Clinical evaluation of an in-house probe-based real-time PCR assay for early diagnosis of leptospirosis compared with Microscopic Agglutination Test
TL;DR
An in-house real-time PCR assay targeting lipL32 gene of pathogenic Leptospira species was developed and validated with known DNA extracts and successfully detected Leptospira DNA positive controls and two clinical samples.
Abstract
Introduction: TaqMan probe-based real-time PCR assay targeting the lipL32 gene offer higher specificity and accuracy than conventional PCR for leptospirosis diagnosis. However, it has not been implemented in Sri Lanka.Methods: An in-house real-time PCR assay targeting lipL32 gene of pathogenic Leptospira species was developed and validated with known DNA extracts. PCR assay was performed for whole blood of 30 clinically suspicious patients within the first five days of illness and compared with Microscopic Agglutination Test (MAT), performed after day six with titers ≥1 : 320 considered positive. Haematological and biochemical results were retrieved.Results: Of 30 patients, 16 (53.3%) were confirmed positive: 14 by MAT alone and 2 by both PCR and MAT. DNA from Leptospira interrogans serovar Icterohaemorrhagiae was consistently amplified across six independent runs with an average CT value of 12. The two clinical PCR-positive samples showed CT values of 28 and 30. Compared with MAT, the PCR assay had a diagnostic sensitivity of 13.3% and specificity of 100%. Haematological findings included neutrophilia (>70%) in 86.7%, lymphopenia (<20%) in 90%, and thrombocytopenia (<100×10⁹/L) in 96.7%. All patients had elevated C-reactive protein (>5 mg/L). Most had raised AST levels (>35 U/L), while ALT and serum creatinine were largely normal.Conclusions: The newly designed primers and probes successfully detected Leptospira DNA positive controls and two clinical samples. Hence, further validation with a larger sample size may enhance the assay’s sensitivity and clinical utility.