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中华实验和临床感染病杂志(电子版) ›› 2016, Vol. 10 ›› Issue (02) : 166 -172. doi: 10.3877/cma.j.issn.1674-1358.2016.02.008

临床论著

获得性免疫缺陷综合征合并结核病患者对结核分枝杆菌二线药物耐药特征分析
张玲1, 孙月2, 陈勇3, 郜桂菊4, 杨思园1, 万康林5, 李兴旺1,†()   
  1. 1. 100015　北京，北京大学地坛医院教学医院
    2. 100016　北京，清华大学第一附属医院
    3. 100015　北京，首都医科大学附属北京地坛医院；075000　张家口市，河北北方学院附属第一医院
    4. 100015　北京，首都医科大学附属北京地坛医院
    5. 102206　北京，中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室
  • 收稿日期:2015-04-27 出版日期:2016-04-15
  • 通信作者: 李兴旺
  • 基金资助:
    国家十二五科技重大专项中医药防治重大传染病临床科研基地与技术平台建设(No. 2012ZX10005010-003)

The second-line drug resistance characterization of Mycobacterium tuberculosis in patients with acquired immune deficiency syndrome and tuberculosis

Ling Zhang1, Yue Sun2, Yong Chen3, Guiju Gao4, Siyuan Yang1, Kanglin Wan5, Xingwang Li1,†()   

  1. 1. Beijing Ditan Hospital, Peking University Teaching Hospital, Beijing 100015, China
    2. First Hospital of Tsinghua University, Beijing 100016, China
    3. Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China; The First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, China
    4. Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China
    5. National Institute for Communicable Diseases Control and Prevention, Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Diseases Prevention and Control/National Reference Laboratory of Tuberculosis, Beijing 102206, China
  • Received:2015-04-27 Published:2016-04-15
  • Corresponding author: Xingwang Li
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引用本文：

张玲, 孙月, 陈勇, 郜桂菊, 杨思园, 万康林, 李兴旺. 获得性免疫缺陷综合征合并结核病患者对结核分枝杆菌二线药物耐药特征分析[J]. 中华实验和临床感染病杂志(电子版), 2016, 10(02): 166-172.

Ling Zhang, Yue Sun, Yong Chen, Guiju Gao, Siyuan Yang, Kanglin Wan, Xingwang Li. The second-line drug resistance characterization of Mycobacterium tuberculosis in patients with acquired immune deficiency syndrome and tuberculosis[J]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2016, 10(02): 166-172.

目的

了解获得性免疫缺陷综合征（AIDS）合并结核病（TB）患者感染结核分枝杆菌二线药物耐药特点。

方法

选取2010年4月至2012年10月于北京大学地坛医院教学医院住院的艾滋病合并结核病患者标本，由中国疾病预防控制中心培养鉴定。进行4种一线药物（异烟肼、利福平、链霉素、乙胺丁醇）和4种二线药物（卷曲霉素、卡那霉素、氧氟沙星、乙硫异烟胺）药敏试验监测，并对所有菌株在gyrA、gyrB、rrs、tlya、eis和ethA基因位点进行DNA测序以检测基因多态性。

结果

经培养鉴定共得到31株结核分枝杆菌，其中12株耐卷曲霉素，8株耐氧氟沙星，4株耐卡那霉素，5株耐乙硫异烟胺，耐药率分别为38.71%、25.81%、12.90%和16.13%。7株菌为耐多药菌株，1株菌为广泛耐药菌株，耐药率分别为22.58%和3.23%。耐药菌株最常见的突变位点是rrs1401，gyrA94和gyrA90。一线敏感菌株中氧氟沙星的耐药率显著低于一线耐药菌株（P = 0.012）。性别与结核分枝杆菌耐药差异无统计学意义（P = 0.533），年龄> 40岁组的氧氟沙星耐药率低于其余两组（P = 0.043）。结核初治组与复治组患者二线耐药率、CD4水平差异无统计学意义（P = 0.333、0.307）。

结论

AIDS合并TB患者存在二线抗结核药物原发耐药，其中卷曲霉素耐药率最高，其次是氧氟沙星。

关键词: 获得性免疫缺陷综合征, 结核病, 耐药特点, 分子生物学特征, 二线药物
Objective

To explore the second-line drug resistance and molecular characterization of Mycobacterium tuberculosis in patients with acquired immune deficiency syndrome (AIDS) and tuberculosis (TB).

