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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 IC50 values to amphotericin B (0.1?±?0.0004 ?g/ml and 0.07?±?0.0019 ?g/ml). One isolate was refractory to SbIII with an IC50 of > 200 ?M while the second isolate was sensitive to SbIII with an IC50 of 36.70?±?3.2 ?M. However, in both the isolates, IC50 against miltefosine was more than 10-fold higher (> 100 ?M) than the standard strain DD8 (6.8?±?0.1181 ?M). Furthermore, genetic analyses demonstrated single nucleotide polymorphisms (SNPs) (354Tyr?Phe and 1078Phe?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.

SUBMITTER: Srivastava S

PROVIDER: S-EPMC5282768 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

Srivastava Saumya S Mishra Jyotsna J Gupta Anil Kumar AK Singh Amit A Shankar Prem P Singh Sarman S

Parasites & vectors 20170131 1

<h4>Background</h4>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.<h4>Methods</h4>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 ...[more]

PMID: 28137296

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Project description:BackgroundWith 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 findingsWe 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<0.0001) to MIL than VL isolates (n = 22, mean IC(50) = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC(50) = 7.05±2.24 µM and 6.18±1.51 µM.ConclusionThe in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.

Project description:BackgroundThere is a continued need to develop effective and safe treatments for visceral leishmaniasis (VL). Preclinical studies on pharmacokinetics and pharmacodynamics of anti-infective agents, such as anti-bacterials and anti-fungals, have provided valuable information in the development and dosing of these agents. The aim of this study was to characterise the pharmacokinetic and pharmacodynamic properties of the anti-leishmanial drugs AmBisome and miltefosine in a preclinical disease model of VL.Methodology / principal findingsBALB/c mice were infected with L. donovani (MHOM/ET/67/HU3) amastigotes. Groups of mice were treated with miltefosine (orally, multi-dose regimen) or AmBisome (intravenously, single dose regimen) or left untreated as control groups. At set time points groups of mice were killed and plasma, livers and spleens harvested. For pharmacodynamics the hepatic parasite burden was determined microscopically from tissue impression smears. For pharmacokinetics drug concentrations were measured in plasma and whole tissue homogenates by LC-MS. Unbound drug concentrations were determined by rapid equilibrium dialysis. Doses exerting maximum anti-leishmanial effects were 40 mg/kg for AmBisome and 150 mg/kg (cumulatively) for miltefosine. AmBisome displayed a wider therapeutic range than miltefosine. Dose fractionation at a total dose of 2.5 mg/kg pointed towards concentration-dependent anti-leishmanial activity of AmBisome, favouring the administration of large doses infrequently. Protein binding was >99% for miltefosine and amphotericin B in plasma and tissue homogenates.Conclusion / significanceUsing a PK/PD approach we propose optimal dosing strategies for AmBisome. Additionally, we describe pharmacokinetic and pharmacodynamic properties of miltefosine and compare our findings in a preclinical disease model to available knowledge from studies in humans. This approach also presents a strategy for improved use of animal models in the drug development process for VL.

Project description:BACKGROUND:Visceral leishmaniasis (VL) is an infectious disease with a variety of clinical signs. The main form of parasite transmission to humans and other mammalian hosts is through the bite of infected arthropod females with Lutzomyia longipalpis serving as the main vector in the Americas. Dogs are the main urban domestic reservoirs of the parasite and the main source of vector infection due to their high prevalence in endemic areas and the large number of parasites in the skin of infected animals. Although miltefosine has been used in Europe since 2002 for treatment of VL infected dogs, in the Americas the treatment of dogs has not been recommended. Therefore, this study aimed to evaluate efficacy of miltefosine observing a reduction of clinical signs in infected dogs and the infectiveness to the vector by Leishmania (L.) infantum. METHODS:To our knowledge, this is the first controlled study using qPCR and xenodiagnosis to evaluate the efficacy of miltefosine (Milteforan®, Virbac) as a single treatment in Brazil. Thirty-five adult dogs with canine visceral leishmaniasis (CVL), confirmed by clinical and laboratory tests, were included in this study. They received miltefosine at a dose of 2 mg/kg every 24 h for 28 days. The dogs were observed over a three-month period, during which clinical evaluations based on a scoring system were conducted at pre-established times. Parasite load was assessed by cytology and real-time polymerase chain reaction (qPCR). Transmissibility to the vector was evaluated by xenodiagnosis. RESULTS:At the end of the period, the following were observed: (i) the remission of clinical signs with a reduction in clinical scores for 94.2% of the animals; (ii) a statistically significant reduction (98.7%) in parasitic load by qPCR; and (iii) a reduction in infectivity to sand flies. After treatment, 74.2% of the animals remained or had become non-infectious. CONCLUSIONS:Our study indicates that the use of miltefosine administered orally for 4 weeks contributes to a clinical improvement and reduction in infectivity of dogs to L. infantum.

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Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

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Laboratory confirmed miltefosine resistant cases of visceral leishmaniasis from India.

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