耐药脊柱结核个体治疗的疗效评价及基因芯片检测结核耐药的应用研究
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摘要
目的：
耐药结核分枝杆菌的传播流行加剧了结核病的全球疫情，而且对脊柱结核也构成了严峻威胁。然迄今为止，国内外关于耐药脊柱结核的临床特征、耐药模式及原因、治疗及预后的报道较少。耐药脊柱结核容易引起初治失败或局部复发，因此早期诊断并采取合理的治疗方案至关重要。传统的基于分枝杆菌液体培养系统及改良罗氏绝对浓度法的药敏试验是诊断耐药结核的金标准，但其耗时较长，容易造成诊断延误。近年来，基因芯片技术被用于在结核分枝杆菌临床分离株或痰液标本中进行分枝杆菌菌种鉴定及MDR-TB检测，具有较高的敏感度和特异度，但其在脊柱结核临床标本中的应用尚未见报道。另外，随着MDR-TB及XDR-TB的日趋流行，对其它一线药物及二线药物耐药的结核病例也逐渐增多，而目前多数分子药敏检测方法只针对一线药物中的INH及RMP设计，因此亟需开发新的结核耐药快速检测方法，用于同时检测常用一线药物及二线药物的耐药性。
因此，本研究拟分析耐药脊柱结核的临床特征、耐药表型及产生原因，并回顾性评价基于药敏试验的个体化化疗联合手术对耐药脊柱结核的临床疗效；此外，针对脊柱结核临床标本，采用晶芯DNA微阵列芯片行平行的分枝杆菌菌种鉴定及INH、RMP耐药基因突变检测，以验证该技术用于耐药脊柱结核诊断的可行性及准确性；最后，研发用于SM/EMB/LVFX/AMK/CPM等五种抗结核药物耐药基因突变检测的新型DNA微阵列芯片，对结核分枝杆菌临床分离株进行检验，并与表型药敏结果对照，以初步评价该技术的可行性及准确性。
方法：
1.回顾性分析2005年9月-2010年5月在第三军医大学西南医院骨科接受手术治疗，术后经病理学检查及药敏试验证实为耐药脊柱结核，且随访时间大于18月的35例患者。术后参考ATT史及药敏结果，行药敏试验指导下的个体化化疗。术后定期随访，观察各随访时相点的临床表现、影像学变化及实验室指标。分析其临床特征、耐药表型及既往ATT史，评价植骨融合情况、手术相关并发症及术后结核复发情况。
2.自2009年3月-2011年12月，在第三军医大学西南医院骨科开展此项前瞻性验证性研究，共153例连续收治的、且经病理学及影像学检查确诊的脊柱结核患者被纳入本研究。术中获得病灶组织标本(如脓液、干酪样或肉芽组织)并分成两等份，一份行BACTEC MGIT960分枝杆菌液体培养系统快速培养，培养阳性者行改良罗氏绝对浓度法药敏试验；另一份采用晶芯基因芯片行分枝杆菌菌种鉴定及RMP、INH耐药相关基因rpoB、inhA及katG的突变检测。以表型药敏试验为金标准，评价基因芯片在脊柱结核临床标本中行分枝杆菌菌种鉴定及RMP、INH耐药检测的敏感度及特异度。
3.自重庆市公共医疗救治中心检验科的结核分枝杆菌菌株库中，收集137株结核分枝杆菌临床分离株样本，其中包括111株耐药菌株及26株敏感菌株。设计并制备用于SM/EMB/LVFX/AMK/CPM耐药基因突变检测的DNA微阵列芯片，对137株临床分离株进行检测，并与表型药敏试验对照，评价基因芯片检测上述五种抗结核药物耐药性的可行性与准确性。
结果：
1.35例耐药脊柱结核患者包括初治病例22例(62%)，复治病例13例(38%)；MDR-TB12例，既往化疗平均持续14.50±2.00(0-60)月；非MDR-TB23例，既往化疗平均持续4.56±1.54(0-74)月；32例接受开放手术，3例接受微创置管灌洗局部化疗术；术后个体化化疗平均持续23.6(18-29)月；术后6例局部复发；末次随访时33例临床治愈，另外2例仍在进行化疗。
2.采用晶芯基因芯片对153份脊柱结核临床标本行分枝杆菌菌种鉴定，结核分枝杆菌的鉴定阳性率为74.51%(114/153)。在培养阳性标本中，基因芯片结核分枝杆菌菌种鉴定的敏感度为93.55%(87/93)。另外，对涂阳、培养阳性标本及涂阴、培养阳性标本，基因芯片菌种鉴定的敏感度分别为97.4%及73.3%。在RMP耐药检测方面，基因芯片的敏感度为88.9%，特异度为90.7%。在INH耐药检测方面，基因芯片的敏感度为80.0%，特异度为91.0%。采用基因芯片行结核分枝杆菌菌种鉴定及耐药基因突变检测平均耗时5.8(4-9)小时。
3.137株结核分枝杆菌临床分离株样本中，包括SM耐药菌株83株，SM敏感菌株54株；EMB耐药菌株63株，EMB敏感菌株74株；LVFX耐药菌株37株，LVFX敏感菌株100株；AMK耐药菌株50株，AMK敏感菌株87株；CPM耐药菌株29株，CPM敏感菌株108株。基因芯片检测SM耐药性的敏感度和特异度分别为90.36%及70.37%；检测EMB耐药性的敏感度和特异度分别为61.90%及58.11%；检测LVFX耐药性的敏感度和特异度分别为94.59%与58.00%；检测AMK/CPM耐药性的敏感度和特异度分别为60.34%与93.67%。
结论：
1.耐药结核分枝杆菌的传播流行、诊断延误及长期不规范化疗是导致耐药脊柱结核产生的主要原因；基于药敏试验的化疗联合手术的个体化治疗方案有助于尽早治愈结核病灶，解决并发症，并避免获得性耐药的产生。此外，传统药敏试验耗时较长，而耐药脊柱结核术后的局部复发率较高。
