title: qPCR detection of Mycobacterium leprae in biopsies and slit skin smear of different leprosy clinical forms.
abstract: Leprosy, whose etiological agent is Mycobacterium leprae, is a chronic infectious disease that mainly affects the skin and peripheral nervous system. The diagnosis of leprosy is based on clinical evaluation, whereas histopathological analysis and bacilloscopy are complementary diagnostic tools. Quantitative PCR (qPCR), a current useful tool for diagnosis of infectious diseases, has been used to detect several pathogens including Mycobacterium leprae. The validation of this technique in a robust set of samples comprising the different clinical forms of leprosy is still necessary. Thus, in this study samples from 126 skin biopsies (collected from patients on all clinical forms and reactional states of leprosy) and 25 slit skin smear of leprosy patients were comparatively analyzed by qPCR (performed with primers for the RLEP region of M. leprae DNA) and routine bacilloscopy performed in histological sections or in slit skin smear. Considering clinical diagnostic as the gold standard, 84.9% of the leprosy patients were qPCR positive in skin biopsies, resulting in 84.92% sensitivity, with 84.92 and 61.22% positive (PPV) and negative (NPV) predictive values, respectively. Concerning bacilloscopy of histological sections (BI/H), the sensitivity was 80.15% and the PPV and NPV were 80.15 and 44.44%, respectively. The concordance between qPCR and BI/H was 87.30%. Regarding the slit skin smear, 84% of the samples tested positive in the qPCR. Additionally, qPCR showed 100% specificity, since all samples from different mycobacteria, from healthy individuals, and from other granulomatous diseases presented negative results. In conclusion, the qPCR technique for detection of M. leprae using RLEP primers proved to be specific and sensitive, and qPCR can be used as a complementary test to diagnose leprosy irrespective of the clinical form of disease.
authors.affiliation: Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas, Bauru, SP, Brazil.
authors.forename: Michelle de Campos Soriani
authors.initials: MC
authors.lastname: Azevedo
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Natália Mortari
authors.initials: NM
authors.lastname: Ramuno
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Luciana Raquel Vincenzi
authors.initials: LR
authors.lastname: Fachin
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Mônica
authors.initials: M
authors.lastname: Tassa
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Patrícia Sammarco
authors.initials: PS
authors.lastname: Rosa
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Andrea de Faria Fernandes
authors.initials: AF
authors.lastname: Belone
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Suzana Madeira
authors.initials: SM
authors.lastname: Diório
authors.affiliation: Instituto Lauro de Souza Lima, Bauru, SP, Brazil.
authors.forename: Cleverson Teixeira
authors.initials: CT
authors.lastname: Soares
authors.affiliation: Universidade de São Paulo, Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas, Bauru, SP, Brazil.
authors.forename: Gustavo Pompermaier
authors.initials: GP
authors.lastname: Garlet
authors.affiliation: Universidade do Sagrado Coração, Centro de Ciências da Saúde, Bauru, SP, Brazil. Electronic address: tromboneap@yahoo.com.br.
authors.forename: Ana Paula Favaro
authors.initials: AP
authors.lastname: Trombone
mesh_terms: D001706:Biopsy; D017931:DNA Primers / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000302:isolation & purification; D006801:Humans; D007918:Leprosy / Q000382:microbiology* / Q000473:pathology; D009166:Mycobacterium leprae / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology* / Q000473:pathology
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial
keywords: Bacilloscopy; Leprosy; Mycobacterium leprae; qPCR