结核分枝杆菌L型检测对结核病诊疗价值的研究
摘要
研究背景
进入21世纪后,我国肺结核防治局面依然十分严峻。结核病疫情呈现感染率高、患病率高、发病率高、耐药率高、死亡率高,结核病控制进程慢等“五高一慢”的特点。结核病疫情农村高于城市、青壮年患病率和死亡比例高,严重危害着人民群众的生命安全。结核分枝杆菌(Mycobacterium Tuberculosis, MTB) L型(L form, L)是结核分枝杆菌抵抗外界环境和赖以生存的重要形式,可在体内长期存活、生长、繁殖,导致结核病缓慢地进展、恶化、复发和耐药。近年来国内外的多项研究结果显示,菌阴肺结核约占活动性肺结核病的70%,而在菌阴肺结核标本中,MTB-L阳性率约为30%；在耐多药结核病(MDR-TB)患者中MTB-L阳性率达50%。因此,探索MTB-L检测方法对确定结核感染的性质和进展,选择治疗策略及其效果评价和判定预后、以及修正预防措施具有非常重要的意义。
研究目的
针对当前结核病防控的严峻形势,以及MTB-L生物学特性和致病性特点,本研究拟系统分析深圳市罗湖辖区结核病感染及耐药状况,建立快速简便、灵敏度高、特异度好的MTB-L的涂片染色和培养药敏的检测方法,并探讨其对结核病诊疗的临床价值。
研究内容
本研究拟开展以下工作：①建立快速简便、灵敏度高、特异度好的MTB-L的涂片染色和培养药敏的检测方法,并进行方法学评价。②采用改进的MTB-L涂片染色方法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间结核病门诊收治的960例结核病确诊患者及其治疗期间的痰标本同时进行MTB和MTB-L涂片染色检查。③采用改进的MTB-L药敏培养方法对上述痰标本进行MTB-L培养及药敏分析。④采用罗氏药敏培养基对上述痰标本进行MTB培养及药敏分析。⑤分析深圳市罗湖辖区结核病流行现状,MTB和MTB-L的耐药情况,以及其对结核病临床诊断和治疗预后的影响。
研究方法
1MTB-L涂片染色方法的改进及其方法学评价
1.1Ziehl-Neelsen(Z-N)抗酸染色法
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：玻片经火焰固定后,滴加石碳酸复红染液盖满痰膜,在火焰高处徐徐加热,切勿沸腾,出现蒸汽即暂时离开,若染液蒸发减少,应再加染液,以免干涸,加热3～5min,待标本冷却后流水自玻片一端轻缓冲洗,洗去染色液,沥去玻片上剩余的水。自痰膜上端外缘滴加3%盐酸酒精,流过痰膜,需脱至痰膜无可视红色为止,脱色应单片进行。流水自玻片一端轻缓冲洗,冲去脱色液,沥去玻片上剩余的水。滴加亚甲蓝复染液,染色30s。流水自玻片一端轻缓冲洗,冲去复染液,沥去标本上剩余的水。
③镜检：待玻片干燥后镜检,记录结果。
1.2改良IK抗酸染色法
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：涂片于空气中自然干燥后,丙酮固定；玻片倾斜,蒸馏水自一端流下清洗。加苯酚品红液后置湿盒内,于室温24h后细流水洗。自痰膜上端外缘滴加0.5%盐酸乙醇脱色剂,脱至无红色为止。加复染剂亚甲蓝,复染0.5～2min,水洗后干燥。
③镜检：待玻片干燥后镜检,镜检方法、结果判断和分级报告标准同Ziehl-Neelsen抗酸染色法。
1.3本研究改进的MTB-L抗酸染色法(以下简称‘'MTB-L抗酸染色法”)
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：玻片经火焰固定后,滴加石碳酸复红染液盖满痰膜,然后加滴2～3滴双氧水,混匀,保持染色1～2min。流水自玻片一端轻缓冲洗,洗去染色液,沥去玻片上剩余的水。自痰膜上端外缘滴加3%盐酸酒精,流过痰膜,需脱至痰膜无可视红色为止,脱色应单片进行。流水自玻片一端轻缓冲洗,冲去脱色液,沥去玻片上剩余的水。滴加亚甲蓝复染液,染色30s。流水自玻片一端轻缓冲洗,冲去复染液,沥去标本上剩余的水。
③镜检：待玻片干燥后镜检,镜检方法、结果判断和分级报告标准同Ziehl-Neelsen抗酸染色法。本改良方法染色合格的玻片呈亮蓝色,且放置于报纸上可透过痰膜分辨报纸上的文字。
1.4荧光定量PCR法
①DNA提取：收集样本,按试剂说明书要求处理样本,提取DNA；
②PCR扩增：按试剂说明书要求进行荧光定量PCR扩增检测；
③结果分析：分析扩增曲线,判定结果。
1.5方法学评价
取经我院肺科门诊确诊的活动性肺结核患者100例和健康体检者20例的痰标本,分别采用荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法,以及改良IK抗酸染色法进行检测,计算并评估荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法和改良IK抗酸染色法的特异度、灵敏度、试验总有效率等指标；分别比较荧光定量PCR法MTB-L阳性检出率与后三种方法MTB-L阳性检出率的差异。
2改进的MTB-L涂片染色法的临床应用
采用MTB-L抗酸染色法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间结核病门诊收治的960例结核病确诊患者及其治疗期间的痰标本同时进行MTB和MTB-L涂片染色检查,分析深圳市罗湖辖区结核病流行现状,MTB和MTB-L的感染情况,以及其对结核病临床诊断和治疗预后(阴转率)的影响。
3MTB-L培养基的改进及其方法学评价
3.1罗氏培养基常规培养法
