Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters
Real-Time PCR Assay for Detection and Quantification of Leishmania (Viannia) Organisms in Skin and Mucosal Lesions: Exploratory Study of Parasite Load and Clinical Parameters
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DOI：
10.1128/jcm.00208-13
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发表时间：
2013-06-01
影响因子：
9.4
通讯作者：
Arevalo, Jorge
中科院分区：
文献类型：
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作者：
Jara, Marlene;Adaui, Vanessa;Arevalo, Jorge
早期的组织病理学研究表明，皮肤利什曼病(CL)和粘膜利什曼病(ML)皮损以及急性和慢性利什曼病之间的寄生虫负荷可能不同。正式的演示需要高度灵敏的检测和准确地量化人体皮损组织中的利什曼原虫。在本研究中，我们建立了一种针对微小环动泡体DNA(KDNA)的实时定量聚合酶链式反应(QPCR)方法来检测和定量检测利什曼原虫(Viannia)寄生虫。我们评估了总共156例CL或ML疑似病例的病变活检标本，并将我们的kDNA qPCR方法与先前验证的基于葡萄糖-6-磷酸脱氢酶(G6PD)基因的qPCR方法的定量性能进行了比较。我们还检查了寄生虫载量和临床参数之间的关系。检测利什曼原虫的灵敏度为97.9%，特异度为87.5%。KDNAqPCR法和G6PDqPCR法测定的寄生虫载量高度相关(r=0.87P<；0.0001)，但前者的敏感性更高(P=0.000)。CL病变的寄生虫载量是ML病变的10倍(P=0.009)。在CL患者中，寄生虫载量与病程呈负相关(P=0.004)，但不同寄生虫种类、患者年龄、病变数目或面积的寄生虫载量无差异。我们的发现证实，CL和最近发病(<；3个月)与高寄生虫负荷有关。我们的kDNA定量聚合酶链式反应方法对利什曼原虫(Viannia)spp的检测和定量具有高度的敏感性和准确性。在病变活检标本中。它作为美国被盖型利什曼病的诊断和随访工具具有潜在的应用价值。
Earlier histopathology studies suggest that parasite loads may differ between cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) lesions and between acute and chronic CL. Formal demonstration requires highly sensitive detection and accurate quantification of Leishmania in human lesional tissue. In this study, we developed a quantitative real-time PCR (qPCR) assay targeting minicircle kinetoplast DNA (kDNA) to detect and quantify Leishmania (Viannia) parasites. We evaluated a total of 156 lesion biopsy specimens from CL or ML suspected cases and compared the quantitative performance of our kDNA qPCR assay with that of a previously validated qPCR assay based on the glucose-6-phosphate dehydrogenase (G6PD) gene. We also examined the relationship between parasite load and clinical parameters. The kDNA qPCR sensitivity for Leishmania detection was 97.9%, and its specificity was 87.5%. The parasite loads quantified by kDNA qPCR and G6PD qPCR assays were highly correlated (r = 0.87; P < 0.0001), but the former showed higher sensitivity (P = 0.000). CL lesions had 10-fold-higher parasite loads than ML lesions (P = 0.009). Among CL patients, the parasite load was inversely correlated with disease duration (P = 0.004), but there was no difference in parasite load according to the parasite species, the patient's age, and number or area of lesions. Our findings confirm that CL and recent onset of disease (< 3 months) are associated with a high parasite load. Our kDNA qPCR assay proved highly sensitive and accurate for the detection and quantification of Leishmania (Viannia) spp. in lesion biopsy specimens. It has potential application as a diagnostic and follow-up tool in American tegumentary leishmaniasis.