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Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic
Ilana Teruszkin Balassiano1, Juliana Magalhães Vital-Brazil, Martha Maria Pereira
1Laboratório de Zoonoses Bacterianas, Centro de Referência Nacional para Leptospirose, WHO/PAHO Centro Colaborador para Leptospirose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil. ilana@ioc.fiocruz.br
Abstract:
The aim of this study was to develop an immunocapture polymerase chain reaction (IC-PCR) protocol for leptospirosis. For the standardization of IC-PCR, polyclonal (AS) and monoclonal (MAb) antibodies against different serogroups and serovars of Leptospira were coupled to polystyrene plates. Human sera were artificially contaminated with leptospires and incubated on plates. The bacterial DNA was obtained and used in a multiplex PCR. Sensitivity was tested using sera contaminated with crescent concentrations of leptospires, while specificity was established using sera contaminated with different bacterial genera and sera obtained from patients positive for viral infections. IC-PCR using AS was able to recognize specific serogroups, although some cross-reactions have been observed. No cross-reactions were observed when MAbs were used; however, the sensitivity in this case was lower than that of IC-PCR using AS. IC-PCR proved to be specific to Leptospira and is a promising tool for early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup.
Insights
This study developed an immunocapture polymerase chain reaction (IC-PCR) for leptospirosis diagnosis. The new IC-PCR method shows specificity for Leptospira, offering potential for early detection and serovar identification.
Area of Science:
- Microbiology
- Immunology
- Molecular Diagnostics
Background:
- Leptospirosis is a significant zoonotic disease with diverse clinical presentations.
- Accurate and early diagnosis is crucial for effective treatment and public health management.
- Current diagnostic methods may have limitations in sensitivity or specificity for certain Leptospira serogroups.
Purpose of the Study:
- To develop and standardize an immunocapture polymerase chain reaction (IC-PCR) protocol for the detection of Leptospira.
- To evaluate the sensitivity and specificity of the developed IC-PCR assay.
- To assess the potential of IC-PCR for identifying infecting Leptospira serogroups or serovars.
Main Methods:
- Development of IC-PCR by coupling polyclonal (AS) and monoclonal (MAb) antibodies to polystyrene plates.
- Incubation of artificially contaminated human sera with leptospires on antibody-coated plates.
- DNA extraction from captured bacteria followed by multiplex PCR for Leptospira detection.
- Sensitivity testing with serial dilutions of Leptospira and specificity testing with other bacterial genera and viral infections.
Main Results:
- IC-PCR using polyclonal antibodies recognized specific Leptospira serogroups but showed some cross-reactivity.
- IC-PCR using monoclonal antibodies demonstrated no cross-reactivity but exhibited lower sensitivity compared to polyclonal antibodies.
- The developed IC-PCR assay proved specific for Leptospira detection in human sera.
Conclusions:
- Immunocapture polymerase chain reaction (IC-PCR) is a specific and promising method for the early diagnosis of leptospirosis.
- The IC-PCR technique has the potential to provide additional information regarding the infecting Leptospira serovar or serogroup.
- Further optimization may be needed to balance sensitivity and specificity for widespread clinical application.
