Margaryan H, Rüsch-Gerdes S, Hayrapetyan A, Mirzoyan A
National TB Control Center SNPO, Abovyan, Armenia.
National Reference Center for Mycobacteria, Forschungsinstitut Borstel, Germany.
Int J Mycobacteriol. 2016 Dec;5 Suppl 1:S50. doi: 10.1016/j.ijmyco.2016.06.007. Epub 2016 Jul 4.
Despite the successes in managing drug-susceptible TB, drug-resistant (DR) tuberculosis is a major challenge to the effectiveness of National Tuberculosis Program in Armenia, placing the country in the list of 18 high-burden countries for MDR-TB in the WHO European Region. Estimated burden of MDR-TB in 2012 was 9.4 (7-12) and 43 (38-49) among retreatment TB cases. A total of 92 laboratory confirmed cases had been reported to the WHO (57 new and 35 previously treated) out of 511 cases tested for MDR-TB. GenoType MTBDRsl is a new molecular kit designed for rapid identification of the resistance to the second-line antituberculosis drugs with a single strip. The aim of this study was to identify the mutation that confers resistance to ethambutol (EMB) in Mycobacterium tuberculosis in comparison with the phenotypic drug susceptibility test (DST). Ethambutol is used in M/XDR-TB regimens only if it is susceptible by DST results.
A set of 173 drug resistances TB isolates during 2011 and 2012 period, being either acid fast bacterium positive or negative but culture positive resistant to isoniazid, rifampin, or both according to the GenoType MTBDR plus assay were consecutively tested, using the GenoType MTBDRsl (MTBDR plus version 1.0 and MTBDRsl version 2.0 Hain Lifescience, Nehren). embB gene analysis and the results were compared to phenotypic EMB testing (DST). The DNA preparation method used as described recommended by the manufacturer.
Genotypic analysis identified mutations at codon 306 of the embB gene in 20.8% (36/173) and miss band of wild type in 12.71% (22/173) of the resistant isolates in comparison to only 14.45% (25/173) of those that were phenotypically resistant to EMB by DST MGIT liquid media. Mutation locus were identified in embB MUT1B and embB MUT1A 8.67% (15/173) and 12.13% (21/173), simultaneously. Phenotypic retesting in MGIT demonstrated that 45 (306 of the embB gene and miss band of wild type together) genotypically resistant isolates were phenotypically resistant to EMB. This implies that 58.62% (33/58) of EMB resistance had been phenotypically missed by routine laboratory procedures. EMB resistance was closely linked to multidrug resistance (MDR); 70.69% (41/58) of the EMB-resistant isolates were resistant to both isoniazid and rifampicin.
Implementation of more accurate diagnosis of EMB resistance may enhance patient management in Armenia, as standardized treatment of MDR-TB with second-line drugs is currently dependent on the outcome of the EMB resistance test.
尽管在治疗药物敏感型结核病方面取得了成功，但耐药结核病对亚美尼亚国家结核病规划的有效性构成了重大挑战，使该国位列世界卫生组织欧洲区域耐多药结核病高负担的18个国家名单之中。2012年，复治结核病病例中耐多药结核病的估计负担分别为9.4（7 - 12）和43（38 - 49）。在接受耐多药结核病检测的511例病例中，共有92例实验室确诊病例报告给了世界卫生组织（57例新发病例和35例既往治疗病例）。GenoType MTBDRsl是一种新型分子试剂盒，设计用于通过单条试纸快速鉴定对二线抗结核药物的耐药性。本研究的目的是与表型药物敏感性试验（DST）相比，鉴定结核分枝杆菌中赋予对乙胺丁醇（EMB）耐药性的突变。仅当根据DST结果显示敏感时，乙胺丁醇才用于耐多药/广泛耐药结核病治疗方案。
在2011年至2012年期间，对一组173株耐药结核病分离株进行连续检测，这些分离株根据GenoType MTBDR plus检测法，无论是抗酸杆菌阳性还是阴性，但培养阳性且对异烟肼、利福平或两者耐药，使用GenoType MTBDRsl（MTBDR plus版本1.0和MTBDRsl版本2.0，海因生命科学公司，内伦）。进行embB基因分析，并将结果与表型EMB检测（DST）进行比较。按照制造商推荐的方法进行DNA制备。
与通过DST MGIT液体培养基表型对EMB耐药的分离株中仅14.45%（25/173）相比，基因分型分析在20.8%（36/173）的耐药分离株中鉴定出embB基因第306位密码子的突变，在12.71%（22/173）的耐药分离株中鉴定出野生型条带缺失。在embB MUT1B和embB MUT1A中同时鉴定出突变位点的比例分别为8.67%（15/173）和12.13%（21/173）。在MGIT中进行的表型复测表明，45株（embB基因第306位密码子突变和野生型条带缺失合计）基因分型耐药的分离株对EMB表型耐药。这意味着58.62%（33/58）的EMB耐药性在常规实验室检测中被表型漏检。EMB耐药性与耐多药密切相关；70.69%（41/58）的EMB耐药分离株对异烟肼和利福平均耐药。
实施更准确的EMB耐药性诊断可能会改善亚美尼亚的患者管理，因为目前耐多药结核病的二线药物标准化治疗依赖于EMB耐药性检测的结果。
