Laboratory Diagnosis of Leptospirosis
The laboratory diagnosis of leptospirosis requires a combination of methods including microscopic agglutination test (MAT), serology, PCR, and culture, with MAT on paired samples remaining the gold standard despite its limitations. 1
Direct Detection Methods
Culture
- Gold standard for definitive diagnosis but has limited utility for early diagnosis 2
- Requires specialized media and expertise
- Takes 1-4 weeks for results, making it impractical for acute management
- Best performed on blood samples during the first week of illness or urine after the first week
Microscopy
- Direct visualization using dark-field microscopy
- Low sensitivity and requires experienced personnel
- Not recommended as a standalone diagnostic method
Nucleic Acid Amplification Tests (NAATs)
- PCR is valuable for early diagnosis before seroconversion 1
- Most useful during the first week of illness when leptospires are present in blood
- Higher sensitivity compared to culture for early diagnosis 2
- Target genes include Lfb1 which has shown promising diagnostic accuracy 3
- Limited availability in many developing countries 2
Serological Methods
Microscopic Agglutination Test (MAT)
- Gold standard serological test despite limitations 3
- Requires:
- Live cultures of different Leptospira serovars
- Specialized equipment and trained personnel
- Paired serum samples (acute and convalescent phases)
- Diagnostic criteria:
- Four-fold rise in agglutination titers between acute and convalescent samples
- Single titer ≥400 is considered positive in endemic areas 1
- Performance characteristics:
IgM ELISA
- More sensitive than MAT during acute phase 5
- Anti-leptospiral IgM ≥20 IU/ml considered positive 5
- Sensitivity: 86.0% by Bayesian latent class model analysis 5
- Specificity: 84.5% 5
- Suitable for early diagnosis when MAT sensitivity is low
- Various antigens used, including Leptospira fainei serovar Hurstbridge 3
Rapid Immunochromatography Tests
- Point-of-care tests like Leptocheck-WB
- Similar sensitivity to IgM-ELISA (87.4%) 5
- Specificity around 82.9% 5
- Ideal for resource-limited settings due to rapid results and minimal equipment requirements
Latex Agglutination Test (LAT)
- Point-of-care test using recombinant LigA/B antigens
- High sensitivity (99.5%) during early phase of infection 4
- Suitable as a bedside test in remote locations 4
Diagnostic Algorithm
Acute phase (first week of illness):
- PCR on blood samples (if available)
- IgM ELISA or rapid immunochromatography test
- Collect first serum sample for MAT
Convalescent phase (2-4 weeks after onset):
- Collect second serum sample for MAT
- Compare paired samples for four-fold rise in titer
Interpretation:
- A confirmed case requires:
- Laboratory confirmation through culture, PCR, or four-fold rise in MAT titer
- A probable case requires:
- Clinically compatible illness with supportive serological findings (MAT titer ≥200) 1
- A confirmed case requires:
Pitfalls and Considerations
- MAT has poor sensitivity during early disease but remains the reference standard
- Single acute-phase MAT has very low sensitivity (14%) and should not be used to rule out disease 3
- Cross-reactivity with other diseases may occur with serological tests
- Antibiotics initiated before testing may reduce sensitivity of culture and PCR
- Recent advances include machine learning models to improve MAT interpretation 6
- In endemic areas, background seropositivity may complicate interpretation of single titers