Belli A, Rodriguez B, Aviles H, Harris E
Department of Parasitology, Centro Nacional de Diagnostico y Referencia, Ministerio de Salud, Managua, Nicaragua.
Am J Trop Med Hyg. 1998 Jan;58(1):102-9. doi: 10.4269/ajtmh.1998.58.102.
Polymerase chain reaction (PCR)-based detection of New World Leishmania from different types of clinical specimens has been further streamlined for field use by simplifying sample preparation and modifying published protocols. A multiplex PCR reaction was developed that allows simultaneous detection of the Leishmania genus and identification of the L. braziliensis complex. For typing isolates in culture, we found that simply boiling diluted cultured strains was sufficient preparation for the PCR. We have demonstrated that Leishmania parasites can be reliably detected from boiled dermal scrapings, instead of the more invasive skin biopsies often used as PCR specimens. The PCR of dermal scrapings yielded a sensitivity of 100% and a specificity of 100%, as compared with microscopic examination. In a study population, PCR was more sensitive than classic diagnostic techniques. The PCR detection of Leishmania in biopsies and peripheral blood mononuclear cells (PBMCs) was investigated. Diluting crude extracts of skin biopsies was sufficient to eliminate sample inhibition yet maintain required sensitivity. Leishmania braziliensis was also detected by PCR in PBMCs of individuals with active cutaneous leishmaniasis. The simplifications described here significantly improve the feasibility of using the PCR in endemic countries as the primary method for detection and preliminary characterization of Leishmania in clinical specimens of cutaneous leishmaniasis.
通过简化样本制备并修改已发表的方案，基于聚合酶链反应（PCR）从不同类型临床标本中检测新大陆利什曼原虫的方法已进一步优化，以便用于现场检测。我们开发了一种多重PCR反应，可同时检测利什曼原虫属并鉴定巴西利什曼原虫复合体。对于培养物中的分离株分型，我们发现简单煮沸稀释后的培养菌株就足以用于PCR。我们已经证明，利什曼原虫寄生虫可以从煮沸的皮肤刮片中可靠地检测到，而不是使用通常用作PCR标本的更具侵入性的皮肤活检。与显微镜检查相比，皮肤刮片的PCR灵敏度为100%，特异性为100%。在一个研究人群中，PCR比经典诊断技术更敏感。我们研究了在活检组织和外周血单核细胞（PBMC）中利什曼原虫的PCR检测。稀释皮肤活检组织的粗提物足以消除样本抑制作用，同时保持所需的灵敏度。在活动性皮肤利什曼病患者的PBMC中也通过PCR检测到了巴西利什曼原虫。本文所述的简化方法显著提高了在流行国家将PCR用作皮肤利什曼病临床标本中利什曼原虫检测和初步鉴定主要方法的可行性。
