title: Development and validation of a real-time PCR for detection of pathogenic leptospira species in clinical materials.
abstract: Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first--for treatment important--4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
authors.affiliation: WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis and Section of Epidemiology, Department of Biomedical Research, Royal Tropical Institute (KIT), Amsterdam, The Netherlands.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Ahmed
authors.forename: Mirjam F M
authors.initials: MF
authors.lastname: Engelberts
authors.forename: Kimberly R
authors.initials: KR
authors.lastname: Boer
authors.forename: Niyaz
authors.initials: N
authors.lastname: Ahmed
authors.forename: Rudy A
authors.initials: RA
authors.lastname: Hartskeerl
mesh_terms: D052160:Benzothiazoles; D015331:Cohort Studies; D017931:DNA Primers / Q000378:metabolism; D003959:Diamines; D005260:Female; D006801:Humans; D007919:Leptospira / Q000235:genetics* / Q000472:pathogenicity*; D007922:Leptospirosis / Q000097:blood* / Q000175:diagnosis*; D008297:Male; D009930:Organic Chemicals / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011804:Quinolines; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D012698:Serologic Tests
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I