2.晶芯基因芯片能准确地对脊柱结核临床标本行结核分枝杆菌菌种鉴定及RMP、INH耐药检测。其操作简便、耗时较短。
3.在SM与LVFX的耐药性检测方面，基因芯片具有较高的敏感度，但基因芯片检测EMB与LVFX耐药结核的特异度较低。在EMB、AMK/CPM耐药性检方面，基因芯片的敏感度均较低。
Objective: Drug-resistant tuberculosis is a major public health concern globally. Asthe most common extrapulmonary form of tuberculosis, spinal tuberculosis has anaggressive behaviour of profound vertebral destruction and severe complications. Theinitial optimism with tuberculosis spondylitis lessens as we see the increased number ofrelapses and initial treatment failures owing to the emergence of drug-resistant tuberculosis.However, clinical characteristics, drug resistant profile and treatment of drug-resistantspinal tuberculosis have been received little attention in the literature. Optimal managementof drug-resistant tuberculosis of the spine relies on the early diagnosis. The methods ofDST using the BACTEC MGIT960system can provide definitive results, but they usuallyrequire at least1-2months to produce the strain-susceptibility profile, leading to thediagnosis delay. DNA microarrays can detect tuberculosis and its multi-drug resistant formin M. tuberculosis isolates and sputum specimens with high sensitivity and specificity.However, no performance data currently exists for its use in spinal tuberculosis specimens.Otherwise, because of the dissemination of other first-line and second-line drug-resistant M.tuberculosis strains, gene mutations that relate to other first-line and second-line drugresistance should be considered for incorporation into the further DNA microarray system.
Therefore, the aim of this study was to investigate the clinical characteristics, drugsusceptibility patterns and the outcomes of management with a combination of surgery andindividualized chemotherapy for drug-resistant spinal tuberculosis. Moreover, to access thefeasibility and accuracy of the DNA microarrays for parallel species identification anddetection of mutations that confer INH and RMP resistance among consecutive patientswith spinal tuberculosis. In the end, to develop a novel DNA microarray system for the detection of the SM/EMB/LVFX/AMK/CPM resistant M. tuberculosis, and to ascess itsaccuracy and feasibility.
