Abstract

Recent studies have highlighted the interest in noninvasive sampling procedures coupled with real-time PCR methods for the detection of Leishmania species in South America. In French Guiana, the sampling method still relied on skin biopsies. Noninvasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of cutaneous leishmaniasis. Therefore, we evaluated the performance of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR green-based real-time PCR of the Hsp70 gene, coupled with Sanger sequencing. Between May 2017 and May 2018, 145 patients with ulcerated lesions compatible with cutaneous leishmaniasis were included in the study at the Cayenne Hospital and its remote health centers. Each patient underwent scrapings for a smear, skin biopsies for parasite culture and PCR-restriction fragment length polymorphism (RFLP) (RNA polymerase II), and sampling with a cotton swab for SYBR green-based PCR. The most accurate diagnostic test was the SYBR green-based PCR on swab samples, showing 98% sensitivity. The mean PCR cycle threshold (CT ) was 24.4 (minimum CT , 17; maximum CT , 36) and was <35 in 97.6% of samples. All samples positive by SYBR green-based real-time PCR were successfully identified at the species level by DNA sequencing. This new method should be considered for routine diagnosis of cutaneous leishmaniasis in South America and especially for remote areas, since noninvasive collection tools are easier to use and require fewer precautions for transportation.

Highlights

  • Leishmaniases are vector-borne diseases caused by parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae)

  • 164 patients were seen with suspected cutaneous leishmaniasis

  • This study validates the use of swab sampling coupled with real-time SYBR greenbased PCR and Hsp70 sequencing for the diagnosis of Cutaneous leishmaniasis (CL) in French Guiana

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Summary

Introduction

Leishmaniases are vector-borne diseases caused by parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae). The aim of this study was to validate the use of cotton swabs coupled with SYBR green-based real-time PCR of the Hsp gene as a new tool for the diagnosis of cutaneous leishmaniasis on the large annual cohort in French Guiana. This new protocol includes the assessment of DNA sequencing of the Leishmania target gene HSP70, which has been recommended for Leishmania typing [20]

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