PCR-RFLP analyses of Leishmania species causing cutaneous and mucocutaneous leishmaniasis revealed distribution of genetically complex strains with hybrid and mito-nuclear discordance in Ecuador

Abstract

PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b ( cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L . (V . ) braziliensis and between L . (V . ) guyanensis and L . (V . ) panamensis , of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L . (V . ) braziliensis and nuclear genes of L . (V . ) guyanensis , L . (V . ) panamensis , or a hybrid between L . (V . ) guyanensis and L . (V . ) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.

Nuclear and kinetoplast DNA analyses reveal genetically complex Leishmania strains with hybrid and mito-nuclear discordance in Peru

“…Multiple targets including mpi , 6-phosphogluconate dehydrogenase ( 6pgd ), heat shock protein 70 ( hsp70 ), and cytochrome oxidase subunit II-NADH dehydrogenase subunit I ( COII-ND1 ) gene fragments were used to confirm species identifications for full details of gene property variants. The primers and PCR conditions were described previously [ 17 ] except for the mpi gene fragment, where new primers were designed to amplify a shorter fragment ( S2 and S3 Figs). For the mpi gene fragment, PCR amplification with a pair of specific primers, L.MPI-AS (5’- TCGATTCGCACGGCTCTGTC-3’) and L.MPI-OR (5’-CTCAAGTCGTTGGTCGACGC-3’), was performed with 30 cycles of denaturation (95°C, 1 min), annealing (55°C, 1 min), and polymerization (72°C, 1 min) using Ampdirect Plus reagent (Shimadzu Biotech, Tsukuba, Japan) and high-fidelity DNA polymerase, KOD plus (Toyobo, Osaka, Japan), or PrimeSTAR HS (Takara Bio, Shiga, Japan).…”

“…Kinetoplast DNA (kDNA) is frequently used as a target for detection and typing of Leishmania due to its multicopy nature and high sensitivity [ 15 , 16 ]. However, combining nuclear DNA (nDNA) and kDNA markers has improved the power of molecular data to detect unexpected genetically complex strains with characteristics of hybrid and mito-nuclear discordance widely distributed in Ecuador [ 17 ]. These recent findings have shown the necessity of updating the available data in its neighbouring country of Peru, with a similar eco-epidemiological situation, and searching for genetic recombination in Peruvian strains that were identified at the species level by kinetoplast cytochrome b ( cyt b) gene sequence analysis in a previous study.…”

“…The identification of hybrid forms was performed using a polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) analysis of a short mannose phosphate isomerase ( mpi ) gene fragment. Hybrid genotypes have been rarely revealed among natural isolates of Leishmania parasites, which conventionally indicated that members of the genus possess the machinery for genetic exchange [ 17 , 19 ]. A hybrid of L .…”

Exploring the Potential and Limitations of PCR-RFLP and PCR-SSCP for SNP Detection: A Review

“…The apparent superiority of PCR-RFLP was observed regarding its broad utilization in several aspects of medical human genetics, such as the diagnosis of carcinogenesis, parasitic infection, gastritis, urinary tract infection, arthrosclerosis, infertility, and blood grouping. [40][41][42][43][44][45][46][47][48] This higher reliability on PCR-RFLP may be attributed to its simplicity compared with PCR-SSCP, because of which it has been used in previous SNP-specified applications. In contrast to medical applications, PCR-RFLP has not exhibited superiority compared with PCR-SSCP.…”

The burden of cutaneous and mucocutaneous leishmaniasis in Ecuador (2014-2018), a national registry-based study

“…L. (V.) braziliensis predominate in the Amazon region, L. panamensis/guyanensis in the subtropical and tropical lowlands of Pacific region, whilst L. (L.) mexicana in the inter-Andean valleys [5]. Most of the MCL cases are infected in the Amazon region associated with the most virulent L. braziliensis [6]. Regarding the proportion of cases of CL and MCL, only 6.9% (18/260 cases) showed the destructive form [7].…”