Materials and Methods:
1. We retrospectively analysed thirty-five patients with drug-resistant tuberculousspondylitis in Southwest Hospital between September2005and January2010. After surgery,individualized chemotherapy was tailored for each patient according to the drug-resistanceprofile and previous history of chemotherapy. The patients were followed up clinically andradiologically for an average period of35.8months.
2. From March2009to December2011,153consecutive patients from SouthwestHospital with clinically and pathologically diagnosed spinal tuberculosis were enrolled intothis study. Specimens collected during surgery from the tuberculosis patients weresubjected to M. tuberculosis species identification and RMP/INH-resistance detection bythe DNA microarray, and results were compared with those obtained from conventionalDST.
3. A set of111drug-resistant M. tuberculosis strains and26drug-susceptible M.tuberculosis stains were collected from Chongqing Infecious Disease Center. DNAmicroarrays were designed and prepared for the detection of rrs/rpsL/gyrA/embB genemutations, and the results were compared to the phenotypic DST. The feasibility andaccuracy of the DNA microarrays for the diagnosis of SM/EMB/LVFX/AMK/CPMresistant M. tuberculosis strains were evaluated.
Results:
1. Among35drug-resistant spinal tuberculosis cases,13were retreatment cases.Twelve were MDR-TB, and23were non-MDR-TB. The patients with MDR-TB andnon-MDR-TB had undergone previous chemotherapy for an average of14.50±2.00(0–60)months and4.56±1.54(0–74) months, respectively. A total of32cases underwent openoperations, and the other3had percutaneous drainage and local chemotherapy. Patientsreceived individualized chemotherapy for an average of23.6months postoperatively. Localrecurrence was observed in6patients. Thirty-three patients had been cured at the finalfollow-up, and the other2were still receiving chemotherapy.
2. The DNA microarray achieved93.55%sensitivity for the correct M. tuberculosisspecies identification of the93specimens that tested positive for spinal tuberculosis through culture. In addition,27additional patients (45.0%) were detected by the DNAmicroarray to be positive for M. tuberculosis among60spinal tuberculosis patients whowere culture negative. Moreover, the DNA microarray had a sensitivity of88.9%and aspecificity of90.7%for RMP resistance, and had a sensitivity of80.0%and a specificity of91.0%for INH resistance. The mean turn-around time of M. tuberculosis speciesidentification and drug resistance detection using the DNA microarray was5.8(range,4-9)hours, which has been significantly shorten compared to that of conventional culture andDST.
3. Among the137M. tuberculosis stains, involving83SM-resistant strains,54SM-susceptible strains,63EMB-resistant strains,74EMB-susceptible strains,37LVFX-resistant strains,100LVFX-susceptible strains,50AMK-resistant strains,87AMK-susceptible strains,29CPM-resistant strains and108CPM-susceptible strains. Thesensitivity and specificity for the detection of drug resistance using DNA microarrays were90.36%and70.37%for SM,61.90%and58.11%for EMB,94.59%and58.00%for LVFX,60.34%and93.67%for AMK/CPM.
Conclusions:
1. Drug-resistant tuberculous spondylitis is mainly acquired through previous irregularchemotherapy and the spreading of drug-resistant M. tuberculosis strains. Management witha combination of surgery and DST-guided individualized chemotherapy is feasible in thetreatment of severe complications and the prevention of acquired drug resistance. Moreover,the traditional DST usually requires at least1to2months to produce the strainsusceptibility profile, leading to inadequate treatment and local relapse during this period.Therefore, developing new, rapid and accurate molecular DST methods is essential for theearly diagnosis of drug-resistant tuberculosis.
