Diagnosis of Human Leptospirosis: High Resolution Melting Analysis for Direct Detection of Leptospira in the Early Stage of the Disease in a Clinical Setting

Abstract

Currently, the direct detection of Leptospira infection can be done in clinical laboratories by a conventional nested polymerase chain reaction method (nested PCR), which is labourious and time-consuming. To overcome these drawbacks, we tested a set of paired samples of serum and urine from 202 patients presenting at a hospital located in an area endemic for leptospirosis using high resolution melting (HRM). The results were compared with those obtained by nested PCR for direct detection of the pathogen in both specimens and with the gold standard test used for indirect detection of anti-Leptospira antibodies in serum (the microscopic agglutination test, MAT). The HRM assay results were positive for 46/202 (22.7%) samples, whereas 47/202 (23.3%) samples were positive by nested PCR. As expected in recently infected febrile patients, MAT results were positive in only 3/46 (6.5%) HRM-positive samples. We did a unique comparative analysis using a robust biobank of paired samples of serum and urine from the same patient to validate the HRM assay for molecular diagnosis of human leptospirosis in a clinical setting. This assay fills a void unmet by serologic assays as it can detect the presence of Leptospira in biological samples even before development of antibody takes place.