收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15-20min,其间振荡2～3次,使痰液化。取前处理消化后痰液0.1ml,无菌操作接种于罗氏培养基斜面上,每份标本同时接种两支培养基,放37℃孵育。接种后3～7d观察,此后每周观察一次菌落生长情况,有菌落生长需经抗酸染色确认是否为结核分支扦菌。若至第8周仍无菌落生长可报告阴性结果。
3.2改良胰胨大豆蛋白胨L型菌培养基(tryptone soybean tryptone ager, TSA-L)培养法[6]
收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15-20min,其间振荡2～3次,使痰液化,再加无菌生理盐水至约40ml,离心10min,去上清,再用无菌生理盐水洗一次,余约0.5ml,取此前处理液0.1ml,无菌操作接种于改良TSA-L上,均匀涂布后置5%CO2恒温培养箱中37℃培养6周。每周肉眼观察1次,若有可疑菌落,显微镜下观察菌落形成情况。平板上一般先出现颗粒状(G型)幼小菌落,由十几个到数十个球状体组成。随时间延长,典型“油煎蛋”状(L型)菌落逐渐增多。培养时有时亦可见丝状菌落(F型菌落)。出现菌落时即可涂片抗酸染色镜检。无菌落生长者盲刮检查可提高阳性率。
3.3本研究改进的MTB-L培养基培养方法(以下简称"MTB-L培养基培养法”)
对TSA-L培养基配方进行改进,加入有利于分枝杆菌生长的胆固醇和卵磷脂等特殊营养成分。收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15～20min,其间振荡2～3次,使痰液化,再加无菌生理盐水至约40ml,离心10min,去上清,再用无菌生理盐水洗一次,余约0.5ml,取此前处理消化后痰液0.1ml,无菌操作接种于改进的MTB-L培养基上,均匀涂布后置5%CO2培养箱中37℃培养6周。接种后第3和7日观察培养情况,此后每周肉眼观察1次,若有可疑菌落,显微镜下观察菌落形成情况。平板上一般先出现颗粒状(G型)幼小菌落,由十几个到数十个球状体组成。随时间延长,典型“油煎蛋”状(L型)菌落逐渐增多。培养时有时亦可见丝状菌落(F型菌落)。出现菌落时即可涂片抗酸染色镜检。无菌落生长者盲刮检查可提高阳性率。
3.4方法学评价
取50例经荧光定量PCR法确诊的MTB-L标本,分别采用罗氏培养基、改良TSA-L培养基和MTB-L培养基进行培养,比较其培养阳性检出率及阳性菌落形成所需时间。
4改进的MTB-L培养基的临床应用
采用MTB-L培养法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间收治的960例结核病确诊患者及其治疗期间MTB-L培养阳性样本进行4个抗结核病一线药的药物敏感试验,分析MTB-L对4个一线药品的耐药情况。
5MTB药物敏感试验
采用罗氏药敏培养基对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间收治的960例结核病确诊患者及其治疗期间MTB培养阳性样本进行4个抗结核病一线药的药物敏感试验,分析MTB对4个一线药品的耐药情况。
统计学处理
1MTB-L抗酸染色法、Z-N抗酸染色法及改良IK抗酸染色法与荧光定量PCR法MTB-L阳性检出率比较,分别采用配对设计的两样本率的卡方检验(McNemar配对法),校正检验水准a'=0.05/3=0.017。统计学软件为SPSS13.0, P<校正检验水准a’为差异有统计学意义。
2MTB-L抗酸染色法分别与Z-N抗酸染色法、改良IK抗酸染色法的MTB、 MTB-L阳性检出率结果比较采用配对设计的非参数检验(McNemar配对法),校正检验水准a'=0.05/2=0.025。初治及复治结核病患者治疗期间MTB、MTB-L阴转率比较,均采用成组设计的两样本率的卡方检验,校正检验水准a'=0.05/3=0.017。统计学软件为SPSS13.0,P<校正检验水准a’为差异有统计学意义。
3MTB-L培养基和TAS-L培养基MTB-L培养阳性率检测结果比较采用配对设计的非参数检验(McNemar配对法)；两种培养法培养MTB-L阳性所需天数采用x士s表示,两者比较采用成组设计t检验。统计学软件为SPSS13.0, P<0.05为差异有统计学意义。
4MTB-L培养基培养法和TAS-L培养基培养法的涂阳培阴率比较采用配对设计的非参数检验(McNemar配对法),初治及复治结核病患者治疗期间MTB、MTB-L耐药率比较,均采用成组设计的两样本率的卡方检验。统计学软件为SPSS13.0, P<0.05为差异有统计学意义。
研究结果
1荧光定量PCR法、MTB-L抗酸染色法、改良IK抗酸染色法和Z-N抗酸染色法特异度均为100%,荧光定量PCR法、MTB-L抗酸染色法和改良IK抗酸染色法的灵敏度依次为30%、26%和28%,均高于Z-N抗酸染色法(14%)。荧光定量PCR法MTB-L抗酸染色法和改良IK抗酸染色法的总有效率依次为41.7%、38.3%和40.0%,均高于Z-N抗酸染色法(28.3%)。
2100例痰标本经荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法和改良IK法分析,MTB-L阳性检出率依次为30%、26%、14%和28%。MTB-L抗酸染色法MTB-L阳性检出率与荧光定量PCR法比较差异无统计学意义(Exact P=0.344>0.017), Z-N抗酸染色法MTB-L阳性检出率显著低于荧光定量PCR法(Exact P=0.000<0.017),改良IK法MTB-L阳性检出率与荧光定量PCR法比较差异无统计学意义(Exact P=0.688>0.017)
