title: 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.
abstract: 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.
authors.affiliation: Division of Dermatology, Department of Medical Clinics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil ciromgomes@gmail.com.|Division of Dermatology, Department of Medical Clinics, University of Brasília, Brasília, Federal District, Brazil.
authors.forename: Ciro Martins
authors.identifier: 0000-0002-3069-6884
authors.initials: CM
authors.lastname: Gomes
authors.affiliation: Division of Dermatology, Department of Medical Clinics, University of Brasília, Brasília, Federal District, Brazil.
authors.forename: Mariana Vicente
authors.initials: MV
authors.lastname: Cesetti
authors.affiliation: Laboratory of Dermatology, University Hospital of Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.
authors.forename: Natália Aparecida
authors.initials: NA
authors.lastname: de Paula
authors.affiliation: Division of Dermatology, Department of Medical Clinics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.|Laboratory of Dermatology, University Hospital of Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.
authors.forename: Sebastián
authors.initials: S
authors.lastname: Vernal
authors.affiliation: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.|Department of Immunology, Max Rady College of Medicine, University of Manitoba, Winnipeg, Canada.
authors.forename: Gaurav
authors.initials: G
authors.lastname: Gupta
authors.affiliation: Division of Dermatology, Department of Medical Clinics, University of Brasília, Brasília, Federal District, Brazil.|Laboratory of Dermatomycology, Faculty of Medicine, University of Brasília, Brasília, Federal District, Brazil.
authors.forename: Raimunda Nonata Ribeiro
authors.initials: RN
authors.lastname: Sampaio
authors.affiliation: Division of Dermatology, Department of Medical Clinics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.|Laboratory of Dermatology, University Hospital of Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo State, Brazil.
authors.forename: Ana Maria
authors.initials: AM
authors.lastname: Roselino
mesh_terms: D000328:Adult; D000368:Aged; D052160:Benzothiazoles; D001706:Biopsy; D003959:Diamines; D019353:Endemic Diseases; D005260:Female; D006801:Humans; D007892:Leishmania braziliensis / Q000302:isolation & purification*; D007897:Leishmaniasis, Mucocutaneous / Q000175:diagnosis* / Q000453:epidemiology; D008297:Male; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009930:Organic Chemicals; D011446:Prospective Studies; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013194:Staining and Labeling
chemical_list: D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I
keywords: diagnosis; kinetoplast DNA; leishmaniasis; mucocutaneous; real-time PCR; real-time polymerase chain reaction