人巨细胞病毒临床分离株对更昔洛韦耐药的表型分析

目的检测分析人巨细胞病毒（HCMV）临床分离株的更昔洛韦（GCV）耐药表型。方法收集临床接受GCV治疗过的各类移植受体的血液或尿液标本以及婴幼儿HCMV感染症患儿的尿液标本,分离HCMV。然后通过噬斑减少试验测试其对GCV药物的耐受性。病毒株对GCV的耐受性程度表示以不加药物孔中病毒致细胞病变效应（CPE）为参照,能够抑制50％CPE的药物浓度（IC50）为GCV的耐受性。GCV敏感型毒株的IC50为≤5．000μmol／L。结果以标本接种人胚肺成纤维细胞（MRC-5）,最终获得病毒株共33株。其中6株来自移植受体,27株来自婴幼儿HCMV感染症患儿。以MRC-5为敏感细胞,调整所测病毒浓度为100×50％组织培养感染量（TCID50）,并加入不同浓度的GCV药物,检测发现32株GCV的IC50均≤5．000μmol／L（1．500～5．000μmol／L）,只有1株GCVIC50为12．500μmoL／L。同步平行检测的标准毒株HCMVAD169的GCVIC50为1．500μmol／L。结论药物耐受表型测定能够了解并评估病毒对GCV药物的敏感与否,但全过程周期较长,技术要求比较高,尤其必须以获得活病毒为前提,尚难以直接推向临床实验室。

Analysis of the ganciclovir-resistant phenotype of Human cytomegalovirus clinical isolates

Objective To study the ganciclovir （GCV） -resistant phenotyping of Human cytomegalovirus （HCMV） clinical isolates. Methods HCMV was isolated from the urine of congenital HCMV infection patients and the anticoagulated blood or the urine of transplant recipients who had been treated with GCV. Drug susceptibility was determined by the plaque reduction assay. The degree of GCV susceptibility based on the viral cytopathic effects （CPE） of drug free plaques, and the concentration of drug reduced the number of plaques by 50% was namely IC50 of GCV. IC50 value of sensitive HCMV isolates was less than or equal to 5. 000 μmol/L. Results Thirty three HCMV isolates were isolated from different materials on MRC-5 ceils. Six HCMV isolates were from six kidney transplant recipients and 27 from patients with congenital HCMV infection. For MRC-5 cells as sensitive cell, 100 × 50% tissue culture infective dose （TCID50） were inoculated on 96-well microtiter plates under increasing concentrations of GCV. Thirty two clinical HCMV isolates showed GCV IC50 value was less than or equal to 5. 000 μmol/L （range 1. 500-5. 000 μmol/L） , only one clinical HCMV isolates showed GCV IC50 value of 12. 500 μmol/L. Parallel examination of HCMV AD169 were performed and showed GCV IC50 value of 1. 500 μmol/L. Conclusions The phenotyping of drug susceptibility could be used for evaluating whether the virus is sensitive to GCV or not. However, it is time-consuming and requires high technology, especially the isolation of the living virus, which impedes the assay application in clinical laboratory.