Saldarriaga Omar A, Castellanos-Gonzalez Alejandro, Porrozzi Renato, Baldeviano Gerald C, Lescano Andrés G, de Los Santos Maxy B, Fernandez Olga L, Saravia Nancy G, Costa Erika, Melby Peter C, Travi Bruno L
Division of Infectious Diseases, Department of Internal Medicine, University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America.
Center for Tropical Diseases (CTD), University of Texas Medical Branch (UTMB), Galveston, Texas, United States of America.
PLoS Negl Trop Dis. 2016 Apr 26;10(4):e0004638. doi: 10.1371/journal.pntd.0004638. eCollection 2016 Apr.
Cutaneous and mucosal leishmaniasis is widely distributed in Central and South America. Leishmania of the Viannia subgenus are the most frequent species infecting humans. L. (V.) braziliensis, L. (V.) panamensis are also responsible for metastatic mucosal leishmaniasis. Conventional or real time PCR is a more sensitive diagnostic test than microscopy, but the cost and requirement for infrastructure and trained personnel makes it impractical in most endemic regions. Primary health systems need a sensitive and specific point of care (POC) diagnostic tool. We developed a novel POC molecular diagnostic test for cutaneous leishmaniasis caused by Leishmania (Viannia) spp. Parasite DNA was amplified using isothermal Recombinase Polymerase Amplification (RPA) with primers and probes that targeted the kinetoplast DNA. The amplification product was detected by naked eye with a lateral flow (LF) immunochromatographic strip. The RPA-LF had an analytical sensitivity equivalent to 0.1 parasites per reaction. The test amplified the principal L. Viannia species from multiple countries: L. (V.) braziliensis (n = 33), L. (V.) guyanensis (n = 17), L. (V.) panamensis (n = 9). The less common L. (V.) lainsoni, L. (V.) shawi, and L. (V.) naiffi were also amplified. No amplification was observed in parasites of the L. (Leishmania) subgenus. In a small number of clinical samples (n = 13) we found 100% agreement between PCR and RPA-LF. The high analytical sensitivity and clinical validation indicate the test could improve the efficiency of diagnosis, especially in chronic lesions with submicroscopic parasite burdens. Field implementation of the RPA-LF test could contribute to management and control of cutaneous and mucosal leishmaniasis.
皮肤利什曼病和黏膜利什曼病广泛分布于中美洲和南美洲。维安亚亚属的利什曼原虫是最常感染人类的物种。巴西利什曼原虫（维安亚亚属）、巴拿马利什曼原虫（维安亚亚属）也会引发转移性黏膜利什曼病。传统或实时聚合酶链反应（PCR）是比显微镜检查更灵敏的诊断测试，但成本以及对基础设施和专业人员的要求使其在大多数流行地区不切实际。基层医疗卫生系统需要一种灵敏且特异的即时检测（POC）诊断工具。我们开发了一种针对由利什曼原虫（维安亚亚属）引起的皮肤利什曼病的新型POC分子诊断测试。使用等温重组酶聚合酶扩增（RPA）技术，以靶向动基体DNA的引物和探针扩增寄生虫DNA。扩增产物通过侧向流动（LF）免疫层析试纸条进行肉眼检测。RPA-LF的分析灵敏度相当于每个反应0.1个寄生虫。该测试扩增了来自多个国家的主要维安亚利什曼原虫物种：巴西利什曼原虫（维安亚亚属）（n = 33）、圭亚那利什曼原虫（维安亚亚属）（n = 17）、巴拿马利什曼原虫（维安亚亚属）（n = 9）。较少见的赖氏利什曼原虫（维安亚亚属）、沙氏利什曼原虫（维安亚亚属）和奈菲氏利什曼原虫（维安亚亚属）也能被扩增。在利什曼原虫亚属的寄生虫中未观察到扩增。在少量临床样本（n = 13）中，我们发现PCR与RPA-LF之间的一致性为100%。高分析灵敏度和临床验证表明该测试可提高诊断效率，尤其是在寄生虫负荷处于亚显微水平的慢性病变中。RPA-LF测试的现场应用有助于皮肤利什曼病和黏膜利什曼病的管理与控制。
