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

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-γ, TNF-α, 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/µg 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/µg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/µg 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

Foxp3 and IL-10 Expression Correlates with Parasite Burden in Lesional Tissues of Post Kala Azar Dermal Leishmaniasis (PKDL) Patients

Post kala azar dermal leishamniasis (PKDL), an unusual dermatosis develops in 5–15% of apparently cured visceral leishmaniasis cases in India and in about 60% of cases in Sudan. PKDL cases assume importance since they constitute a major human reservoir for the parasite. Inadequate treatment of VL, genetics, nutrition and immunological mechanisms that allow renewed multiplication of latent parasites or reinfection predispose to PKDL. Immunopathogenesis of PKDL is poorly understood. IL-10 is widely accepted as an immuno-suppressive cytokine and produced by diverse cell populations including, B cells, macrophages and CD4+ T cells. Natural T regulatory (nTreg) cells are subpopulation of CD4+ T cells that inhibit the response of other T cells. In this study we reported the accumulation of nTreg cells in lesion tissues of PKDL patients. Further correlation of Treg markers and IL-10 with parasite load in lesion tissues suggested a role of IL-10 and Treg in parasite establishment or persistence. Further studies are warranted to explore antigen specific IL-10 source in lesion tissues and unravel the concerted induction or accumulation of Treg in PKDL

Evidence for Involvement of Th17 Type Responses in Post Kala Azar Dermal Leishmaniasis (PKDL)

Post kala azar dermal leishamniasis (PKDL), an unusual dermatosis, develops in 5–15% of apparently cured visceral leishmaniasis cases in India and in about 60% of cases in Sudan. PKDL cases assume importance since they constitute an important human reservoir for the parasite. Host immunological responses, considered as major factors in PKDL development, are poorly understood. Limited studies have been performed to explore the host immune responses and that too, restricted to a few immune parameters. The present study employed cDNA array technique that identified various host immuno-determinants including cytokines, chemokines, apoptotic and signaling molecules which were not reported previously in PKDL. In addition, we showed for the first time that Th17 responses are present during L. donovani infection in PKDL which possibly contributes significantly to disease pathogenesis by inducing TNF-α and nitric oxide production. Our findings lead to improved understanding of the host parasite interaction in terms of immune responses and pathology in tissue lesions of PKDL

Immunohistochemical analysis of Foxp3 in tissue lesions of PKDL patients.

<p>Distribution of Foxp3 in dermal lesion tissue sections at pre treatment, post treatment stages and normal skin of healthy individuals. Panel A (10×), Panel B (40×) magnification.</p

Levels of cytokine nitric oxide and in PBMCs supernatants stimulated with TSLA or recIL-17.

<p>(a) IL-17 and IL-23 levels in PBMCs of PKDL pre (n = 8), post (n = 6) and control subjects (n = 6) stimulated with total soluble <i>Leishmania</i> antigen (TSLA). (b) Release of TNF-α (pg/ml) or (c) NO (ng/ml) from PBMCs of same set of subjects following incubation with TSLA (10 µg/ml) or recombinant IL-17 (50 ng/ml) for 72 h at 37°C. Cytokine levels were determined by ELISA and NO was quantified by Griess reagent method in culture supernatants. The concentrations shown are the values in the stimulated cultures minus the unstimulated controls. Individual values (pg/ml) are presented and the mean±SE are shown. **P<0.01, and ***P<0.001.</p

Genes showing altered expression in tissue lesions of PKDL (P) compared to human normal skin (HC).

<p>Genes showing altered expression in tissue lesions of PKDL (P) compared to human normal skin (HC).</p

Major characteristics of the study population.

<p>Abbreviations: M = Male, F = Female, PKDL = Post kala azar dermal leishmaniasis, KA = Kala azar.</p

mRNA expression of TNF-α and its correlation with IL-17.

<p>Relative mRNA levels of TNF-α in lesion tissues of PKDL patients determined by real time PCR (a) at pretreatment (n = 19) or post treatment (n = 12), or control tissues (n = 5) or (b) in paired samples (n = 12) and (c) Correlation between IL-17 and TNF-α expression in tissue lesions of PKDL. (r) represents Spearman's rank correlation coefficient. **P<0.01, and ***P<0.001.</p