Laboratório Interdisciplinar de Pesquisas Médicas, Instituto Oswaldo Cruz (IOC), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
Laboratório de Pesquisa Clínica e Vigilância em Leishmanioses, Instituto Nacional de Infectologia Evandro Chagas (INI), Fundação Oswaldo Cruz, Rio de Janeiro, RJ, Brasil.
PLoS One. 2021 Jan 21;16(1):e0243978. doi: 10.1371/journal.pone.0243978. eCollection 2021.
Cutaneous leishmaniasis (CL) is an infectious vector-borne disease caused by protozoa of the Leishmania genus that affects humans and animals. The distribution of parasites in the lesion is not uniform, and there are divergences in the literature about the choice of the better sampling site for diagnosis-inner or outer edge of the ulcerated skin lesion. In this context, determining the region of the lesion with the highest parasite density and, consequently, the appropriate site for collecting samples can define the success of the laboratory diagnosis. Hence, this study aims to comparatively evaluate the parasite load by qPCR, quantification of amastigotes forms in the direct exam, and the histopathological profile on the inner and outer edges of ulcerated CL lesions.
Samples from ulcerated skin lesions from 39 patients with confirmed CL were examined. We performed scraping of the ulcer inner edge (base) and outer edge (raised border) and lesion biopsy for imprint and histopathological examination. Slides smears were stained by Giemsa and observed in optical microscopy, the material contained on the smears was used to determine parasite load by quantitative real-time PCR (qPCR) with primers directed to the Leishmania (Viannia) minicircle kinetoplast DNA. The histopathological exam was performed to evaluate cell profile, tissue alterations and semi-quantitative assessment of amastigote forms in inner and outer edges.
Parasite loads were higher on the inner edge compared to the outer edge of the lesions, either by qPCR technique (P<0.001) and histopathological examination (P< 0.003). There was no significant difference in the parasite load between the imprint and scraping on the outer edge (P = 1.0000).
CONCLUSION/SIGNIFICANCE: The results suggest that clinical specimens from the inner edge of the ulcerated CL lesions are the most suitable for both molecular diagnosis and direct parasitological examination.
皮肤利什曼病（CL）是一种由原生动物利什曼属引起的传染性媒介传播疾病，影响人类和动物。寄生虫在病变中的分布不均匀，文献中对于选择诊断的最佳采样部位——溃疡皮肤病变的内缘或外缘存在分歧。在这种情况下，确定病变中寄生虫密度最高的区域，以及因此适合采集样本的部位，可以确定实验室诊断的成功与否。因此，本研究旨在通过 qPCR 比较评估寄生虫负荷，直接检查中无鞭毛体形式的定量以及溃疡性 CL 病变的内缘和外缘的组织病理学特征。
检查了 39 例确诊为 CL 的溃疡皮肤病变样本。我们对内缘（溃疡底部）和外缘（隆起边缘）进行刮取，并对病变进行活检以进行印模和组织病理学检查。载玻片涂片用吉姆萨染色，在光学显微镜下观察，载玻片上的材料用于通过针对 Leishmania（Viannia）微环动质体 DNA 的定量实时 PCR（qPCR）确定寄生虫负荷。进行组织病理学检查以评估细胞形态、组织改变以及内缘和外缘中无鞭毛体形式的半定量评估。
通过 qPCR 技术（P<0.001）和组织病理学检查（P<0.003），病变内缘的寄生虫负荷均高于外缘。在外缘的印迹和刮取之间，寄生虫负荷没有差异（P = 1.0000）。
结论/意义：结果表明，溃疡性 CL 病变内缘的临床标本最适合用于分子诊断和直接寄生虫检查。
