the first time, a fingerprinting approach – amplified fragment length polymorphism (AFLP). Altogether 2338 informative AFLP bands were generated using 10 selective primer combinations. Percentage of polymorphism was 55.35%. A number of unique AFLP markers (217) were also identified in these strains. It was deduced that a higher rate of variations occurred among Leishmania clinical isolates which indicate the shifting of drug sensitive nature of parasite towards resistant condition.