Reevaluation of treatment guidelines for Old and New World leishmaniasis is urgently needed on a global basis because treatment failure is an increasing problem. Drug resistance is a fundamental determinant of treatment failure, although other factors also contribute to this phenomenon, including the global HIV/AIDS epidemic with its accompanying impact on the immune system. Pentavalent antimonials have been used successfully worldwide for the treatment of leishmaniasis since the first half of the 20th century, but the last 10 to 20 years have witnessed an increase in clinical resistance, e.g., in North Bihar in India. In this review, we discuss the meaning of “resistance” related to leishmaniasis and discuss its molecular epidemiology, particularly for Leishmania donovani that causes visceral leishmaniasis. We also discuss how resistance can affect drug combination therapies. Molecular mechanisms known to contribute to resistance to antimonials, amphotericin B, and miltefosine are also outlined.

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Drug resistance and treatment failure in leishmaniasis: A 21st century challenge

A rapid and accurate method to detect and quantify Leishmania parasite is urgently needed to facilitate early diagnosis of leishmaniasis and monitoring of antileishmania therapy. In this study, real-time assay was applied to estimate parasite load in clinical samples of visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients. The mean parasite load in blood of VL patients (n = 31) was 8,372 parasites/ml, while the mean parasite load in bone marrow aspirate (BMA) was 194,962 parasites/million nucleated cells (n = 12). Parasite load was undetectable after treatment with amphotericin B (n = 9) in VL, while a residual parasite burden was detected in 2 of 6 patients following treatment with sodium antimony gluconate. Further, circulating levels of IFN-gamma, TNF-alpha, IL-10, IL-6, IL-4 and IL-2 were analysed in VL patients (n = 29) by Cytometric Bead Array to evaluate correlation with parasitic load. Interestingly, IL-10 levels correlated significantly with parasite load (r = 0.82, P<0.0001). The mean parasite load in dermal lesions of PKDL patients was 9,502 parasites/microg tissue DNA at pre-treatment stage (n = 25), with no detectable parasites after therapy (n = 5). Parasite burden was distinctly higher (P<0.0001) in nodular lesions (n = 12) (19,586 parasites/microg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/microg tissue DNA). Further, chronic PKDL lesions showed significantly (P = 0.0166) higher parasite load in comparison with acute lesions. Results indicate that chronic, nodular cases constitute the major parasite reservoir for anthroponotic transmission. Our results establish that the high parasite load in VL is strongly correlated with a high level of IL-10, implicating IL-10 as a marker of disease severity. The assay is applicable for diagnosis as well as prognosis of both VL and PKDL, providing a simple molecular tool to monitor the efficacy of antileishmanial drugs or vaccines.

Quantification of Parasite Load in Clinical Samples of Leishmaniasis Patients: IL-10 Level Correlates with Parasite Load in Visceral Leishmaniasis

The leishmaniases are diseases caused by pathogenic protozoan parasites of the genus Leishmania. Infections are initiated when a sand fly vector inoculates Leishmania parasites into the skin of a mammalian host. Leishmania causes a spectrum of inflammatory cutaneous disease manifestations. The type of cutaneous pathology is determined in part by the infecting Leishmania species, but also by a combination of inflammatory and anti-inflammatory host immune response factors resulting in different clinical outcomes. This review discusses the distinct cutaneous syndromes described in humans, and current knowledge of the inflammatory responses associated with divergent cutaneous pathologic responses to different Leishmania species. The contribution of key hematopoietic cells in experimental cutaneous leishmaniasis in mouse models are also reviewed and compared with those observed during human infection. We hypothesize that local skin events influence the ensuing adaptive immune response to Leishmania spp. infections, and that the balance between inflammatory and regulatory factors induced by infection are critical for determining cutaneous pathology and outcome of infection.

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Cutaneous Manifestations of Human and Murine Leishmaniasis.

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.

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Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis.

Visceral Leishmaniasis: Recent Advances in Diagnostics and Treatment Regimens.

