title: Application of a real time Polymerase Chain Reaction (PCR) assay for the early diagnosis of human leptospirosis in Sri Lanka.
abstract: Leptospirosis has a major impact on health in Sri Lanka but is probably grossly under-recognized due to difficulties in clinical diagnosis and lack of diagnostic laboratory services. The objective of this study was to establish and evaluate a SYBR Green-based real-time Polymerase Chain Reaction (rt-PCR) assay for early, rapid and definitive laboratory diagnosis of leptospirosis in Sri Lanka. The rt-PCR assay was established and analytical specificity and sensitivity were determined using reference DNA samples. Evaluation of the assay for diagnosis of clinical samples was performed using two panels of serum samples obtained from 111 clinically suspected adult patients. Patients were confirmed as leptospirosis (n = 65) and non-leptospirosis (n = 30) by the Patoc - MAT. Other 16 samples gave ambiguous results. The analytical sensitivity of the rt-PCR was approximately 60 genome copies and no cross-reactivity was observed with saprophytic Leptospira spp. and other pathogenic microorganisms. Based on confirmation with Patoc-MAT on paired samples this corresponds to a diagnostic sensitivity and specificity of 67.7% (44/65) and 90.0% (27/30), respectively. This study showed that rt-PCR has the potential to facilitate rapid and definitive diagnosis of leptospirosis during early phase of infection in Sri Lanka.
authors.affiliation: Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, P.O. Box 06, Ragama, Sri Lanka; Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03, Sri Lanka. Electronic address: thanujaddenipitiya@yahoo.com.
authors.forename: D T H
authors.initials: DT
authors.lastname: Denipitiya
authors.affiliation: Department of Chemistry, Faculty of Science, University of Colombo, Colombo 03, Sri Lanka. Electronic address: vishvanathc@hotmail.com.
authors.forename: N V
authors.initials: NV
authors.lastname: Chandrasekharan
authors.affiliation: Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, P.O. Box 06, Ragama, Sri Lanka. Electronic address: wabeyewickreme@yahoo.com.
authors.forename: W
authors.initials: W
authors.lastname: Abeyewickreme
authors.affiliation: WHO/FAO/OIE Collaborating Centre on Leptospirosis, KIT Biomedical Research, Meibergdreef 39, Amsterdam, Netherlands. Electronic address: cedrichartskeerl@gmail.com.
authors.forename: C M
authors.initials: CM
authors.lastname: Hartskeerl
authors.affiliation: WHO/FAO/OIE Collaborating Centre on Leptospirosis, KIT Biomedical Research, Meibergdreef 39, Amsterdam, Netherlands. Electronic address: r.hartskeerl@kit.nl.
authors.forename: R A
authors.initials: RA
authors.lastname: Hartskeerl
authors.affiliation: Postgraduate Institute of Medicine, University of Colombo, Sri Lanka. Electronic address: ajiffriy@yahoo.com.
authors.forename: A M
authors.initials: AM
authors.lastname: Jiffrey
authors.affiliation: Molecular Medicine Unit, Faculty of Medicine, University of Kelaniya, P.O. Box 06, Ragama, Sri Lanka. Electronic address: menakaha@yahoo.com.
authors.forename: M D
authors.initials: MD
authors.lastname: Hapugoda
mesh_terms: D000328:Adult; D004269:DNA, Bacterial / Q000097:blood / Q000235:genetics; D005260:Female; D006801:Humans; D007919:Leptospira / Q000235:genetics*; D007922:Leptospirosis / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D008297:Male; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013188:Sri Lanka
chemical_list: D004269:DNA, Bacterial
keywords: Diagnosis; Leptospirosis; MAT; Real-time PCR; SYBR Green-I