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

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.

Figure 1

High resolution melting curve analysis profiles of cultured Leptospira spp., and Leptospira isolates from human leptospirosis patients. (a) HRM profiles using the LFB1 primer pair. (b) HRM profiles using the G1/G2 primer pair. Abbreviations: HI1, Human isolate 1; HI6, Human isolate 6.

Figure 2

High resolution melting curve analysis profiles of human clinical samples (serum and urine) from patients with suspected leptospirosis. (a) HRM profiles using the LFB1 primer pair. (b) HRM profiles using the G1/G2 primer pair.

Figure 3

Sensitivity of molecular assays in function of the kinetics of disease progression. Comparative analysis of positive samples by nested PCR and qRT-PCR-HRM through the three early leptospirosis stages: onset (serum positive), dissemination and kidney colonization (serum and urine positive) and excretion (urine positive). (a) Comparison with primer pair LFB1. (b) Comparison with primer pair G1/G2. Statistics by Fisher Exact test, A p = 0.0033 and B p = 0.0763.

Figure 4

Confirmation of the qRT-PCR-HRM analysis by Sanger sequencing. Alignment of the consensus sequences of the clinical samples, Leptospira spp. and human isolates. Only the sequences showing differences from the first sequence are shown. Nucleotides identical to those in the first sequence are indicated by dots.