Validation of swab sampling and SYBR Green-based real-time PCR for the diagnosis of Cutaneous Leishmaniasis in French Guiana

Recent studies have highlighted the interest of non-invasive sampling procedures coupled to real-time PCR methods for detection of Leishmania species in South America. In French Guiana, sampling method still relied on skin biopsies. Non-invasive protocols should be tested on a large annual cohort to improve routine laboratory diagnosis of Cutaneous Leishmaniasis. Therefore, we evaluated the performances of a new Leishmania detection and species identification protocol involving cotton swabs and SYBR Green real-time PCR of 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 Cayenne Hospital and its remote health centres. Each patient underwent scrapings for smear, skin biopsies for parasite culture and PCR-RFLP (RNA pol II) and cotton swabs for SYBR Green PCR. The most accurate diagnostic test was the SYBR Green PCR on swab sampling, showing a 98% sensitivity. Mean PCR cycle threshold (Ct) was of 24.4 Ct (min=17 Ct, max=36 Ct) and was inferior to 35 Ct in 97.6% of samples. All samples positive with real-time PCR SYBR Green 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, as non-invasive collection tools are easier to use and require less precautions for transportation.