PP7 -Validation of an in-house TaqMan probe based real time PCR assay for early detection of leptospirosis in clinically suspected patients at National Hospital Galle

Abstract

Introduction

The TaqMan probe-based real-time PCR assay targeting the lipL32 gene exhibits higher specificity and accuracy compared to conventional PCR assays used for the diagnosis of leptospirosis. However, it has not yet been implemented as a diagnostic test. In contrast, the gold standard test, the Microscopic Agglutination Test (MAT), is currently restricted to a few reference laboratories in Sri Lanka.

Objective

This study aimed to develop a probe-based real-time PCR assay for the early detection of leptospirosis and to evaluate its performance in comparison with MAT.

Design, setting, and methods

An in-house PCR assay targeting the lipL32 gene, which is specific to pathogenic Leptospira species, was developed using a modified amplification protocol. Species-specific primers and probes were confirmed using DNA extracts (13.2ng/µL and 12.62ng/µL) of Leptospira interrogans serovar Icterohaemorrhagiae. Extracted nuclease-free water and human RNaseP gene were used as negative and internal controls, respectively. Whole blood samples from 30 clinically suspicious patients in the early acute phase admitted to the National Hospital Galle were tested. PCR test results were compared with MAT results, which were performed at the Medical Research Institute in Colombo, using serum samples collected during the late acute phase of the illness.

Results

Out of the total, sixteen patients (53.3%) were confirmed to have leptospirosis via either the MAT test alone (N=14) or a combination of PCR and MAT (N=2). A MAT titer of ≥ 1:320 was considered positive. The aforementioned Leptospira DNA extracts were tested a total of six times in six different runs, and each yielded amplification curves with an average cycle threshold (CT) value of 12. Two clinical samples were positive for both PCR and MAT, with amplification of the Leptospira target at CT values of 28 and 30. All internal controls showed amplification, and the negative controls failed to show any. Compared to MAT, the diagnostic performance of PCR showed a sensitivity of 13.33%, specificity of 100%, positive predictive value of 100% and negative predictive value of 53.57%.

Conclusion

While MAT remains the gold standard for leptospirosis diagnosis, the PCR assay offers promise as a complementary tool for early detection, especially before antibody titers rise. Further validation with a larger sample size and optimization of sample collection timing may enhance the assay’s diagnostic sensitivity and clinical applicability.

Acknowledgments

Financial assistance by Avon Pharma Chem (Pvt) Ltd and resources provided by the Department of Community Medicine, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka, is acknowledged.