32010年度468例结核患者经检测,MTB-L抗酸染色法MTB、MTB-L阳性检出率分别为59.0%、26.9%,改良IK抗酸染色法MTB、MTB-L阳性检出率分别为57.5%、27.6%,Z-N抗酸染色法MTB、MTB-L阳性检出率分别为38.5%、16.0。MTB-L抗酸染色法MTB、MTB-L阳性检出率与改良IK抗酸染色法比较差异均无统计学意义(P=0.092>0.025; P=0.549>0.025); MTB-L抗酸染色法MTB、MTB-L阳性检出率均显著高于Z-N抗酸染色法,差异均有统计学意义(χ2=85.142, P=0.000<0.025;χ2=40.984, P=0.000<0.025)。
42011年度492例结核患者经检测,MTB-L抗酸染色法MTB、MTB-L阳性检出率分别为60.8%、27.8%,Z-N抗酸染色法MTB、MTB-L阳性检出率分别为39.2%、15.4%。MTB-L抗酸染色法MTB、MTB-L阳性检出率均显著高于Z-N抗酸染色法,差异均有统计学意义(χ2=96.711, P=0.000<0.025;χ2=50.704, P=0.000<0.025)。
52010年1月至2011年12月间初治结核病患者痰液经涂片抗酸染色检查,MTB阳性患者411例,MTB+MTB-L阳性患者130例,MTB-L阳性患者129例。经规范化治疗5个月后,除1例MTB-L阳性患者外,其余全部转阴。按《中国结核病防治规划实施工作指南》(2008年版)规范要求计算结核病涂阳患者2、3月末痰菌阴转率(痰菌阴转率=某月末累计痰菌阴转患者数/涂阳患者登记数×100%)。其中MTB阳性患者2、3月末阴转率均分别高于MTB+MTB-L阳性患者(χ2=115.330,P=0.000<0.017；校正χ2=32.889,P=0.000<0.017)及MTB-L阳性患者(χ2=120.031,P=0.000<0.017；校正χ2=33.178P=0.000<0.017)；而MTB+MTB-L阳性患者2、3月末阴转率与MTB-L阳性患者无显著性差异(χ2=0.037,P=0.848>0.017；χ2=0.000,P=0.983>0.017)。
62010年1月至2011年12月间复治结核病患者痰液经涂片抗酸染色检查,MTB阳性患者12例,MTB+MTB-L阳性患者19例,MTB-L阳性患者13例。经规范化治疗4个月后,除2例MTB+MTB-L阳性患者和1例MTB-L阳性患者外,其余全部转阴。按《中国结核病防治规划实施工作指南》(2008年版)规范要求计算结核病涂阳患者2、3月末痰菌阴转率(痰菌阴转率=某月末累计痰菌阴转患者数/涂阳患者登记数×100%)。其中MTB阳性患者2月末阴转率分别略高于MTB+MTB-L阳性患者及MTB-L阳性患者,但无统计学差异(Exact P=0.032>0.017;Exact P=0.041>0.017);MTB阳性患者3月末阴转率分别略高于MTB+MTB-L阳性患者及MTB-L阳性患者,但无统计学差异(Exact P=0.363>0.017;Exact P=0.593>0.017);MTB+MTB-L阳性患者2、3月末阴转率与MTB-L阳性患者均无显著差异(均exact P=1.000>0.017)。
750例MTB-L阳性标本在含不同卵磷脂和胆固醇浓度的MTB-L培养基上生长情况不一致,以卵磷脂加入量≥0.6g和胆固醇加入量≥0.6g时生长状况最佳,因此,卵磷脂和胆固醇的最小加入量均为0.6g。
8MTB-L培养法MTB-L阳性检出率为30.6%,改良TAS-L培养法MTB-L阳性检出率为23.9%,MTB-L培养法MTB-L阳性检出率显著高于改良TAS-L培养法(χ2=23.077,P=0.000<0.05),同时其培养MTB-L阳性所需时间显著缩短(t=14.851,P=0.000<0.05)。
9分别采用改良TAS-L培养法和MTB-L培养法对281例涂片染色MTB-L阳性标本进行分离培养,两种方法(?)培阴率[涂阳培阴率=(涂阳培阴例数/涂阳总例数)×100%]分别为9.3%和1.8(?)MTB-L培养法涂阳培阴率显著低于改良TAS-L培养法(P=0.000<0.05)
10深圳市罗湖辖区MTB初治耐药率为22.4%,其中单耐药率为：INH4.7%、 RFP3.3%、EB1.7%、SM5.8%,多耐率为3.4%,耐多药率为3.7%；复治耐药率为33.3%,其中单耐药率为：INH5.6%、RFP5.6%、SM5.6%,多耐率为5.6%,耐多药率为11.1%。初治结核病患者治疗2月末结核分枝杆菌耐药率为61.5%,其中单耐药率为：：INH23.1%、RFP5.1%、EB12.8%、SM10.2%,多耐率为5.1%,耐多药率为5.1%；复治结核病患者治疗2月末结核分枝杆菌耐药率为40%,其中单耐药率为：INH20%,多耐率为20%,耐多药率为13.6%。
11深圳市罗湖辖区MTB-L初治耐药率为46.7%,其中单耐药率为：INH11.9%、 RFP1.2%、EB11.0%、SM2.0%,多耐率为16.3%,耐多药率为4.5%；复治耐药率为46.7%,其中单耐药率为:INH10.0%、EB10.0%、SM3.3%,多耐率为16.7%,耐多药率为6.7%。初治结核病患者治疗2月末结核分枝杆菌耐药率为46.6%,其中单耐药率为INH11.6%、RFP1.0%、SM1.9%,多耐率为17.5%,耐多药率为3.9%；复治结核病患者治疗2月末结核分枝杆菌耐药率为50%,其中单耐药率为：INH16.7%, EB16.7%,多耐率为8.3%,耐多药率为8.3%。
