Department of Public Health, Arba Minch University, Arba Minch, Ethiopia.
Evolutionary Ecology Group, University of Antwerp, Antwerp, Belgium.
PLoS Negl Trop Dis. 2021 Jan 12;15(1):e0008903. doi: 10.1371/journal.pntd.0008903. eCollection 2021 Jan.
In most low-resource settings, microscopy still is the standard method for diagnosis of cutaneous leishmaniasis, despite its limited sensitivity. In Ethiopia, the more sensitive molecular methods are not yet routinely used. This study compared five PCR methods with microscopy on two sample types collected from patients with a suspected lesion to advise on optimal diagnosis of Leishmania aethiopica. Between May and July 2018, skin scrapings (SS) and blood exudate from the lesion spotted on filter paper (dry blood spot, DBS) were collected for PCR from 111 patients of four zones in Southern Ethiopia. DNA and RNA were simultaneously extracted from both sample types. DNA was evaluated by a conventional PCR targeting ITS-1 and three probe-based real-time PCRs: one targeting the SSU 18S rRNA and two targeting the kDNA minicircle sequence (the 'Mary kDNA PCR' and a newly designed 'LC kDNA PCR' for improved L. aethiopica detection). RNAs were tested with a SYBR Green-based RT-PCR targeting spliced leader (SL) RNA. Giemsa-stained SS smears were examined by microscopy. Of the 111 SS, 100 were positive with at least two methods. Sensitivity of microscopy, ITS PCR, SSU PCR, Mary kDNA PCR, LC kDNA PCR and SL RNA PCR were respectively 52%, 22%, 64%, 99%, 100% and 94%. Microscopy-based parasite load correlated well with real-time PCR Ct-values. Despite suboptimal sample storage for RNA detection, the SL RNA PCR resulted in congruent results with low Ct-values. DBS collected from the same lesion showed lower PCR positivity rates compared to SS. The kDNA PCRs showed excellent performance for diagnosis of L. aethiopica on SS. Lower-cost SL RNA detection can be a complementary high-throughput tool. DBS can be used for PCR in case microscopy is negative, the SS sample can be sent to the referral health facility where kDNA PCR method is available.
在大多数资源匮乏的环境中，尽管显微镜检查的敏感性有限，但它仍然是诊断皮肤利什曼病的标准方法。在埃塞俄比亚，更敏感的分子方法尚未常规使用。本研究比较了 5 种聚合酶链反应（PCR）方法与显微镜检查，以评估其在两种样本类型（从疑似病变处采集的皮肤刮片[SS]和滤纸斑点处采集的血液渗出液[干血斑，DBS]）上对埃塞俄比亚利什曼原虫（Leishmania aethiopica）的诊断效果。2018 年 5 月至 7 月，从埃塞俄比亚南部四个地区的 111 名患者中采集 SS 和 DBS 用于 PCR，同时从两种样本类型中提取 DNA 和 RNA。对 DNA 进行了针对 ITS-1 的常规 PCR 检测和三种基于探针的实时 PCR 检测：一种针对 SSU 18S rRNA，两种针对 kDNA 微环序列（“Mary kDNA PCR”和新设计的“LC kDNA PCR”，用于提高 L. aethiopica 的检测）。RNA 采用针对剪接先导（SL）RNA 的 SYBR Green 实时 RT-PCR 检测。用 Giemsa 染色 SS 涂片进行显微镜检查。111 个 SS 中有 100 个至少有两种方法呈阳性。显微镜检查、ITS PCR、SSU PCR、Mary kDNA PCR、LC kDNA PCR 和 SL RNA PCR 的灵敏度分别为 52%、22%、64%、99%、100%和 94%。显微镜检查的寄生虫载量与实时 PCR Ct 值相关性良好。尽管 RNA 检测的样本储存条件不理想，但 SL RNA PCR 仍能获得低 Ct 值的一致结果。与 SS 相比，来自同一病变的 DBS 显示出较低的 PCR 阳性率。kDNA PCR 对 SS 上 L. aethiopica 的诊断具有出色的性能。成本较低的 SL RNA 检测可作为一种补充高通量工具。如果显微镜检查结果为阴性，可以使用 DBS 进行 PCR，并且可以将 SS 样本送到可提供 kDNA PCR 方法的转诊医疗机构。
