Validation of a leishmania RNA virus-1 detection assay in a non-endemic setting

Abstract

Background: Tegumentary leishmaniasis is an emerging disease among travelers, and case reports suggest that TL among travelersmaybeparticularly severe. LeishmaniaRNAvirus-1 (LRV1) has been implicated in the pathogenesis of severe TL in murine models, though whether this extends to humans is unknown. We sought to validate a LRV-1 detection assay. Methods & Materials: ATCC reference and clinical strains of Leishmania identified to species level were screened for LRV1. Two real time PCR assays for detection of LRV1 were performed with LRV1 set A and set B primers. Amplification of Leishmania kinetoplastid membrane protein 11 (kmp11) served as a quantification and extraction control. ATCC L. (V.) guyanensis strain MHOM/BR/75/M4147 was used as a positive control. Results: We screened 5 New World ATCC strains of Leishmania (L. amazonensis, L. mexicana, L. (V.) braziliensis, L. (V.) panamensis, and L. (V.) guyanensis) and 12 clinical strains from primary clinical samples including: bone marrow (N=1), whole blood (N=2), skin scraping (N=2), skin biopsy (N=2), cytology brush (N=4) and unspecified (N=1). Causative Leishmania species in clinical specimens were as follows: L. chagasi/infantum (N=3), L. (V.) braziliensis (N=3), L. tropica (N=2), L. (V.) panamensis (N=1), L. major (N=1), and L. (V.) guyanensis (N=1). Amplification of LRV1 primer sets A and B along with kmp11 only occurred with the ATCC strain of L. (V.) guyanensis known to harbour LRV1. Kmp11 alone was amplified in the remaining ATCC strains, and in 6 of 12 (50%) clinical strains, thus 80% of ATCC strains and 50% of clinical strains could be deemed negative for LRV1. Neither LRV1 setA or B primers nor kmp11 were amplified in 6 of 12 (50%) clinical strains, leading to an indeterminant result regarding LRV1 co-infection status. Conclusion: Detection of LRV1 from clinical strains of Leishmaniamaybecome relevant to clinicians in the future. Performance of the LRV1 assay was superior in cultured ATCC strains, which reflects high parasite concentrations in culture compared to primary clinical specimens. In 50% of clinical isolates, we were able to assign an LRV1 result, thus, further optimization of assay performance in primary clinical specimens is warranted.