12深圳市罗湖辖区MTB-L初治始耐药率(46.7%)显著高于MTB初治始耐药率(22.5%)(χ2=47.148,P=G.000<0.05), MTB-L复治耐药率(46.7%)略高于MTB复治耐药率(33.3%),但无统计学差异(χ2=0.823,P=0.364>0.05)。结果提示MTB-L的存在与耐药严重程度密切相关。
研究结论
1本研究改进的MTB-L抗酸染色方法具有良好的灵敏度、特异度,操作简便易行,适合于痰MTB-L常规检测,能够在基层结核病实验室推广应用。
2本研究改进的MTB-L培养基配方简单、成本低廉,培养MTB-L所需时间短且细菌生长良好,适合MTB-L培养鉴定及其药物敏感试验,能够在基层结核病实验室推广应用。
3本辖区初治和复治结核病患者中痰MTB阳性者转阴率高于MTB+MTB-L阳性者及MTB-L阳性者,MTB+MTB-L阳性者和MTB-L阳性者转阴率无明显差异。结果表明MTB-L是结核病患者阴转率低的重要原因,因此加强结核病患者痰MTB-L的检测是当前结核病防控的重要策略。
4深圳市罗湖辖区MTB-L初治耐药率和复治耐药率均为46.7%,结果表明本辖区MTB-L耐药形势依然严峻,因此有必要加强结核病患者MTB-L的耐药性监测。
5深圳市罗湖辖区MTB初治耐药率为22.5%,复治耐药率为33.3%,较2010年第5次全国结核病流行病学抽样调查初始耐药率42.7%,复治耐药率38.5%的水平低,结果表明深圳市罗湖辖区结核病防控维持在一个良好的水平。
6深圳市罗湖辖区MTB-L初治耐药率和复治耐药率均分别高于MTB初治耐药率和复治耐药率,结果表明MTB-L的存在与耐药严重程度密切相关,因此有必要强结核病患者MTB-L感染及其耐药性监测。
Research background
The prevention and cure of pulmonary tuberculosis remain severe extremely in21century in our country. The epidemic situation of tuberculosis shows5characters: high infection rate, high prevalence, high incidence, high resistance rate, high death rate and slow tuberculosis control process. The epidemic situation of tuberculosis in countryside is higher than that in city. The prevalence and death rate of young adults are higher, which harm the life safety of the people severely. Mycobacterium tuberculosis (MTB) can form cell wall-deficient mutants (L forms) easily, and M. tuberculosis L forms (MTB-L) are able to survive under harsh environmental conditions. MTB-L can survive over long periods in vivo and can be isolated and cultivated from blood samples of patients with active sarcoidosis. The biological characteristics, pathogenicity, and drug susceptibility of MTB-L differ from those of MTB, and these differences might be important factors underlying tardy progression, aggravation, recurrence, and drug resistance in tuberculosis. Approximately70%of active pulmonary tuberculosis cases are reported to be smear and culture negative. However, the MTB-L positive rate exceeds30%for sputum specimens from smear- and culture-negative pulmonary tuberculosis cases, and the MTB-L positive rate exceeds50%for sputum specimens from multidrug-resistant tuberculosis cases. Thus, a fast and convenient method to detect MTB and MTB-L would be useful in determining the nature of a tuberculosis infection, selecting a treatment strategy, and identifying preventive measures.
