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An immunomagnetic separation-PCR method for detection of pathogenic Leptospira in biological fluids
Cláudia Pinho Hartleben Fernandes1, Fabiana Kömmling Seixas, Mariana Loner Coutinho
1Centro de Biotecnologia, Universidade Federal de Pelotas, Pelotas, Brazil.
Abstract:
Abstract Leptospirosis is a zoonotic disease that occurs worldwide and is caused by pathogenic bacteria of the genus Leptospira. Clinical manifestations of leptospirosis are similar to other febrile illnesses and this fact frequently retards the beginning of antibiotic therapy. Thus, early and accurate diagnosis is a prerequisite for proper treatment of leptospirosis. Antigen and DNA-based detection tests offer potential advantage over tests based on antibody detection for early diagnosis of leptospirosis since antibodies only reach detectable levels several days after the onset of the infection. This work describes a method for detection of pathogenic Leptospira that associates an immunoseparation step with a PCR assay and uses an internal amplification control (IAC) to ensure accuracy of the test. The immunoseparation was performed with protein A-magnetic beads in house coated with an MAb specific for LipL32, the major outer membrane protein of pathogenic Leptospira; PCR was performed using lipL32 specific primers. The IMS-PCR method enhanced detection of Leptospira in experimentally contaminated human sera and urine when compared to PCR performed alone. IMS-PCR was able to detect 10(2) Leptospira cells per mL of human sera and urine, corresponding to 25 genomic copies per PCR reaction.
Insights
Early diagnosis of leptospirosis is crucial. A new immunoseparation combined with PCR (IMS-PCR) method improves detection of pathogenic Leptospira bacteria in human samples, enabling faster treatment.
Area of Science:
- Microbiology
- Infectious Diseases
- Diagnostic Technology
Background:
- Leptospirosis, a global zoonotic disease caused by Leptospira, presents with non-specific febrile symptoms, often delaying diagnosis and treatment.
- Early diagnosis is critical for effective antibiotic therapy, as antibody detection methods are only reliable several days post-infection.
- Antigen and DNA-based tests offer advantages for early leptospirosis detection over antibody-based methods.
Purpose of the Study:
- To develop and validate an enhanced diagnostic method for early detection of pathogenic Leptospira.
- To improve the sensitivity and accuracy of Leptospira detection in clinical samples like human sera and urine.
- To establish a reliable method for early diagnosis that overcomes limitations of existing techniques.
Main Methods:
- Development of an immunoseparation technique using protein A-magnetic beads coated with a monoclonal antibody (MAb) specific to the outer membrane protein LipL32.
- Integration of immunoseparation with a Polymerase Chain Reaction (PCR) assay targeting the lipL32 gene.
- Inclusion of an internal amplification control (IAC) to ensure the accuracy and reliability of the IMS-PCR assay.
Main Results:
- The developed Immunoseparation-PCR (IMS-PCR) method significantly enhanced the detection of pathogenic Leptospira in experimentally contaminated human sera and urine.
- IMS-PCR demonstrated a detection limit of 10(2) Leptospira cells per mL in human sera and urine.
- The method corresponds to detecting as few as 25 genomic copies per PCR reaction, indicating high sensitivity.
Conclusions:
- The IMS-PCR method provides a sensitive and accurate approach for the early diagnosis of leptospirosis.
- This technique overcomes the limitations of traditional diagnostic methods by enabling earlier detection of Leptospira.
- The enhanced detection capabilities of IMS-PCR are crucial for timely initiation of antibiotic treatment and improved patient outcomes.
