Validation of Swab Sampling and SYBR Green-Based Real-Time PCR for the Diagnosis of Cutaneous Leishmaniasis in French Guiana

Abstract

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.

Outbreak of Cutaneous Leishmaniasis among military personnel in French Guiana, 2020: Clinical, phylogenetic, individual and environmental aspects

“…Similar results were observed during the small L. braziliensis outbreak in Sau ¨l in 2013 with 5 distinct and non-clustered genotypes [13]. We used the Hsp70 as a target gene for sequencing, as this gene is deemed the best marker for New World CL phylogenetics [33] and is routinely used for species identification in French Guiana [9]. RNA Pol II was once used as a target for routine diagnosis of CL in French Guiana but replaced with Hsp 70 in February 2020.…”

“…A phylogenetic comparison was made between the different strains isolated during the outbreak. PCR on cotton swabs was performed according to previously published protocols [9]. QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany), was used to extract the parasite genomic DNA from cotton swabs, according to the manufacturer's instructions.…”

“…Smear is commonly used in French Guiana but does not provide species identification. As treatment differs between L. guyanensis and L. braziliensis, this identification is paramount for proper clinical care [3] Species identification is usually performed through Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) or Polymerase Chain Reaction (PCR) followed by DNA Sanger Sequencing obtained from parasites cultured from skin biopsy or impregnate on cotton swabs [9]. Most contaminations occur during professional forest activities in farmers, gold miners and soldiers [3].…”

Cutaneous Leishmaniasis: A 2022 Updated Narrative Review into Diagnosis and Management Developments

“…Adams and co-workers found that a lesional swab processed with Qiagen ® DNA extraction was the most efficient, sensitive, and specific recovery method for Leishmania DNA, when compared with aspirate samples, which were also more painful and more complicated to obtain [61]. This approach has now been further validated in several other studies that support the use of cotton swabs for the non-invasive collection of diagnostic material for subsequent PCR analysis [62][63][64][65]. Although rubbing over the lesion with cotton swabs potentially yields a low parasitic load, several studies have indicated that the choice of the subsequent nucleic acid extraction and amplification method influences the results obtained with cotton swab collection [61,63].…”

Leishmaniasis epidemiology in endemic areas of metropolitan France and its overseas territories from 1998 to 2020

“…As previously reported [29], patients infected by L. guyanensis were more likely to develop multiple lesions compared to those infected by L. braziliensis. The precise species identification is crucial for therapeutic adaptation because treatment differs for the two main species in this region: L. guyanensis (pentamidine) and L. braziliensis (meglumine antimoniate or amphotericin B) [31]. L. naiffi was associated with unique lesions, as previously described [17], and with a mild clinical course.…”