Evaluation of blood based quantitative PCR as a molecular diagnostic tool for post kala-azar dermal leishmaniasis (PKDL).

BACKGROUND: Post kala-azar dermal leishmaniasis (PKDL) is a consequential dermal manifestation of visceral leishmaniasis (VL), serving as a parasite reservoir. The traditional diagnostic approach, which requires an invasive skin biopsy is associated with inherent risks and necessitates skilled healthcare practitioners in sterile settings. There is a critical need for a rapid, less invasive method for Leishmania detection. The main objective of this study was to evaluate and compare the diagnostic efficacy of PCR and qPCR in detecting PKDL, utilizing both skin and blood samples and to assess the utility of blood samples for molecular diagnosis. METHODS AND RESULTS: 73 individuals exhibiting clinical symptoms of PKDL and who had tested positive for rK39 rapid diagnostic test (RDT) were enrolled in this study. For the diagnosis of PKDL, both PCR and real-time quantitative PCR (qPCR), employing SYBR Green and TaqMan assays, were performed on blood and skin matched samples. qPCR results using both TaqMan and SYBR Green assay, indicated higher parasite loads in the skin compared to blood, as evident by the Ct values. Importantly, when blood samples were used for PKDL diagnosis by qPCR, an encouraging sensitivity of 69.35% (TaqMan assay) and 79.36% (SYBR Green) were obtained, compared to 8.2% with conventional PCR. CONCLUSION: The findings of the study suggest the potential utility of blood for molecular diagnosis by qPCR, offering a less invasive alternative to skin biopsies in field setting for the early detection of parasitaemia in PKDL patients and effective management and control of the disease.

Apolipoprotein E Is Upregulated in Blood and Circulating Monocytes of Indian Patients With Visceral Leishmaniasis.

Apolipoprotein E (ApoE) has been associated with several diseases including Parkinson's disease, Alzheimer's and multiple sclerosis. ApoE also has documented immunomodulatory functions. We investigated gene expression in circulating monocytes and in bone marrows of patients with visceral leishmaniasis (VL) living in an endemic area in Bihar, India, and contrasted these with control healthy subjects or other diagnostic bone marrows from individuals in the same region. Samples from VL patients were obtained prior to initiating treatment. Our study revealed significant upregulated expression of the apoE transcript in patients with VL. Furthermore, the levels of ApoE protein were elevated in serum samples of subjects with VL compared with healthy endemic controls. These observations may provide clues regarding the complex interactions between lipid metabolism and immunoregulation of infectious and inflammatory diseases.

Phenotypic and functional characteristics of monocyte subsets in the blood and bone marrow of Indian subjects with Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic infection caused by Leishmania donovani in India. L. donovani is an obligate intracellular protozoan residing mostly in macrophages of the reticuloendothelial system throughout chronic infection. Monocytic phagocytes are critical in the pathogenesis of different forms of leishmaniasis. Subsets of monocytes are distinguished by their surface markers into CD14+CD16- classical monocytes, CD14+CD16+ intermediate monocytes, and CD16++CD14low non-classical monocyte subsets. During cutaneous leishmaniasis (CL), intermediate monocyte are reported to be a source of inflammatory cytokines IL-1ß and TNF, and they express CCR2 attracting them to sites of inflammatory pathology. We examined monocyte subsets in the blood and bone marrow of patients with VL from an endemic site in Bihar, India, and found these contrasted with the roles of monocytes in CL. During VL, intermediate and non-classical CD16+ monocyte subsets expressed instead a non-inflammatory phenotype with low CCR2, high CX3CR1 and low microbicidal oxidant generation, making them more similar to patrolling monocytes than inflammatory cells. Bone marrow CD16+ monocyte subsets expressed a phenotype that might be more similar to the inflammatory subsets of CL, although our inability to obtain bone marrow from healthy donors in the endemic region hampered this interpretation Overall the data suggest that CD16+ intermediate monocyte subsets in VL patients express a phenotypes that contributes to an immunosuppressed pathologic immune state, but in contrast to CL, these do not mediate localized inflammatory responses.