A simplified and standardized polymerase chain reaction format for the diagnosis of leishmaniasis.

BACKGROUND: Definite diagnosis of Leishmania infections is based on demonstration of the parasite by microscopic analysis of tissue biopsy specimens or aspirate samples. However, microscopy generally shows low sensitivity and requires invasive sampling. METHODS: We describe here the development of a simple and rapid test for the detection of polymerase chain reaction-amplified Leishmania DNA. A phase 1 evaluation of the text was conducted in clinical samples from 60 nonendemic and 45 endemic control subjects and from 44 patients with confirmed cutaneous leishmaniasis (CL), 12 with mucocutaneous leishmaniasis (MCL), and 43 with visceral leishmaniasis (VL) from Peru, Kenya, and Sudan. RESULTS: The lower detection limits of the assay are 10 fg of Leishmania DNA and 1 parasite in 180 microL of blood. The specificity was 98.3% (95% confidence interval [CI], 91.1%-99.7%) and 95.6% (95% CI, 85.2%-98.8%) for nonendemic and endemic control samples, respectively, and the sensitivity was 93.2% (95% CI, 81.8%-97.7%), 91.7% (95% CI, 64.6%-98.5%), and 86% (95% CI, 72.7%-93.4%) for lesions from patients with CL or MCL and blood from patients with VL, respectively. CONCLUSIONS: The Leishmania OligoC-TesT showed high specificity and sensitivity in clinical samples and was able to detect the parasite in samples obtained by less invasive means, such as blood, lymph, and lesion scrapings. The assay is a promising new tool for simplified and standardized molecular detection of Leishmania parasites.

C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite survival or macrophage responses.

C-reactive protein (CRP) is an acute phase protein that binds to surface structures of a number of different organisms. Leishmania donovani express CRP ligand when first entering the mammalian host and CRP has been shown to alter macrophage function. The aim of this study was to investigate the functional significance of CRP-mediated uptake of L. donovani on survival of the parasite within human macrophages and macrophage cell responses to the infection. CRP opsonized L. donovani uptake was inhibitable by including excess CRP in the fluid phase, suggesting Fc receptor usage rather than indirect complement-mediated uptake. Comparing equivalent initial infection loads, parasite survival over 72 h within peripheral blood derived macrophages (PBMs) and differentiated U937 cells was unaltered by CRP. Whereas CRP increased macrophage responses to phosphorylcholine coated erythrocytes, no significant alteration in tumour necrosis factor-alpha, interleukin (IL)-10 or IL-12 production from PBMs was observed between CRP opsonized or unopsonized L. donovani promastigotes. Thus, in contrast to other systems, where CRP opsonization results in macrophage activation, Leishmania can use CRP to improve infection without inducing detrimental macrophage activation.