Abstract
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.
Highlights
The protozoan parasites of the genus Leishmania are the causative agents of a group of diseases called leishmaniasis, endemic in more than 88 countries and affecting 12 million people worldwide
This burden is further compounded in India as about 5–15% of apparently cured visceral leishmaniasis (VL) patients develop an unusual dermal form of the disease termed Post kala azar dermal leishmaniasis (PKDL) [3]
We demonstrate a strong correlation of parasite burden with the host immune response in VL and the pattern of parasite load with respect to the clinical presentation in PKDL
Summary
The protozoan parasites of the genus Leishmania are the causative agents of a group of diseases called leishmaniasis, endemic in more than 88 countries and affecting 12 million people worldwide. In India, the state of Bihar, parts of Eastern Uttar Pradesh, and West Bengal are endemic foci of VL where periodic epidemics are common claiming the lives of thousands and causing severe morbidity to hundreds of thousands [2]. This burden is further compounded in India as about 5–15% of apparently cured VL patients develop an unusual dermal form of the disease termed Post kala azar dermal leishmaniasis (PKDL) [3]. The need to search for cases of PKDL and treat them as a part of VL control program has been emphasized since patients with PKDL provide the only known reservoir for the parasite during inter-epidemic periods of VL in India [4]
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