Research purpose
To aim directly at severe situation of the prevention and cure of pulmonary tuberculosis, and the bionomics and pathogenic characters of Mycobacterium tuberculosis L forms; we planed:①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms;②to analyze the infection and drug fast of tuberculosis in Luohu district of Shenzhen city systematically and to evaluate the diagnosis and treatment value of the modified method.
Research contents
①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms, and to evaluate the diagnosis and treatment value of the modified method.
②the sputum specimens of dubious tuberculosis patients and directly observed therapy, short-course tuberculosis patients from Luohu Chronic Disease Prevention and Cure Hospital during January,2010to December,2011were detected for Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms by the modified acid-fast staining.
③the drug sensitivity test of MTB-L of the above samples were carried out by the modified MTB-L culture medium.
④the drug sensitivity test of MTB of the above samples were carried out by the modified Roche medium.
⑤the epidemic situation of tuberculosis in Luohu district was analyzed, the affection of the drug fast to Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms on the clinical diagnosis and treatment prognosis of tuberculosis was evaluated.
Research methods
1The improvement and methodological evaluation of MTB-L acid-fast staining method
1.1Procedures of our modified acid-fast staining method (modified MTB-L acid-fast staining method), modified IK acid-fast staining method and traditional acid-fast staining method
Modified IK acid-fast staining method and traditional Ziehl-Neelsen acid-fast staining method were executed as previously reported. Colorants should be added onto the slide repeatedly and heating was needed in the process of traditional acid-fast staining method. Heating was not needed in the process of modified IK acid-fast staining method; but the process should be sustained for24hours. However, heating was not needed in the process of our modified acid-fast staining method either and the process should be sustained for about5minutes only. The procedures of the three methods were presented in the table below.
