Leptospira Serology

Leptospirosis is a zoonosis caused by pathogenic Leptospira (L. interrogans and others) shed in the urine of rodents, livestock, dogs and wildlife. Humans are infected through broken skin or mucous membranes in contact with contaminated water or soil (floods, rice and sugar cane fields, sewers, recreational water) or animals.

The reference serological test is the microscopic agglutination test (MAT), using live serovars and reading agglutination under dark field microscopy. IgM ELISA, lateral flow assays and macroscopic slide agglutination are used for rapid screening. PCR on blood (first week) and urine (after the first week) detects leptospiral DNA. Culture takes weeks and is rarely helpful.

Leptospirosis causes about 1 million cases and 60 000 deaths each year, mostly in tropical and subtropical regions, with outbreaks after floods and heavy rain. The illness has a leptospiraemic phase (1 week, with sudden fever, myalgia especially of the calves, headache, conjunctival suffusion) followed by an immune phase.

About 5 to 15% develop severe disease (Weil disease) with jaundice, acute kidney injury, haemorrhage and pulmonary haemorrhage syndrome, with mortality of 5 to 15% in severe disease and over 50% with pulmonary haemorrhage.

Indications. Acute febrile illness with myalgia, conjunctival suffusion and exposure to floodwater, rodents, farm animals or fresh water. Fever with jaundice, renal failure and bleeding (Weil disease). Haemoptysis with fever (pulmonary haemorrhage). Aseptic meningitis. Fever of unknown origin in endemic areas. Differential diagnosis of dengue, malaria, typhoid, hantavirus and viral hepatitis. Occupational screening.

Specimen. Acute and convalescent serum 1 to 2 mL (collected at presentation and 7 to 14 days later) for MAT. Blood (EDTA or serum) in the first 10 days and urine (after day 7) for PCR. CSF for PCR in meningitis.

Reference values.

• MAT: negative under 1 in 100 (cut off set by region, 1 in 50 to 1 in 400 in endemic areas).
• IgM ELISA: negative under the cut off.
• Leptospira DNA PCR: not detected.

Interpretation.

• MAT four fold rise in titre between paired sera, or seroconversion from negative to 1 in 100 or higher, confirms leptospirosis. This is the reference method.
• A single MAT titre of 1 in 800 or more with a compatible illness is highly suggestive in non-endemic areas. Single titres of 1 in 100 to 1 in 400 in endemic areas may reflect past exposure.
• IgM ELISA positive from days 5 to 10 of illness (sensitivity about 70% early, rising to over 90% after 10 days). It is useful as a screening test, with confirmation by MAT.
• Antibodies are usually undetectable in the first 5 to 7 days, so the first sample is often negative. Treatment is not delayed for results.
• PCR on blood is positive in the first week (sensitivity 50 to 90%), and urine PCR is positive from week 2 to week 4 and sometimes longer.
• The highest titre often reacts with a serovar different from the infecting one (paradoxical reaction), and the serogroup rather than the serovar is reported.
• Titres decline over months to years, and low persistent titres remain after infection.
• Laboratory features supporting severe disease: neutrophilia, thrombocytopenia, raised creatinine, conjugated hyperbilirubinaemia with only mildly raised transaminases (differentiating from viral hepatitis), raised creatine kinase and hypokalaemia.

Critical values and findings needing immediate action.

• Fever with jaundice, oliguria and bleeding: Weil disease, urgent intravenous antibiotics (benzylpenicillin, ceftriaxone or doxycycline), fluid and electrolyte management, dialysis if needed, without waiting for serology.
• Haemoptysis, dyspnoea and falling oxygen saturation with infiltrates: leptospiral pulmonary haemorrhage syndrome, intensive care, ventilation and high mortality.
• Hypotension and arrhythmia with myocarditis: supportive care and cardiac monitoring.
• Jarisch-Herxheimer reaction within hours of the first antibiotic dose: supportive care.
• Exposure of a laboratory worker or a high risk contact: doxycycline prophylaxis (200 mg once weekly) in selected high risk exposures.

Interferences and pitfalls.

• Early serology is negative. A single sample in the first week cannot exclude leptospirosis, and paired sera are required.
• MAT needs a panel of live local serovars and experienced staff, and is available only in reference laboratories. Results vary between laboratories.
• Cross-reactions occur in syphilis, Lyme disease, relapsing fever, legionellosis, autoimmune disease and viral infections, particularly with IgM tests.
• Antibiotics given early may blunt the antibody response.
• Cut off titres must be set for the local population, as background titres are high in farmers, abattoir workers and flood prone communities.
• A negative PCR does not exclude infection, and sensitivity depends on timing and sample type.
• Dark field microscopy of blood is unreliable.
• Dengue, malaria, scrub typhus, typhoid, hantavirus infection, viral hepatitis, sepsis and Zika or chikungunya infection are common co-infections or differentials.
• Treatment given empirically in endemic regions with compatible illness is life saving, and confirmation can follow.

Special groups.

• Children: usually mild, with anicteric illness, and Weil disease is less common.
• Pregnancy: may cause abortion and foetal death, treated with penicillin or ceftriaxone.
• Older adults and alcohol misuse: higher risk of severe disease and renal failure.
• Occupational and recreational: farmers, sewer and abattoir workers, military, triathletes and travellers after freshwater exposure.
• Regions: high burden in South and Southeast Asia, Latin America, the Caribbean, Pacific islands and sub-Saharan Africa, with seasonal peaks after rainfall and flooding.

Follow up tests. Paired MAT, IgM ELISA, blood and urine PCR, full blood count with platelets, urea, creatinine and electrolytes, liver function tests (bilirubin, ALT, AST), creatine kinase, coagulation screen, urinalysis, chest radiograph, ECG, blood cultures, malaria smear, dengue NS1 and serology, hepatitis serology, CSF analysis in meningitis, and renal replacement monitoring.