Sensitive real-time PCR detection of pathogenic Leptospira spp. and a comparison of nucleic acid amplification methods for the diagnosis of leptospirosis.
BACKGROUND
Bacteria of the genus Leptospira, the causative agents of leptospirosis, are categorized into pathogenic and non-pathogenic species. However, the benefit of using a clinical diagnostic that is specific for pathogenic species remains unclear. In this study, we present the development of a real-time PCR (rtPCR) for the detection of pathogenic Leptospira (the pathogenic rtPCR), and we perform a comparison of the pathogenic rtPCR with a published assay that detects all Leptospira species [the undifferentiated febrile illness (UFI) assay] and a reference 16S Leptospira rtPCR, which was originally designed to detect pathogenic species.


RESULTS
For the pathogenic rtPCR, a new hydrolysis probe was designed for use with primers from the UFI assay, which targets the 16S gene. The pathogenic rtPCR detected Leptospira DNA in 37/37 cultured isolates from 5 pathogenic and one intermediate species. Two strains of the non-pathogenic L. biflexa produced no signal. Clinical samples from 65 patients with suspected leptospirosis were then tested using the pathogenic rtPCR and a reference Leptospira 16S rtPCR. All 65 samples had tested positive for Leptospira using the UFI assay; 62 (95.4%) samples tested positive using the pathogenic rtPCR (p = 0.24). Only 24 (36.9%) samples tested positive in the reference 16S rtPCR (p<0.0001 for comparison with the pathogenic rtPCR and UFI assays). Amplicon sequencing confirmed the detection of pathogenic Leptospira species in 49/50 cases, including 3 cases that were only detected using the UFI assay.


CONCLUSIONS
The pathogenic rtPCR displayed similar sensitivity to the UFI assay when testing clinical specimens with no difference in specificity. Both assays proved significantly more sensitive than a real-time molecular test used for comparison. Future studies are needed to investigate the clinical and epidemiologic significance of more sensitive Leptospira detection using these tests.
Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, California, United States of America.
Jesse J
JJ
Waggoner
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.
Ilana
I
Balassiano
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
Janaki
J
Abeynayake
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
Malaya K
MK
Sahoo
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
Alisha
A
Mohamed-Hadley
Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.
Yuanyuan
Y
Liu
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.
Juliana Magalhães
JM
Vital-Brazil
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.
Benjamin A
BA
Pinsky
D001483:Base Sequence; D006801:Humans; D007919:Leptospira / Q000235:genetics* / Q000302:isolation & purification*; D007922:Leptospirosis / Q000097:blood / Q000175:diagnosis* / Q000382:microbiology; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
D012336:RNA, Ribosomal, 16S