Abstract

Leptospirosis is recognized as the most globally widespread reemerging zoonosis and represents a serious threat for both human and animal health. Indeed, leptospirosis is linked to more than 60,000 human deaths per year and to incalculable economic burden as consequence of medical treatment costs and livestock loss. The increasing number of reports from species of pathogenic Leptospira spp. group II causing disease in both humans and animals constitutes an additional concern to the complex epidemiology of this zoonotic agent. Diagnostic methods based on qPCR have improved the diagnosis of Leptospira spp. in terms of cost, time, and reliability, but most of the validated assays fail to detect species from the pathogenic group II. Hence, the current study was aimed to develop and validate a novel multiplex qPCR to enable the specific and selective detection of the whole group of infectious Leptospira spp., including both pathogenic groups I and II and moreover, selectively discriminate between them. To fit the “fitness of purpose” for the specific detection of infectious Leptospira spp. and further discrimination between both pathogenic groups three target regions on the 16S RNA gene were selected. These targets facilitated a broad and selective spectrum for the detection of all infectious Leptospira spp. with the exclusion of all saprophytic groups and the novel clade of environmental Leptospira spp. The analytical sensitivity (ASe) showed by the new assay also enables a wide window of detection for the agent at different stages of infection since the assay was able to efficiently detect at 95% of confidence ∼5 leptospires/reaction. From the evaluation of the analytical specificity (ASp) by in silico and in vitro approaches, it was congruently revealed that the primers and probes selected only recognized the specific targets for which the assay was intended. Bayesian latent class analysis of performance of the new assay on 684 clinical samples showed values of diagnostic sensitivity of 99.8% and diagnostic specificity of 100%. Thus, from the evaluation of the analytical and diagnostic parameters, the new multiplex qPCR assay is a reliable method for the diagnosis of Leptospira spp.

Highlights

  • Leptospirosis is globally recognized as the most widespread reemerging zoonosis (Adler, 2015)

  • Considering the 48 sequences belonging to infectious Leptospira spp. group, it was observed that on the region of the forward primer infL-78F22, 25 sequences from the Leptospira spp. pathogenic group I were 100% identical, 15 sequences presented one mismatch (18C × T), two sequences from the pathogenic group I presented two mismatches (17T × C/18C × T), five sequence from the pathogenic group II presented two mismatches (18C × T/21A × G), and one sequence from the pathogenic group II presented two mismatches

  • For the region targeted by the probe infL-179U20, it was found that 36 sequences from the pathogenic group I were 100% identical, two sequences from pathogenic group I presented one mismatch (19C × T), one sequence from the pathogenic group I presented two mismatches (8C × A/19C × T), four sequences from the pathogenic group II presented two mismatches (3T × G/19G × T), and five sequences from the pathogenic group II presented two other mismatches) (1T × C/19G × T) (Figure 1B)

Read more

Summary

Introduction

Leptospirosis is globally recognized as the most widespread reemerging zoonosis (Adler, 2015). This severe infectious disease has an estimated incidence of 1.03 million cases in humans every year, with approximately 59,000 deaths (Costa et al, 2015; Torgerson et al, 2015). Leptospirosis presents a variety of clinical manifestations, from asymptomatic subclinical infection to severe potentially fatal disease (Vk et al, 2018). Leptospirosis is considered a global lifethreatening disease in public health which can have a major impact on animal health

Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call