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Identification and Functional Validation of a Biomarker for the Diagnosis of Miltefosine Relapse during Visceral Leishmaniasis.

ABSTRACT: Miltefosine is the only orally administrable drug for the treatment of leishmaniasis. But in recent years, a decline in its efficacy points toward the emergence of resistance to this drug. Knowledge of biomarkers for miltefosine resistance may be beneficial for proper selection of treatment regimen. Splenic aspirates were collected and parasites cultured from patients relapsed after initial cure (N = 15) and successfully treated (N = 15) with miltefosine. Differential expression of genes in miltefosine-resistant strains was examined by DNA microarray and validated by real-time reverse transcription polymerase chain reaction and Western blotting. Of 669 upregulated genes, the cysteine protease-like protein of calpain family (GenBank: CBZ34784) was found to be significantly overexpressed in resistant parasite strains and only anti-calpain antibodies showed its presence in the sera of relapse patients through Western blotting. Calpain family cysteine protease-like protein can be useful as a potential biomarker of miltefosine unresponsiveness.

SUBMITTER: Tiwary P

PROVIDER: S-EPMC5929172 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Identification and Functional Validation of a Biomarker for the Diagnosis of Miltefosine Relapse during Visceral Leishmaniasis.

Tiwary Puja P Kumar Dinesh D Sundar Shyam S

The American journal of tropical medicine and hygiene 20171221 2

Miltefosine is the only orally administrable drug for the treatment of leishmaniasis. But in recent years, a decline in its efficacy points toward the emergence of resistance to this drug. Knowledge of biomarkers for miltefosine resistance may be beneficial for proper selection of treatment regimen. Splenic aspirates were collected and parasites cultured from patients relapsed after initial cure (<i>N</i> = 15) and successfully treated (<i>N</i> = 15) with miltefosine. Differential expression of ...[more]

PMID: 29280431

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Project description:Leishmania donovani is an intracellular protozoan parasite that causes leishmaniasis, which can range from a self-healing cutaneous disease to a fatal visceral disease depending on the infecting species. Miltefosine is currently the latest and only oral antileishmanial that came out of drug discovery pipelines in the past few decades, but recent reports indicate a significant decline in its efficacy against visceral leishmaniasis (also known as kala-azar) in the Indian subcontinent. This relapse rate of up to 20% within 12 months after treatment was shown not to be related to reinfection, drug quality, drug exposure, or drug-resistant parasites. We therefore aimed to assess other phenotypes of the parasite that may affect treatment outcome and found a significant association between the number of metacyclic parasites, parasite infectivity, and patient treatment outcome in the Indian subcontinent. Together with previous studies on resistance of L. donovani against pentavalent antimonials, these data suggest that the infectivity of the parasite, or related phenotypes, might be a more determinant factor for treatment failure in visceral leishmaniasis than drug susceptibility, warranting a reassessment of our current view on treatment failure and drug resistance in leishmaniasis and beyond.The high miltefosine relapse rate poses a major challenge for the current Kala-Azar Elimination Program in the Indian subcontinent and other leishmaniasis control programs worldwide. This relapse rate could not be related to reinfection, drug-resistant parasites, or reduced treatment quality. Here we report that an increased infectivity of the parasite is associated with miltefosine relapse of visceral leishmaniasis (VL) patients. These results supplement those obtained with antimonial-resistant L. donovani where an increased infectivity was also observed. This challenges the current view of Leishmania drug susceptibility being the biggest parasitic factor that contributes to treatment failure in leishmaniasis. These selected more infectious parasites may pose an additional burden to leishmaniasis control programs, highlighting the importance of multifaceted control measures to achieve leishmaniasis elimination in the Indian subcontinent and other regions where leishmaniasis is endemic.

Project description: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.

