Usefulness of real-time PCR assay targeting lipL32 gene for diagnosis of human leptospirosis in Uruguay

Abstract

Introduction: Assays based on DNA amplification can provide information that contributes to the initial management of patients with leptospirosis. However, these have not been adopted in Uruguay. Our aim was to evaluate the performance of the lipL32 real-time PCR (qPCR) for diagnosis of leptospirosis. Methodology: We analyzed by microscopic agglutination test (MAT) and lipL32 qPCR serum samples from 183 patients with suspected leptospirosis. To establish the analytical sensitivity of the qPCR, experimentally spiked samples with known amounts of Leptospira interrogans were analyzed. Results: The analytical sensitivity of the qPCR was 10 2 leptospires/mL. In 98 patients MAT results were negative meanwhile 85 showed positive reactions, revealing acute infections. Twenty six acute-phase sera of these 85 patients showed a positive signal by qPCR (diagnostic sensitivity 30%). In these patients the average time between onset of symptoms and collection of the first sample was 8 days. In patients with negative results for qPCR and positive MAT results (n=59) the average interval between onset of symptoms and collection of the first sample was 13 days. The qPCR did not yield false positive results. Conclusions: The qPCR had a lower diagnostic sensitivity than MAT and a higher cost. However, it allowed to make an early diagnosis in 26 patients. In patients with confirmed acute infections and negative results by qPCR, more than 8 days had elapsed between the onset of the illness and extraction of the first serum sample. Our data support that the qPCR from sera have clinical utility within the first week of illness.

First Real-Time PCR in Morocco for Human Leptospirosis Using TaqMan Probes Targeting the LipL32 Gene

“…PCR has more sensitivity in the first days of illness due to its ability to detect 2 -20 genomics of leptospires from sera and 10 genomics from urine (11,16). However, low positivity during the course of the disease was observed (11,(18)(19)(20).…”

“…urea, creatinine, and hemoglobin derivates) that may inhibit DNA amplification with leptospiral primers (18). Moreover, other possible reasons include the absence of the organisms in the blood (26), the microbial counts of about five cells that were too low to be detected (20,26), and degradation of DNA due to prolonged sample storage and several sample thawing (26,27).…”

Leptospiral major outer membrane protein

“…As the latency increased, the MAT was found to be more reliable than PCR. The authors suggest that a PCR-mediated diagnosis is more useful in the first week of infection [37]. Cermakova et al [38] also reported a real-time PCR method that could detect one to five genome copies/ml of liquid biological material and differentiate pathogenic strains from nonpathogenic strains [38].…”

“…PCR-assisted diagnostic methods are adopted. González et al [37] developed a PCR-based diagnostic method targeting the LipL32 gene to detect leptospirosis in Uruguay. Compared with agglutination, the PCR method could detect the disease early.…”

Comparative genomic analyses of transport proteins encoded within the genomes of Leptospira species

“…Consequently, serotyping has resulted in the identification of over 230 different serovars among Leptospira with serovars crossing species lines [10]. The limited ability of sequencing technology at the advent of the identification and characterization of members of Leptospira was a driving force for this convention, but rapid sequencing technology such as qPCR has begun to enable scientists and clinicians to rapidly and effectively identify infecting leptospires and their phylogenetic relationships [11]. …”