2. The DNA microarray is a simple and accurate tool for M. tuberculosis identificationand for the diagnosis of RMP and INH resistance, demonstrating that the system is likely tobe applicable to spinal tuberculosis specimens. The automated readout and short turnaroundtime make this assay suitable for testing and decreases the diagnosis delay, without the needto build large numbers of advanced biosafety facilities. Otherwise, the gene mutations thatrelate to other first-line and second-line drug resistance should be considered forincorporation into the further DNA microarray system because of the dissemination of other first-line and second-line drug-resistant M. tuberculosis strains. Additionally, it will benecessary to explore whether early and individualized treatment based on the DNAmicroarray can influence the clinical outcome of drug-resistant spinal tuberculosis patients.
3. The DNA microarrays can detect SM-resistant strains with high sensitivity andspecificity, having a great prospect of clinical application. For the detection of EMBresistance, the sensitivity and specificity were rather low. For the detection of LVFXresistance, the sensitivity was as high as the previous molecular DST technologies, but thespecificity was rather low. Moreover, for the detection of AMK and CPM resistance, thesensitivity was much lower, but has a high sensitivity. Thus, there’s a need to improve thedesign of the DNA chip and methods of the investigation. Otherwise, the limitation of thisstudy was the small scale samples, the selection of gold standard and samples.
耐药结核分枝杆菌的传播流行加剧了结核病的全球疫情，而且对脊柱结核也构成了严峻威胁。然迄今为止，国内外关于耐药脊柱结核的临床特征、耐药模式及原因、治疗及预后的报道较少。耐药脊柱结核容易引起初治失败或局部复发，因此早期诊断并采取合理的治疗方案至关重要。传统的基于分枝杆菌液体培养系统及改良罗氏绝对浓度法的药敏试验是诊断耐药结核的金标准，但其耗时较长，容易造成诊断延误。近年来，基因芯片技术被用于在结核分枝杆菌临床分离株或痰液标本中进行分枝杆菌菌种鉴定及MDR-TB检测，具有较高的敏感度和特异度，但其在脊柱结核临床标本中的应用尚未见报道。另外，随着MDR-TB及XDR-TB的日趋流行，对其它一线药物及二线药物耐药的结核病例也逐渐增多，而目前多数分子药敏检测方法只针对一线药物中的INH及RMP设计，因此亟需开发新的结核耐药快速检测方法，用于同时检测常用一线药物及二线药物的耐药性。
因此，本研究拟分析耐药脊柱结核的临床特征、耐药表型及产生原因，并回顾性评价基于药敏试验的个体化化疗联合手术对耐药脊柱结核的临床疗效；此外，针对脊柱结核临床标本，采用晶芯DNA微阵列芯片行平行的分枝杆菌菌种鉴定及INH、RMP耐药基因突变检测，以验证该技术用于耐药脊柱结核诊断的可行性及准确性；最后，研发用于SM/EMB/LVFX/AMK/CPM等五种抗结核药物耐药基因突变检测的新型DNA微阵列芯片，对结核分枝杆菌临床分离株进行检验，并与表型药敏结果对照，以初步评价该技术的可行性及准确性。
方法：
1.回顾性分析2005年9月-2010年5月在第三军医大学西南医院骨科接受手术治疗，术后经病理学检查及药敏试验证实为耐药脊柱结核，且随访时间大于18月的35例患者。术后参考ATT史及药敏结果，行药敏试验指导下的个体化化疗。术后定期随访，观察各随访时相点的临床表现、影像学变化及实验室指标。分析其临床特征、耐药表型及既往ATT史，评价植骨融合情况、手术相关并发症及术后结核复发情况。
2.自2009年3月-2011年12月，在第三军医大学西南医院骨科开展此项前瞻性验证性研究，共153例连续收治的、且经病理学及影像学检查确诊的脊柱结核患者被纳入本研究。术中获得病灶组织标本(如脓液、干酪样或肉芽组织)并分成两等份，一份行BACTEC MGIT960分枝杆菌液体培养系统快速培养，培养阳性者行改良罗氏绝对浓度法药敏试验；另一份采用晶芯基因芯片行分枝杆菌菌种鉴定及RMP、INH耐药相关基因rpoB、inhA及katG的突变检测。以表型药敏试验为金标准，评价基因芯片在脊柱结核临床标本中行分枝杆菌菌种鉴定及RMP、INH耐药检测的敏感度及特异度。
