高寒缺氧地区结核分枝杆菌耐药的分子特征及其与基因型关系的研究

Mycobacterium tuberculosis probably has formed stable adaptation with special geographical region or unique population in the long process of evolution. Isolates of mycobacterium tuberculosis from Qinghai Plateau maybe changed genetically because of extreme harsh natural environment and application of anti-TB drugs. Current research on tuberculosis in area of high altitude and hypoxia is almost blank and a few oversea studies available found that mortality and morbidity of TB decreased with altitude increase. Qinghai Plateau is very high in altitude, but prevalence and incidence rates of tuberculosis are higher than the national average. Qinghai is unique geographically with high altitude and low oxygen and majority of patients with tuberculosis are rural people and Tibetan. This project will study drug resistance spectrum, frequency and genetic characteristics among 300-500 Mycobacterium tuberculosis strains which circulating in region with high altitude and hypoxia by using traditional methods, molecular techniques and gene sequencing and study relationship between drug resistance and genotypes base on VNTR typing, RD/LSP deligotyping and plc gene deletion. All these studies will uncover the profile of drug resistant TB epidemic and reveal geographical features, which will be of great significance for choosing sensitive drugs for clinical treatment and provide basis for tuberculosis control strategies.

结核分枝杆菌在长期的进化过程中很可能和某一地域或人群之间形成了较为稳定的适应关系。青海高原高寒缺氧的极端环境以及抗结核杆菌药物的应用，很可能使结核杆菌基因发生了一定变异。目前高海拔地区结核方面的研究很少，国外仅有的研究发现，结核病病死率、患病率随海拔升高而下降。青海地处高原，但结核的患病率、发病率却高于全国平均水平。青海高寒缺氧，地域独特，结核患者以农牧民及藏族居多。本研究立足高原，对高海拔缺氧地区分离的300-500株结核分枝杆菌进行分离、鉴定，采用传统方法、分子生物学技术及基因测序等手段，研究高海拔缺氧地区结核分枝杆菌的耐药谱、耐药频率、耐药的基因特征及其与VNTR分型、RD基因型、plc基因型之间的关系，揭示高寒缺氧地区耐药结核菌的流行特征及地域特点，为结核病临床治疗选择敏感药物及制定防治策略提供依据。

耐药结核已成为中国结核病防治的巨大挑战。课题组四年来开展了高寒缺氧地区结核分枝杆菌的耐药特征、多位点可变数量串联重复序列分析（MLVA）基因分型与耐药关系、差异片段（regions of difference，RD）基因分型与耐药关系、磷脂酶C（PLC）基因多态性与耐药关系、耐药基因突变特征等5个方面的研究。主要结果：（1）青海耐药结核高于全国，耐药谱广、耐药类型多，1531株结核杆菌显示总耐药率为32.98%，单耐药率为12.34%，多耐药率为5.75%，耐多药率为14.89%，广泛耐药率为1.11%，利福平耐药率为18.16%；复治患者总耐药率及利福平耐药率高于初治患者。（2）249株结核菌株MLVA基因分型显示，青海地区北京基因型菌株占73.90%，非北京基因型菌株占26.10%，除利福平任一耐药率外，其他一线抗结核药物任一耐药率、单耐药率、耐多药率在北京/非北京基因型菌株中无差异。（3）不同RDs基因将249株结核菌株分为北京基因型190株（现代型172株/古代型18株）及非北京基因型59株，研究未发现北京型菌株与耐药有关，现代型较古代型趋于获得耐药。（4）结核杆菌PLC编码基因plcABCD研究发现，青海地区结核菌株plc基因呈多态性，plcD基因突变率高达97.19%，北京/非北京型与plcABC基因突变有关，而与plcD基因突变无关。（5）对196株结核菌株一线抗结核药耐药相关基因特征研究发现：①katG基因突变率为30.10%，异烟肼（INH）耐药株与敏感株中katG315突变率分别为61.80%和3.74%，katG315位点突变与INH耐药有关，另外INH耐药与oxyR-ahpC间隔区发生突变有关。②rpoB基因突变率为36.74%，利福平（RFP）耐药株与敏感株中rpoB突变率分别为81.18%和2.70%，rpoB基因突变与RFP耐药有关。③embB基因突变率为20.41%，乙胺丁醇（EMB）耐药株与敏感株中embB突变率分别为45.76%和9.49%，embB基因突变与EMB耐药有关。④rpsL基因突变率为34.69%，链霉素（SM）耐药株与敏感株中rpsL突变率分别为56.25%和19.83%, rpsL基因突变与SM耐药有关;尽管gidB基因突变率较高，但未发现与SM耐药有关。本课题发表论文11篇，培养研究生2名，获国内先进成果1项。