Reyes-Montes María Del Rocío, Acosta-Altamirano Gustavo, Duarte-Escalante Esperanza, Salazar Eduardo García, Martínez-Herrera Erick, Arenas Roberto, González Gloria, Frías-De-León María Guadalupe
Universidad Nacional Autónoma de México, Facultad de Medicina, Departamento de Microbiología y Parasitología, Ciudad de México, México.
Hospital Regional de Alta Especialidad de Ixtapaluca, Unidad de Investigación, Ixtapaluca, México.
Rev Inst Med Trop Sao Paulo. 2019 Aug 8;61:e37. doi: 10.1590/S1678-9946201961037.
Candida glabrata complex includes three species identified through molecular biology methods: C. glabrata sensu stricto , C. nivariensis and C. bracarensis . In Mexico, the phenotypic methods are still used in the diagnosis; therefore, the presence of C. nivariensis and C. bracarensis among clinical isolates is still unknown. The aim of this study was to evaluate the utility of a multiplex PCR for the identification of the C. glabrata species complex. DNA samples from 92 clinical isolates that were previously identified through phenotypic characteristics as C. glabrata were amplified by four oligonucleotides (UNI-5.8S, GLA-f, BRA-f, and NIV-f) that generate amplicons of 397, 293 and 223-bp corresponding to C. glabrata sensu stricto , C. nivariensis , and C. bracarensis , respectively. The amplicon sequences were used to perform a phylogenetic analysis through the Maximum Likelihood method (MEGA6), including strains and reference sequences of species belonging to C. glabrata complex. In addition, recombination and linkage disequilibrium were estimated (DnaSP version 5.0) for C. glabrata sensu stricto isolate s . Eighty-eight isolates generated a 397-bp fragment and only in one isolate a 223-bp amplicon was observed. In the phylogenetic tree, the sequences of 397-bp were grouped with C. glabrata reference sequences , and the sequence of 223-bp was grouped with C. bracarensis reference sequences, corroborating the PCR identification. The number of recombination events for the isolates of C. glabrata sensu stricto was zero, suggesting a clonal population structure. Three isolates that did not amplify any of the expected fragments were identified as Saccharomyces cerevisiae through the sequencing of the D1/D2 domain region within the 28S rDNA gene. The multiplex PCR is a fast, cost-effective and reliable tool that can be used in clinical laboratories to identify C. glabrata complex species.
狭义光滑念珠菌、尼瓦念珠菌和布拉卡念珠菌。在墨西哥，诊断仍采用表型方法；因此，临床分离株中尼瓦念珠菌和布拉卡念珠菌的存在情况仍不清楚。本研究的目的是评估多重聚合酶链反应（PCR）在鉴定光滑念珠菌复合体物种中的实用性。对92株先前通过表型特征鉴定为光滑念珠菌的临床分离株的DNA样本，用四种寡核苷酸（UNI - 5.8S、GLA - f、BRA - f和NIV - f）进行扩增，这些寡核苷酸分别产生对应于狭义光滑念珠菌、尼瓦念珠菌和布拉卡念珠菌的397、293和223碱基对的扩增子。扩增子序列通过最大似然法（MEGA6）用于进行系统发育分析，包括属于光滑念珠菌复合体的菌株和物种参考序列。此外，对狭义光滑念珠菌分离株估计了重组和连锁不平衡（DnaSP 5.0版）。88株分离株产生了397碱基对的片段，仅在一株分离株中观察到223碱基对的扩增子。在系统发育树中，397碱基对的序列与光滑念珠菌参考序列聚在一起，223碱基对的序列与布拉卡念珠菌参考序列聚在一起，证实了PCR鉴定结果。狭义光滑念珠菌分离株的重组事件数为零，表明其群体结构为克隆型。通过对28S rDNA基因内D1/D2结构域区域进行测序，将三株未扩增出任何预期片段的分离株鉴定为酿酒酵母。多重PCR是一种快速、经济高效且可靠的工具，可用于临床实验室鉴定光滑念珠菌复合体物种。
