title: Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.
abstract: Miltefosine unresponsive and relapse cases of visceral leishmaniasis (VL) are increasingly being reported. However, there has been no laboratory confirmed reports of miltefosine resistance in VL. Here, we report two laboratory confirmed cases of VL from India.
  
  
  
  Two patients with VL were referred to us with suspected VL. The first patient was a native of the VL endemic state of Bihar, but residing in Delhi, a VL non-endemic area. He was treated with broad-spectrum antibiotics and antipyretics but was unresponsive to treatment. The second patient was from Jharkhand state in eastern India (adjoining Bihar), another endemic state for VL. He was refractory to anti-leishmanial treatment, which included administration of miltefosine. Following investigation, both patients were serologically positive for VL, and blood buffy coat from both patients grew Leishmania donovani. The isolates derived from both cases were characterized for their drug susceptibility, genetically characterised, and SNPs typed for LdMT and LdROS gene expression. Both patients were successfully treated with amphotericin B.
  
  
  
  The in vitro drug susceptibility assays carried out on both isolates showed good IC50values to amphotericin B (0.1 ± 0.0004 μg/ml and 0.07 ± 0.0019 μg/ml). One isolate was refractory to SbIIIwith an IC50of > 200 μM while the second isolate was sensitive to SbIIIwith an IC50of 36.70 ± 3.2 μM. However, in both the isolates, IC50against miltefosine was more than 10-fold higher (> 100 μM) than the standard strain DD8 (6.8 ± 0.1181 μM). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr↔Phe and1078Phe↔Tyr) in the LdMT gene of the parasites.
  
  
  
  Here, we document two laboratory confirmed cases of miltefosine resistant VL from India. Our finding highlights the urgent need to establish control measures to prevent the spread of these strains. We also propose that LdMT gene mutation analysis could be used as a molecular marker of miltefosine resistance in L. donovani.
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Saumya
authors.initials: S
authors.lastname: Srivastava
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Jyotsna
authors.initials: J
authors.lastname: Mishra
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Anil Kumar
authors.initials: AK
authors.lastname: Gupta
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Amit
authors.initials: A
authors.lastname: Singh
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India.
authors.forename: Prem
authors.initials: P
authors.lastname: Shankar
authors.affiliation: Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi, India. sarman_singh@yahoo.com.
authors.forename: Sarman
authors.initials: S
authors.lastname: Singh
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics; D000328:Adult; D000913:Antibodies, Protozoan / Q000097:blood; D000981:Antiprotozoal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002648:Child; D019411:Clinical Laboratory Techniques; D004351:Drug Resistance; D006801:Humans; D007194:India / Q000453:epidemiology; D007893:Leishmania donovani / Q000187:drug effects* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007898:Leishmaniasis, Visceral / Q000175:diagnosis / Q000188:drug therapy / Q000453:epidemiology* / Q000469:parasitology*; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D015800:Protozoan Proteins / Q000097:blood / Q000235:genetics; D012008:Recurrence
chemical_list: D000913:Antibodies, Protozoan; D000981:Antiprotozoal Agents; D026901:Membrane Transport Proteins; D015800:Protozoan Proteins; D010767:Phosphorylcholine; C039128:miltefosine; D000251:Adenosine Triphosphatases; C480230:miltefosine transporter, Leishmania donovani
keywords: Bihar; Drug resistance; Jharkhand; Miltefosine; Visceral leishmaniasis