Field Validation of SYBR Green- and TaqMan-Based Real-Time PCR Using Biopsy and Swab Samples To Diagnose American Tegumentary Leishmaniasis in an Area Where Leishmania (Viannia) braziliensis Is Endemic

• Published in: Journal of clinical microbiology Vol. 55; no. 2; pp. 526 - 534
• Main Authors: Gomes, Ciro Martins, Cesetti, Mariana Vicente, de Paula, Natália Aparecida, Vernal, Sebastián, Gupta, Gaurav, Sampaio, Raimunda Nonata Ribeiro, Roselino, Ana Maria
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.02.2017
• Subjects: Adult; Aged; Biopsy; Endemic Diseases; Female; Humans; Leishmania; Leishmania braziliensis - isolation & purification; Leishmaniasis, Mucocutaneous - diagnosis; Leishmaniasis, Mucocutaneous - epidemiology; Male; Middle Aged; Molecular Diagnostic Techniques - methods; Organic Chemicals; Parasitology; Prospective Studies; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity; Staining and Labeling; Viannia braziliensis; diagnosis; kinetoplast DNA; mucocutaneous; real-time PCR; leishmaniasis; real-time polymerase chain reaction
• ISSN: 0095-1137; 1098-660X
• DOI: 10.1128/JCM.01954-16

The precise diagnosis of American tegumentary leishmaniasis (ATL) is an essential task due to the disease's associated morbidity. A noninvasive, extremely sensitive, and highly specific exam is critical, particularly for mucosal leishmaniasis (ML), in which a low parasite quantity is expected. We aimed to compare the diagnostic accuracy of swab and biopsy sample analysis using SYBR Green- and TaqMan-based real-time PCR (qPCR) assays with that of a composite reference standard consisting of the Montenegro skin test, serology, histopathology, smears, culture, and conventional PCR. In total, 55 patients with ATL (ML, 18 patients; cutaneous leishmaniasis [CL], 37 patients) and 36 patients without ATL were studied. qPCR analysis of swabs was more accurate when using SYBR Green (87.88%; 95% confidence interval [CI], 77.86 to 93.73 patients) than when using TaqMan (78.79%; 95% CI, 67.49 to 86.92%) (P = 0.031). SYBR Green (84.72%; 95% CI, 74.68 to 91.25%) was also more accurate than TaqMan (73.61%; 95% CI, 62.42 to 82.41%) for biopsy samples (P = 0.008). All qPCR methods were 100% specific. Swabs and biopsy specimens had similar sensitivity when using the same chemistry (P = 0.125 for SYBR Green and P = 0.625 for TaqMan). Moreover, qPCR achieved better performance than most existing techniques used for the diagnosis of ATL and also detected the Leishmania parasite in a greater proportion of patients than the associated histopathology, smear, culture, and conventional PCR techniques did. Swabs therefore represent a useful diagnostic tool because they not only are noninvasive but also can achieve an accuracy similar to that of biopsy samples. The high accuracy of SYBR Green-based qPCR may also reduce the requirement for associated parasitological tests for ATL diagnosis.