title: Reverse-Transcriptase PCR Detection of Leptospira: Absence of Agreement with Single-Specimen Microscopic Agglutination Testing.
abstract: BACKGROUND
  Reference diagnostic tests for leptospirosis include nucleic acid amplification tests, bacterial culture, and microscopic agglutination testing (MAT) of acute and convalescent serum. However, clinical laboratories often do not receive paired specimens. In the current study, we tested serum samples using a highly sensitive real-time nucleic acid amplification test for Leptospira and compared results to MAT performed on the same specimens.
  
  
  RESULTS
  478 serum samples from suspected leptospirosis cases in Rio de Janeiro were tested using a real-time RT-PCR for the diagnosis of leptospirosis, malaria and dengue (the Lepto-MD assay). The Lepto-MD assay detects all species of Leptospira (saprophytic, intermediate, and pathogenic), and in the current study, we demonstrate that this assay amplifies both Leptospira RNA and DNA. Dengue virus RNA was identified in 10 patients, and no cases of malaria were detected. A total of 65 samples (13.6%) were positive for Leptospira: 35 samples (7.3%) in the Lepto-MD assay, 33 samples (6.9%) by MAT, and 3 samples tested positive by both (kappa statistic 0.02). Poor agreement between methods was consistent regardless of the titer used to define positive MAT results or the day of disease at sample collection. Leptospira nucleic acids were detected in the Lepto-MD assay as late as day 22, and cycle threshold values did not differ based on the day of disease. When Lepto-MD assay results were added to the MAT results for all patients in 2008 (n=818), the number of detected leptospirosis cases increased by 30.4%, from 102 (12.5%) to 133 (16.3%).
  
  
  CONCLUSIONS
  This study demonstrates a lack of agreement between nucleic acid detection of Leptospira and single-specimen MAT, which may result from the clearance of bacteremia coinciding with the appearance of agglutinating antibodies. A combined testing strategy for acute leptospirosis, including molecular and serologic testing, appears necessary to maximize case detection.
authors.affiliation: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Waggoner
authors.affiliation: Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.
authors.forename: Ilana
authors.initials: I
authors.lastname: Balassiano
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Alisha
authors.initials: A
authors.lastname: Mohamed-Hadley
authors.affiliation: Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, Coleção de Leptospira, WHO/PAHO Centro Colaborador para Leptospirose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil.
authors.forename: Juliana Magalhães
authors.initials: JM
authors.lastname: Vital-Brazil
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Malaya K
authors.initials: MK
authors.lastname: Sahoo
authors.affiliation: Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America; Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Pinsky
mesh_terms: D000293:Adolescent; D000328:Adult; D000372:Agglutination Tests / Q000379:methods*; D000907:Antibodies, Bacterial / Q000097:blood; D002648:Child; D002675:Child, Preschool; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D003715:Dengue / Q000097:blood / Q000175:diagnosis / Q000821:virology; D005260:Female; D006801:Humans; D007919:Leptospira / Q000235:genetics*; D007922:Leptospirosis / Q000097:blood / Q000175:diagnosis* / Q000235:genetics / Q000382:microbiology*; D008297:Male; D008875:Middle Aged; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D055815:Young Adult
chemical_list: D000907:Antibodies, Bacterial; D004269:DNA, Bacterial