Diagnosis of Human Leptospirosis in a Clinical Setting: Real-Time PCR High Resolution Melting Analysis for Detection of Leptospira at the Onset of Disease

Lisa M Esteves,Maria L Vieira,Maria Gomes-Solecki,Claudia C Branco,Sara M Bulhões,Luisa Mota-Vieira,Teresa Carreira

doi:10.1038/s41598-018-27555-2

Abstract

Currently, direct detection of Leptospira can be done in clinical laboratories by conventional and by real-time PCR (qRT-PCR). We tested a biobank of paired samples of serum and urine from the same patient (202 patients) presenting at the hospital in an area endemic for leptospirosis using qRT-PCR followed by high resolution melting (HRM) analysis. The results were compared with those obtained by conventional nested PCR and with the serologic gold standard microscopic agglutination test (MAT). Differences were resolved by sequencing. qRT-PCR-HRM was positive for 46 of the 202 patients (22.7%, accuracy 100%) which is consistent with known prevalence of leptospirosis in the Azores. MAT results were positive for 3 of the 46 patients (6.5%). Analysis of paired samples allowed us to identify the illness point at which patients presented at the hospital: onset, dissemination or excretion. The melting curve analysis of Leptospira species revealed that 60.9% (28/46) of patients were infected with L. interrogans and 39.1% (18/46) were infected with L. borgpetersenii, both endemic to the Azores. We validated the use of qRT-PCR-HRM for diagnosis of leptospirosis and for identification of the Leptospira species at the earliest onset of infection in a clinical setting, in less than 2 hours.

Highlights

Leptospirosis is a worldwide zoonotic and neglected infectious disease caused by pathogenic bacteria of the Leptospira genus from the family Leptospiraceae[1]
We used a robust biobank of paired serum and urine samples collected from febrile patients at admission at the emergency room and evaluated the accuracy of qRT-PCR-high resolution melting (HRM) analysis as a clinical diagnostic tool for direct detection of Leptospira in the very early stages of human leptospirosis
Clinical diagnosis by the attending physician was based on signs and symptoms of leptospirosis, as previously described[23,26]

Summary

Introduction

Leptospirosis is a worldwide zoonotic and neglected infectious disease caused by pathogenic bacteria of the Leptospira genus from the family Leptospiraceae[1]. HRM uses affordable SYBR green chemistries and is performed with a real-time PCR instrument immediately after PCR; its underlying principle is the generation of different melting curve profiles due to sequence variations in double-stranded DNA4 Advantages of this method include a rapid turn-around time (less than 2 hr), a closed-tube format that significantly reduces contamination risk, high sensitivity and specificity, low cost and, unlike other methods, no sample www.nature.com/scientificreports/. We used a robust biobank of paired serum and urine samples collected from febrile patients at admission at the emergency room and evaluated the accuracy of qRT-PCR-HRM analysis as a clinical diagnostic tool for direct detection of Leptospira in the very early stages of human leptospirosis

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Conclusion