Molecular tools confirm natural Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids causing cutaneous leishmaniasis in the Amazon region of Brazil

Abstract Seven isolates from patients with American cutaneous leishmaniasis in the Amazon region of Brazil were phenotypically suggestive of Leishmania (Viannia) guyanensis/L. (V.) shawi hybrids. In this work, two molecular targets were employed to check the hybrid identity of the putative hybrids. Heat shock protein 70 (hsp70) gene sequences were analyzed by three different polymerase chain reaction (PCR) approaches, and two different patterns of inherited hsp70 alleles were found. Three isolates presented heterozygous L. (V.) guyanensis/L. (V.) shawi patterns, and four presented homozygous hsp70 patterns involving only L. (V.) shawi alleles. The amplicon sequences confirmed the RFLP patterns. The high-resolution melting method detected variant heterozygous and homozygous profiles. Single-nucleotide polymorphism genotyping/cleaved amplified polymorphic site analysis suggested a higher contribution from L. (V.) guyanensis in hsp70 heterozygous hybrids. Additionally, PCR-RFLP analysis targeting the enzyme mannose phosphate isomerase (mpi) gene indicated heterozygous and homozygous cleavage patterns for L. (V.) shawi and L. (V.) guyanensis, corroborating the hsp70 findings. In this communication, we present molecular findings based on partial informative regions of the coding sequences of hsp70 and mpi as markers confirming that some of the parasite strains from the Brazilian Amazon region are indeed hybrids between L. (V.) guyanensis and L. (V.) shawi.摘要：从巴西亚马逊地区美洲皮肤利什曼病（American cutaneous leishmaniasis）患者体内分离得到的7株虫株，其表型提示为圭亚那利什曼原虫（Leishmania (Viannia) guyanensis）与沙氏利什曼原虫（Leishmania (V.) shawi）的杂交株。本研究采用两类分子靶标，对该疑似杂交株的杂交身份进行验证。通过三种不同的聚合酶链反应（polymerase chain reaction, PCR）策略分析热休克蛋白70（heat shock protein 70, hsp70）基因序列，发现两种不同的遗传hsp70等位基因模式：3株虫株呈现圭亚那利什曼原虫/沙氏利什曼原虫杂合模式，剩余4株仅携带沙氏利什曼原虫等位基因，呈现纯合hsp70模式。扩增子序列验证了限制性片段长度多态性（restriction fragment length polymorphism, RFLP）的分析结果；高分辨率熔解曲线法检测到变异的杂合与纯合熔解图谱。单核苷酸多态性基因分型/酶切扩增多态性位点分析提示，在hsp70杂合杂交株中，圭亚那利什曼原虫的遗传贡献占比更高。此外，针对磷酸甘露糖异构酶（mannose phosphate isomerase, mpi）基因的PCR-限制性片段长度多态性（PCR-RFLP）分析，也检测到沙氏利什曼原虫与圭亚那利什曼原虫的杂合与纯合酶切模式，与hsp70的分析结果相符。本通讯基于hsp70与mpi编码序列的部分信息区域作为分子标记，证实巴西亚马逊地区的部分寄生虫虫株确实为圭亚那利什曼原虫与沙氏利什曼原虫的杂交株。