Project description:Immuno-modulators in combination with antileishmanial drug miltefosine is a better therapeutic approach for treatment of Visceral Leishmaniasis (VL) as it not only reduces the dose of miltefosine but also shortens the treatment regimen. However, immunological mechanisms behind the perceived benefits of this combination therapy have not been investigated in detail. In the present study, we hypothesized that potential use of drugs that target the host in addition to the parasite might represent an alternative strategy for combination therapy. We investigated immune responses generated in Leishmania donovani infected animals (hamsters and mice) treated with combination of CpG-ODN-2006 and miltefosine at short dose regimen. Infected animals were administered CpG-ODN-2006 (0.4 mg/kg, single dose), as free and liposomal form, either alone or in combination with miltefosine for 5 consecutive days and parasite clearance was evaluated at day 4 and 7 post treatment. Animals that received liposomal CpG-ODN-2006 (lipo-CpG-ODN-2006) and sub-curative miltefosine (5 mg/kg) showed the best inhibition of parasite multiplication (∼97%) which was associated with a biased Th1 immune response in. Moreover, compared to all the other treated groups, we observed increased mRNA expression levels of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-12) and significantly suppressed levels of Th2 cytokines (IL-10 and TGF-β) on day 4 post treatment in animals that underwent combination therapy with lipo-CpG-ODN-2006 and sub-curative miltefosine. Additionally, same therapy also induced heightened iNOS mRNA levels and NO generation, increased IgG2 antibody level and strong T-cell response in these hamsters compared with all the other treated groups. Collectively, our results suggest that combination of lipo-CpG-ODN-2006 and sub-curative miltefosine generates protective T-cell response in an animal model of visceral leishmaniasis which is characterized by strong Th1 biased immune response thereby underlining our hypothesis that combination therapy, at short dose regimen can be used as a novel way of treating visceral leishmaniasis.

Project description:Introduction:To sustain the achievement of kala-azar elimination program (KEP), early detection and treatment of the visceral leishmaniasis (VL) cases and associated modalities such as treatment failure (TF), relapse VL (RVL), and Post-kala-azar dermal leishmaniasis (PKDL) is the cornerstone. A predictive biomarker for VL development and related complications could also play a crucial role in curtailing disease incidence and transmission. Investigations to find a biomarker with prospective capabilities are, however, scarce. Using samples and known clinical outcomes generated within two previous longitudinal cohort studies, we aimed to determine if fluctuations in serum anti-rK39 antibody levels could provide such predictive value. Materials and Methods:Serum samples collected at four different time points (Baseline, 12, 18, and 24 months) from 16 patients who had developed VL within the monitoring period and 15 of their asymptomatic healthy controls counterparts were investigated. To investigate potential prediction of VL related complications, serum samples of 32 PKDL, 10 RVL, 07 TF, and 38 cured VL from a single dose AmBisome trial were analyzed. Of this second panel, all patients were monitored for 5 years and sera were collected at four time points (Baseline then 1, 6, and 12 months after treatment). The level of anti-rK39 antibodies in archived samples was measured by a semi-quantitative ELISA. Results:The mean antibody level was significantly higher in VL patients compared to their asymptomatic healthy counterparts at each time point. Likewise, we observed a trend toward elevations in antibody levels for PKDL, RVL, TF relative to the reducing levels observed in cured VL. Receiver operating characteristic (ROC) analysis found a promising predictive power of rK39 antibody levels to reveal progression from asymptomatic Leishmania donovani infection stage to VL, defined as 87.5% sensitive and 95% specific. Following treatment, rk39 antibody notably showed 100% sensitivity and 95% specificity in predicting TF. Conclusion:Our data indicate that the relative quantity of serum anti-rK39 antibody has promise within either a predictive or prognostic algorithm for VL and VL-related modalities. These could enable VL control programs to implement more effective measures to eliminate the disease. Further research is, however, imperative to standardize the rK39 antibody ELISA between sites prior to broader use.

Dataset Information

Identification and Functional Validation of a Biomarker for the Diagnosis of Miltefosine Relapse during Visceral Leishmaniasis.

Publications

Identification and Functional Validation of a Biomarker for the Diagnosis of Miltefosine Relapse during Visceral Leishmaniasis.

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