COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

Abstract

SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM), serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT) and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP) of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively). The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01%) than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

Characterization of Leptospira isolates from humans and the environment in Uruguay

“…To amplify Leptospira 16S ribosomal RNA coding sequences, primers were LeptoA F and LeptoB R 14 . Saprophytic Leptospira can yield positive 16S results, but not LipL32 amplicons 15 .…”

Leptospira detection in flood‐prone environment of Jakarta, Indonesia

“…The same sample did not show any amplification band when the lipl32 ‐PCR assay was applied. This is might due to lipl32 ‐PCR could only specific for identification of the pathogenic group, particularly L. interrogans group (Gokmen, Soyal, Kalayci, Onlen, & Koksal, ).…”

Culture-Independent Detection and Identification of Leptospira Serovars

“…Despite these recognized benefits, lipL32 -based assays are not yet routine in clinical or public health settings, presumably because the best options employ fluorescent probes inflating setup and operational costs, which renders them ill-suited to the low-tech’ realities of most leptospirosis endemic regions. To expand access, cheaper PCR-based assays have been developed (18) but these by their nature suffer from inferior (i.e., higher) detection limits and are tedious compared to qPCR-based alternatives. Regrettably, lipl32 -based NAATs provide little insight WRT infecting Leptospira species or serotype.…”