Methods

Specimens of patients with AIDS and TB, hospitalized in Beijing Ditan Hospital, Peking University Teaching Hospital from April 2010 to Octomber 2012, were collected and sent to CDC for identification. Four first-line drugs (isoniazid, rifampicin, streptomycin and ethambutol) and four second-line drugs (capreomycin, kanamycin, ofloxacin and ethionamide) susceptibility tests were taken and gyrA, gyrB, rrs, tlya, eis, ethA loci were detected for genetic polymorphisms.

Results

Total of 31 Mycobacterium tuberculosis strains were identified, while 12 isolates showed resistance to capreomycin, 8 strains resistance to ofloxacin, 4 strains resistance to kanamycin and 5 strains resistance to ethionamide. The drug resistance rates were 38.71%, 25.81%, 12.90% and 16.13%, respectively. Besides, 7 multidrug-resistant isolates and one extensively drug-resistant isolate were identified and the drug resistance rates were 22.58% and 3.23%, respectively. The most frequent mutation loci were rrs1401, gyrA94 and gyrA90. Ofloxacin drug resistance rate was significantly higher in isolates resistant to first-line drugs than first-line drugs sensitive isolates (P = 0.012). Gender was not associated with drug resistance (P = 0.533). Ofloxacin drug resistance rate was significantly lower in > 40 years old group than the other two groups (P = 0.043). There was no drug resistance and CD4 level difference between initial treated group and retreated group (P = 0.333, 0.307).

Conclusions

There existed primary drug resistance of second-line anti-tuberculosis drugs in patients with AIDS and TB. Capreomycin drug resistance rate was the highest followed by ofloxacin.