3.自重庆市公共医疗救治中心检验科的结核分枝杆菌菌株库中，收集137株结核分枝杆菌临床分离株样本，其中包括111株耐药菌株及26株敏感菌株。设计并制备用于SM/EMB/LVFX/AMK/CPM耐药基因突变检测的DNA微阵列芯片，对137株临床分离株进行检测，并与表型药敏试验对照，评价基因芯片检测上述五种抗结核药物耐药性的可行性与准确性。
结果：
1.35例耐药脊柱结核患者包括初治病例22例(62%)，复治病例13例(38%)；MDR-TB12例，既往化疗平均持续14.50±2.00(0-60)月；非MDR-TB23例，既往化疗平均持续4.56±1.54(0-74)月；32例接受开放手术，3例接受微创置管灌洗局部化疗术；术后个体化化疗平均持续23.6(18-29)月；术后6例局部复发；末次随访时33例临床治愈，另外2例仍在进行化疗。
2.采用晶芯基因芯片对153份脊柱结核临床标本行分枝杆菌菌种鉴定，结核分枝杆菌的鉴定阳性率为74.51%(114/153)。在培养阳性标本中，基因芯片结核分枝杆菌菌种鉴定的敏感度为93.55%(87/93)。另外，对涂阳、培养阳性标本及涂阴、培养阳性标本，基因芯片菌种鉴定的敏感度分别为97.4%及73.3%。在RMP耐药检测方面，基因芯片的敏感度为88.9%，特异度为90.7%。在INH耐药检测方面，基因芯片的敏感度为80.0%，特异度为91.0%。采用基因芯片行结核分枝杆菌菌种鉴定及耐药基因突变检测平均耗时5.8(4-9)小时。
3.137株结核分枝杆菌临床分离株样本中，包括SM耐药菌株83株，SM敏感菌株54株；EMB耐药菌株63株，EMB敏感菌株74株；LVFX耐药菌株37株，LVFX敏感菌株100株；AMK耐药菌株50株，AMK敏感菌株87株；CPM耐药菌株29株，CPM敏感菌株108株。基因芯片检测SM耐药性的敏感度和特异度分别为90.36%及70.37%；检测EMB耐药性的敏感度和特异度分别为61.90%及58.11%；检测LVFX耐药性的敏感度和特异度分别为94.59%与58.00%；检测AMK/CPM耐药性的敏感度和特异度分别为60.34%与93.67%。
结论：
1.耐药结核分枝杆菌的传播流行、诊断延误及长期不规范化疗是导致耐药脊柱结核产生的主要原因；基于药敏试验的化疗联合手术的个体化治疗方案有助于尽早治愈结核病灶，解决并发症，并避免获得性耐药的产生。此外，传统药敏试验耗时较长，而耐药脊柱结核术后的局部复发率较高。
2.晶芯基因芯片能准确地对脊柱结核临床标本行结核分枝杆菌菌种鉴定及RMP、INH耐药检测。其操作简便、耗时较短。
3.在SM与LVFX的耐药性检测方面，基因芯片具有较高的敏感度，但基因芯片检测EMB与LVFX耐药结核的特异度较低。在EMB、AMK/CPM耐药性检方面，基因芯片的敏感度均较低。
Objective: Drug-resistant tuberculosis is a major public health concern globally. Asthe most common extrapulmonary form of tuberculosis, spinal tuberculosis has anaggressive behaviour of profound vertebral destruction and severe complications. Theinitial optimism with tuberculosis spondylitis lessens as we see the increased number ofrelapses and initial treatment failures owing to the emergence of drug-resistant tuberculosis.However, clinical characteristics, drug resistant profile and treatment of drug-resistantspinal tuberculosis have been received little attention in the literature. Optimal managementof drug-resistant tuberculosis of the spine relies on the early diagnosis. The methods ofDST using the BACTEC MGIT960system can provide definitive results, but they usuallyrequire at least1-2months to produce the strain-susceptibility profile, leading to thediagnosis delay. DNA microarrays can detect tuberculosis and its multi-drug resistant formin M. tuberculosis isolates and sputum specimens with high sensitivity and specificity.However, no performance data currently exists for its use in spinal tuberculosis specimens.Otherwise, because of the dissemination of other first-line and second-line drug-resistant M.tuberculosis strains, gene mutations that relate to other first-line and second-line drugresistance should be considered for incorporation into the further DNA microarray system.
