Department of Dermatology, Andrée Rosemon Hospital, Cayenne, French Guiana
Tropical Biome and Immunophysiopathology (TBIP), Université de Lille, CNRS, INSERM, Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d'Infection et d'Immunité de Lille, Centre Hospitalier de Cayenne, Université de Guyane, Cayenne, French Guiana.
J Clin Microbiol. 2021 Jan 21;59(2). doi: 10.1128/JCM.02218-20.
Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold ( ) was 24.4 (minimum , 17; maximum , 36) and was <35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.
最近的研究强调了人们对非侵入性采样程序与实时 PCR 方法结合，用于检测南美的 物种的兴趣。在法属圭亚那，采样方法仍然依赖于皮肤活检。应该在一个大型年度队列中测试非侵入性方案，以改善皮肤利什曼病的常规实验室诊断。因此，我们评估了一种新的检测和种属鉴定方案的性能，该方案涉及棉拭子和基于 SYBR 绿色的 Hsp70 基因实时 PCR，结合 Sanger 测序。2017 年 5 月至 2018 年 5 月，在卡宴医院及其远程医疗中心，对 145 例溃疡病变符合皮肤利什曼病的患者进行了研究。每位患者都接受了刮片检查、寄生虫培养和 PCR-限制性片段长度多态性（RNA 聚合酶 II）皮肤活检，以及棉拭子采样进行基于 SYBR 绿色的 PCR。最准确的诊断测试是基于拭子样本的 SYBR 绿色 PCR，显示出 98%的灵敏度。平均 PCR 循环阈值（ ）为 24.4（最小值 ，17；最大值 ，36），97.6%的样本<35。所有通过基于 SYBR 绿色的实时 PCR 阳性的样本均通过 DNA 测序成功鉴定为种属水平。由于非侵入性采集工具更易于使用并且对运输的要求较少，因此该新方法应考虑用于南美的皮肤利什曼病的常规诊断，特别是在偏远地区。