Key words: Acquired immune deficiency syndrome, Tuberculosis, Drug resistance, Molecular characterization, Second-line drug
表1 多位点PCR引物序列（擎科生物公司合成）
引物名称 引物序列（5'→3'）
16s rRNAF ACGGTGGGTACTAGGTGTGGGTTTC
16s rRNAR TCTGCGATTACTAGCGACTCCGACTTCA
IS1561’F CGTGGGTGGGCCCTGGATACGTGAACTCT
IS1561’R AACTGCTCACCCTGGCCACCACCATTGACT
RV1510F GTGCGCTCCACCCAAATAGTTGC
RV1510R TGTCGACCTGGGGCACAAATCAGTC
RV1970F GCGCAGCTGCCGGATGTCAAC
RV1970R CGCCGGCAGCCTCACGAAATG
RV3877/8F CGACGGGTCTGACGGCCAAACTCATC
RV3877/8R CTTGCTCGGTGGCCGGTTTTTCAGC
RV3120F GTCGGCGATAGACCATGAGTCCGTCTCCAT
RV3120R GCGAAAAGTGGGCGGATGCCAGAATAGT
RV0577F ATGCCCAAGAGAAGCGAATACAGGCAA
RV0577R CTATTGCTGCGGTGCGGGCTTCAA
表2 本研究检测基因突变所用的引物（擎科生物公司合成）
引物名称 引物序列（5'→3'） 退火温度（℃） 目的片段长度（bp）
rrs-F GCCGCGAGGTTAAGCGAAT 66 352
rrs-R CGCCCACTACAGACAAGAAC    
tlya1-F AGGCGCACGAGGTGTTGTTG 66 527
tlya1-R AACGACAGGTCGGCCACTACCAGGT    
tlya2-F ATGTCGGATACGGCCAGCTG 68 555
tlya2-R ACTTTTTCTACGCGCCGTGC    
eis-F GCGTAACGTCACGGCGAAATTC 59 567
eis-R GTCAGCTCATGCAAGGTG    
gyrA-F ATGCAGCGCAGCTACATC 60 509
gyrA-R TTGTGCGGCGGGATATTG    
gyrB-F CCGCTGTGATCTCGGTGAAG 64 775
gyrB-R AGACCCTTGTACCGCTGAATG    
ETH1-F ACTACAACCCCTGGGACC 65 667
ETH1-R ATCATCGTCGTCTGACTATGG    
ETH2-F CCTCGAGTACGTCAAGAGCAC 65 692
ETH2-R CCTCGACCTTCCCGTGA    
ETH3-F CGTTGACGGCCTCGACATTAC 68 342
ETH3-R GGTGGAACCGGATATGCCTG    
表3 31例AIDS-TB患者病原学分布
标本类型 阳性例数 百分比（%）
血培养阳性 13 41.94
淋巴结分泌物 11 35.48
痰培养 10 32.26
肺泡灌洗液培养 4 12.90
腹水 2 6.45
脑脊液 1 3.23
表4 31株结核分枝杆菌二线药物耐药特点
耐药情况 例数 百分比（%）
耐1种药物    
  CM 6 19.35
  OFX 1 3.23
  KM 0 0.00
  ETH 0 0.00
耐2种药物    
  CM + KM 0 0.00
  CM + OFX 3 9.68
  KM + OFX 0 0.00
  CM + ETH 0 0.00
  KM + ETH 0 0.00
  OFX + ETH 1 3.23
耐3种药物    
  CM + KM + OFX 0 0.00
  CM + KM + ETH 1 3.23
  KM + OFX + ETH 1 3.23
  CM + OFX + ETH 0 0.00
耐4种药物    
  CM + OFX + KM + ETH 2 6.45
表5 不同一线耐药模式下二线药物耐药比较[例（%）]
抗菌药物 一线敏感菌株（n = 12） 一线耐药菌株（n = 19） P值
OFX 0（0.00） 8（42.11） 0.012
CM 2（16.67） 8（42.11） 0.24
KM 0（0.00） 4（21.05） 0.139
ETH 0（0.00） 5（26.32） 0.128
表6 二线耐药相关的人口统计学和临床特点
特征 菌株数 耐药菌株数量
CM P值 OFX P值 KM P值 ETH P值
性别     1.000   0.550   1.000   1.000
  男 28 11   8   4   5  
  女 3 1   0   0   0  
年龄（岁）     0.563   0.043   0.387   0.145
  ≤ 25 5 3   3   1   2  
  25～40 17 6   5   3   3  
  > 40 9 3   0   0   0  
一线抗结核药物的使用                  
  否 26 11 0.624 8 0.291 4 1.000 5 0.560
  是 5 1   0   0   0  
表7 二线药物耐药的基因特征
药物 位点 耐药株数量 敏感株数量 敏感性（%） 特异性（%）
突变 无突变 突变 无突变
OFX gyrA94（Asp/Gly） 4 4 0 23 50.0 100.00
  gyrA94（Asp/Tyr） 1 7 0 23 12.50 100.00
  gyrA90（Ala/Val） 1 7 0 23 12.50 100.00
  gyrB 0 8 0 23 0.00 100.00
KM rrs1401（A/G） 2 2 0 27 50.00 100.00
  eis 0 4 0 27 0.00 100.00
CM rrs1401（A/G） 2 10 0 19 16.67 100.00
ETH ETHA44（C/T） 1 4 0 26 20.00 100.00
  ETHA266（Ser/Arg） 0 5 2 24 0.00 7.69
  ETHA371（Gly/Cys） 0 5 1 25 0.00 3.85
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