Imaging Leishmania major Antigens in Experimentally Infected Macrophages and Dermal Scrapings from Cutaneous Leishmaniasis Lesions in Tunisia

Abstract

Leishmania major cutaneous leishmaniasis (CL) lesions are characterized by an intense process of parasite destruction and antigen processing that could limit microscopic amastigote detection. The aim of our study was to develop a direct immunofluorescence (DIF) assay for in situ visualization of L. major antigens and access its reliability in the routine diagnosis of CL. The developed DIF assay used IgG polyclonal antibodies produced in rabbits by intravenous injections of live L. major metacyclic promastigotes chemically coupled to fluorescein isothiocyanate. Applied to L. major infected RAW macrophages, corresponding macrophage-derived amastigotes and dermal scrapings from CL lesions, the immunofluorescence assay stained specifically Leishmania amastigotes and showed a diffuse Leishmania antigen deposit into cytoplasm of phagocytic cells. Reliability of DIF in CL diagnosis was assessed on 101 methanol-fixed dermal smears from 59 positive and 42 negative CL lesions diagnosed by direct microscopy and/or kDNA real-time PCR. Sensitivity and specificity of DIF was 98.3% and 100%, respectively, being more sensitive than microscopy (p < 0.001) and as sensitive as ITS1-PCR. ITS1-PCR-RFLP allowed Leishmania species identification in 56 out of the 58 DIF-positive smears, identifying 52 L. major, two L. infantum and two L. tropica cases, which indicates antigenic cross-reactivity between Leishmania species.

Diagnostic Accuracy of Cd1a Immuno Histochemical Evaluation in Comparison with H & E and Giemsa for Cutaneous Leishmaniasis

“…The findings corroborate the widespread use and dependability of Hematoxylin and Eosin (H&E) staining in identifying parasitic structures, echoing its efficacy in visualizing leishmania amastigotes (11). While affirming the traditional utility of H&E staining, the research also illuminated instances where amastigotes remained undetected, aligning with observations by Garcia et al about the variability in sensitivity and the necessity of additional methods for a comprehensive evaluation (12). The study's demonstration of the high sensitivity of CD1a immunohistochemical staining reinforces recent research underscoring the role of immunohistochemistry in enhancing diagnostic accuracy (13).…”

Clinical and immunological spectra of human cutaneous leishmaniasis in North Africa and French Guiana

“…Concerning L. major, this species is the most frequent in NA countries, where it remains a major public health problem, with more than 1000 to 8 000 cases yearly in Tunisia and 10,000 to 25,000 in Algeria (9,40,51,52). Lesion can be single or multiple, usually with ulcerative nodules.…”

Special Issue: “Parasitic Infection and Host Immunity”: Editorial

“…In another contribution, Saïdi et al [21] developed a direct immunofluorescence (DIF) technique that, instead of identifying Leishmania amastigotes, marks the presence of scattered antigens, as well as their degraded residues and soluble components, via the cytoplasm and plasma membrane of host cells. The need for new and more accurate methods for the diagnosis of cutaneous leishmaniasis is necessary since these gold methods have a detection margin of amastigotes between 60 and 80% [22,23].…”