Identification of new foci of cutaneous leishmaniasis (CL), along with reports of Leishmania donovani causing the disease, is an issue of concern. Clinico-epidemiologic analysis of 98 cases in the endemic regions of Rajasthan state, India, suggested the preponderance of infection in men (62.24%) compared with women (37.75%). Species characterization by internal transcribed spacer 1 (ITS1) polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP), kDNA-PCR, and immunofluorescence assay established L. tropica as the causative organism. When applied directly to 32 clinical samples, kDNA PCR had a sensitivity of 96.6%, whereas ITS1 PCR had a sensitivity of 82.75%, thus facilitating diagnosis and species identification. Either parasite culture or direct microscopy alone detected 48.2% and 65.5% of the positive samples, respectively, whereas culture and microscopy together improved overall sensitivity to 89.3% (25/28). Except for the kDNA PCR, all other assays were 100% specific. This study provides the first comprehensive molecular and immunologic studies of CL in India.

Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite identification and characterization using molecular and immunologic tools.

Leishmaniasis is a spectrum of diseases with great relevance to public health. Conventional diagnostic methods are time consuming, needing trained personnel. A robust, rapid and cost effective diagnostic test is warranted for on-time diagnosis and field application. We have developed a loop mediated isothermal amplification (LAMP) assay with primers (n = 6) based on Leishmania donovani kDNA for detection of Leishmania infection, using a closed tube to prevent cross-contamination. The assay was used to detect Leishmania infection in biological samples obtained from patients of visceral leishmaniasis (VL), post kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL). The assay was positive for L. donovani, L. tropica and L. major parasites, with the highest sensitivity towards L. donovani (1 fg DNA). The high sensitivity of the assay for detection of L. donovani was reflected in its ability to detect parasite DNA within 30 min of amplification time with a threshold detection limit of ≥25 copies per reaction. The assay detected parasite in 64 of 66 VL blood samples (sensitivity, 96.9%; 95% CI: 89.6-99.2%), 15 of 15 VL bone marrow aspirate samples (sensitivity, 100%; 95% CI:79.6-100%), 65 of 67 PKDL tissue biopsy samples (sensitivity, 97%; 95% CI:89.7-99.2%). The assay was evaluated in a few cases of CL wherein it was found positive in 8 of 10 tissue biopsies (sensitivity, 80%; 95% CI: 49-94.3%). The assay was negative in all control blood (n = 76) and tissue biopsy (n = 24) samples (specificity, 100%; 95% CI: 96.3-100%). Further, the assay was evaluated for its utility in assessment of cure in treated VL and PKDL patients. The assay detected parasite DNA in 2 of 20VL blood samples and 2 of 21 PKDL tissue samples. Out of 4 cases that were positive for parasite DNA at post treatment stage, 2 patients (1VL and 1 PKDL) returned with relapse. The study demonstrated a Leishmania genus specific closed tube LAMP assay for reliable and rapid molecular diagnosis of VL and PKDL with potential for application in assessment of cure.

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Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

Cutaneous leishmaniasis (CL) is endemic in the Bikaner region situated in the Thar Desert of Rajasthan, India. This study describes clinicoepidemiological data of pediatric CL in pre-school children (0–5 years of age) from this region during 2001–2012. In total, 151 patients with 217 lesions were reported during the study period. The mean age of the study group was 3.29 ± 1.43 years (0.25–5 years), with many (41.7%) cases being in the age group of 2–4 years. Face was the most common site involved, and morphologically, the lesions were either plaque type or papulonodular. Smear for parasitologic examination was positive in 84 (70%) of 120 cases, and histopathologic examination confirmed CL in 10 (55.55%) of 18 cases. Parasite species identification conducted for 13 randomly selected patients by polymerase chain reaction identified Leishmania tropica as the causative species. Intralesional sodium stibogluconate was the most commonly used treatment and found to be well-tolerated. Other therapies that were effective included oral rifampicin, oral dapsone, radiofrequency heat therapy (RFHT), and combinations of the three therapies.

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Ram Avtar Bumb

Papers

Cutaneous leishmaniasis caused by Leishmania tropica in Bikaner, India: parasite identification and characterization using molecular and immunologic tools.

Development of a rapid loop-mediated isothermal amplification assay for diagnosis and assessment of cure of Leishmania infection

Pediatric cutaneous leishmaniasis in an endemic region in India.