Therefore, the aim of this study was to investigate the clinical characteristics, drugsusceptibility patterns and the outcomes of management with a combination of surgery andindividualized chemotherapy for drug-resistant spinal tuberculosis. Moreover, to access thefeasibility and accuracy of the DNA microarrays for parallel species identification anddetection of mutations that confer INH and RMP resistance among consecutive patientswith spinal tuberculosis. In the end, to develop a novel DNA microarray system for the detection of the SM/EMB/LVFX/AMK/CPM resistant M. tuberculosis, and to ascess itsaccuracy and feasibility.
Materials and Methods:
1. We retrospectively analysed thirty-five patients with drug-resistant tuberculousspondylitis in Southwest Hospital between September2005and January2010. After surgery,individualized chemotherapy was tailored for each patient according to the drug-resistanceprofile and previous history of chemotherapy. The patients were followed up clinically andradiologically for an average period of35.8months.
2. From March2009to December2011,153consecutive patients from SouthwestHospital with clinically and pathologically diagnosed spinal tuberculosis were enrolled intothis study. Specimens collected during surgery from the tuberculosis patients weresubjected to M. tuberculosis species identification and RMP/INH-resistance detection bythe DNA microarray, and results were compared with those obtained from conventionalDST.
3. A set of111drug-resistant M. tuberculosis strains and26drug-susceptible M.tuberculosis stains were collected from Chongqing Infecious Disease Center. DNAmicroarrays were designed and prepared for the detection of rrs/rpsL/gyrA/embB genemutations, and the results were compared to the phenotypic DST. The feasibility andaccuracy of the DNA microarrays for the diagnosis of SM/EMB/LVFX/AMK/CPMresistant M. tuberculosis strains were evaluated.
Results:
1. Among35drug-resistant spinal tuberculosis cases,13were retreatment cases.Twelve were MDR-TB, and23were non-MDR-TB. The patients with MDR-TB andnon-MDR-TB had undergone previous chemotherapy for an average of14.50±2.00(0–60)months and4.56±1.54(0–74) months, respectively. A total of32cases underwent openoperations, and the other3had percutaneous drainage and local chemotherapy. Patientsreceived individualized chemotherapy for an average of23.6months postoperatively. Localrecurrence was observed in6patients. Thirty-three patients had been cured at the finalfollow-up, and the other2were still receiving chemotherapy.
2. The DNA microarray achieved93.55%sensitivity for the correct M. tuberculosisspecies identification of the93specimens that tested positive for spinal tuberculosis through culture. In addition,27additional patients (45.0%) were detected by the DNAmicroarray to be positive for M. tuberculosis among60spinal tuberculosis patients whowere culture negative. Moreover, the DNA microarray had a sensitivity of88.9%and aspecificity of90.7%for RMP resistance, and had a sensitivity of80.0%and a specificity of91.0%for INH resistance. The mean turn-around time of M. tuberculosis speciesidentification and drug resistance detection using the DNA microarray was5.8(range,4-9)hours, which has been significantly shorten compared to that of conventional culture andDST.
