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

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.针对多个核基因建立的聚合酶链式反应-限制性片段长度多态性（PCR-Restriction Fragment Length Polymorphism, PCR-RFLP）分析方法，旨在无需使用昂贵设备即可简便、实用地鉴定利什曼原虫（Leishmania）物种。该方法被应用于厄瓜多尔14个省份33个采样点采集的92份临床样本，这些样本此前已通过动基体细胞色素b（cytochrome b, cyt b）基因序列分析完成了物种水平鉴定，研究人员对两种分析方法得到的结果进行了比较。 尽管两种分析的多数结果一致，但PCR-RFLP分析揭示了两类杂交株的分布：一类为圭亚那利什曼原虫（Leishmania (Viannia) guyanensis）与巴西利什曼原虫（Leishmania (Viannia) braziliensis）之间的杂交株，另一类为圭亚那利什曼原虫与巴拿马利什曼原虫（Leishmania (Viannia) panamensis）之间的杂交株，其中后者在厄瓜多尔尚属首次检出。 此外，研究还鉴定出一类异常寄生虫株：其动基体cyt b基因来自巴西利什曼原虫（Leishmania (Viannia) braziliensis），而核基因则属于圭亚那利什曼原虫、巴拿马利什曼原虫，或是二者的杂交株。这是全球首次报道存在线粒体-核基因不协调（mito-nuclear discordance）的原生动物寄生虫分布情况。 研究结果证实，厄瓜多尔境内存在遗传结构复杂的利什曼原虫株。鉴于已有研究表明，杂交形成等遗传交换事件可增强利什曼原虫的致病力，且可能通过更多种类的白蛉进行传播，因此后续有必要在全国范围内开展针对临床症状及传播媒介的流行病学研究。