3. Among the137M. tuberculosis stains, involving83SM-resistant strains,54SM-susceptible strains,63EMB-resistant strains,74EMB-susceptible strains,37LVFX-resistant strains,100LVFX-susceptible strains,50AMK-resistant strains,87AMK-susceptible strains,29CPM-resistant strains and108CPM-susceptible strains. Thesensitivity and specificity for the detection of drug resistance using DNA microarrays were90.36%and70.37%for SM,61.90%and58.11%for EMB,94.59%and58.00%for LVFX,60.34%and93.67%for AMK/CPM.
Conclusions:
1. Drug-resistant tuberculous spondylitis is mainly acquired through previous irregularchemotherapy and the spreading of drug-resistant M. tuberculosis strains. Management witha combination of surgery and DST-guided individualized chemotherapy is feasible in thetreatment of severe complications and the prevention of acquired drug resistance. Moreover,the traditional DST usually requires at least1to2months to produce the strainsusceptibility profile, leading to inadequate treatment and local relapse during this period.Therefore, developing new, rapid and accurate molecular DST methods is essential for theearly diagnosis of drug-resistant tuberculosis.
2. The DNA microarray is a simple and accurate tool for M. tuberculosis identificationand for the diagnosis of RMP and INH resistance, demonstrating that the system is likely tobe applicable to spinal tuberculosis specimens. The automated readout and short turnaroundtime make this assay suitable for testing and decreases the diagnosis delay, without the needto build large numbers of advanced biosafety facilities. Otherwise, the gene mutations thatrelate to other first-line and second-line drug resistance should be considered forincorporation into the further DNA microarray system because of the dissemination of other first-line and second-line drug-resistant M. tuberculosis strains. Additionally, it will benecessary to explore whether early and individualized treatment based on the DNAmicroarray can influence the clinical outcome of drug-resistant spinal tuberculosis patients.
3. The DNA microarrays can detect SM-resistant strains with high sensitivity andspecificity, having a great prospect of clinical application. For the detection of EMBresistance, the sensitivity and specificity were rather low. For the detection of LVFXresistance, the sensitivity was as high as the previous molecular DST technologies, but thespecificity was rather low. Moreover, for the detection of AMK and CPM resistance, thesensitivity was much lower, but has a high sensitivity. Thus, there’s a need to improve thedesign of the DNA chip and methods of the investigation. Otherwise, the limitation of thisstudy was the small scale samples, the selection of gold standard and samples.
引文
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10. Chan T., Huang H., Jou R., Yang Y., Lu P., Huang S., Chang T., et al. Detection of genemutations causing antibiotic resistance in Mycobacterium tuberculosis complex by anarray [J]. Clinical Microbiology and Infection2011,17(4suppl):600s.
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12. Catharina C, Boehme, Pamela Nabeta, et al. Rapid molecular detection of tuberculosisand rifampin resistance[J]. N Engl J Med2010,363(11):1005-1015.
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18. L Zhu, G Jiang, S Wang, Y Guo, et al. Biochip system for rapid and accurateidentification of Mycobacterial species from isolates and sputum [J]. J Clin Microbiol2010,48(10):3654-3660
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20. Mukherjee JS, Rich ML, Socci AR, et al. Programmes and principles in treatment ofmulti drug resistant tuberculosis [J]. Lancet2004,363(7):474.
21. Bridwell KH, Lenke LG, McEmery KW, et al. Anterior structural allografts in thethoracic and lumbar spine [J]. Spine1995,20:1410-1418.
22.吴铮,张泽华,许建中.重庆地区脊柱结核耐药情况流行病学调查初步报告[J].重庆医学2010,39(11):1420-1421.
23. Jain A K. Tuberculosis of the spine: A fresh look at an old disease [J]. J Bone Joint Surg[Br]2010,92(7):905-913.
24.许建中.对脊柱结核手术指征和手术方式的再认识[J].中国脊柱脊髓杂志2006,16(12):889-890.
25. Litao Li, Zehua Zhang, Jianzhong Xu. Management of drug-resistant spinaltuberculosis with a combination of surgery and individualized chemotherapy: aretrospective analysis of thirty-five patients [J].International Orthopaedics (SICOT)2012,36(2):277-283.
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