1.2Fluorescent quantitation PCR method
MTB DNA was extracted from50sputum samples from patients with definite tuberculosis by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da'an Gene) according to the manufacturer's instructions. Extracted DNA was stored at-20℃in extract buffer until analysis.
MTB DNA was analyzed by using a previously reported real-time PCR method. Briefly, real-time PCR was carried out by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da'an Gene) in a40-μl reaction mixture containing PCR buffer, deoxynucleoside triphosphates (i.e., dATP, dTTP, dCTP, and dGTP), forward and reverse primers (5'-TCGCCCGTCTACTTGGTGTT-3';5'-TGATGTGGTCGTAGTAGGTC-3'), and the fluorescence probe (5'-ACAACGCCGAATTGCGAAGGGC-3'), along with3μl Taq DNA polymerase and2μl of the extracted DNA sample. The thermal cycler was programmed as follows:one cycle of93℃×2min,10cycles of93℃×45s and55℃×1min, and30cycles of93℃×30s and55℃×45s. The fluorescence signals of FAM were acquired at the end of the third program (i.e., after55℃×45s). Fluorescence data analysis was conducted using SDS software (Version1.4; Applied Biosystems). The threshold fluorescence level, used to derive threshold cycle (Ct) values, was automatically determined by the SDS software.
1.3Evaluation of our modified acid-fast staining method
The sensitivity, specificity, positive predictive value, negative predictive value, positive rate, and diagnostic efficiency of the modified MTB-L acid-fast staining method were evaluated by using the PCR method as the comparison's gold standard. Briefly, fifty sputum samples from patients with definite tuberculosis were analyzed by comparing results from our modified acid-fast staining method and the PCR method.
2Clinical application of the modified MTB-L acid-fast staining method
Nine hundred and sixty sputum samples from patients with definite tuberculosis were smeared onto individual glass slides to form egg-shaped sputum membranes of size10×20mm (6smears per sample). Two of the six smears were analyzed using the traditional acid-fast staining method; two, using the modified IK acid-fast staining method; and two, using our modified acid-fast staining method. The positive detection rates for MTB and MTB-L in the smears of the960samples treated with each of the three methods were compared. The epidemic situation of tuberculosis in Luohu district, the infection status of MTB and MTB-L, and the affection of MTB and MTB-L on clinical diagnosis and treatment prognosis to tuberculosis were analyzed.
3The improvement and methodological evaluation of MTB-L medium
3.1The routine cultivation using Roche medium
The sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the Roche medium at37℃. After3～7days, the colonies were observed once a week and defined using acid-fast staining method. Negative results were reported when no colonies were found till8th week.
3.2The cultivation using modified tryptone soybean tryptone ager for MTB-L (TSA-L medium)
The sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the TSA-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.
3.3The cultivation using our modified medium for MTB-L (MTB-L medium)
The prescription of TSA-L were improved, that is to say, cholesterol and lecithin in favor of mycobacteria growth were added into TSA-L. Then the sputum samples were collected from clinically diagnosed patients with tuberculosis. The samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the MTB-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.
3.4Evaluation of our modified acid-fast staining method
Fifty MTB-L positive samples defined by Fluorescent quantitation PCR method were cultivated using Roche medium, TSA-L medium and MTB-L medium respectively. The positive rates and required time of colonies growth were analyzed.
4Clinical application of the modified MTB-L medium
Drug sensitivity test for MTB-L positive samples were carried out using the modified MTB-L medium. The resistant states of MTB-L to first line drug were analyzed.
Drug sensitivity test for MTB positive samples were carried out using Roche medium. The resistant states of MTB to first line drug were analyzed.
Statistical treatment
①Differences between values of MTB-L acid-fast staining method, Z-N acid-fast staining method, modified IK acid-fast staining method and PCR method were assessed by using McNemar's chi square test.
②Differences among values of MTB-L acid-fast staining method, modified IK acid-fast staining method, and Z-N acid-fast staining method, and differences among values of negative conversion rate of MTB、MTB-L were assessed by using group design chi-square test.
③Differences among values of culture positive rate of MTB-L culture method and TAS-L culture method were assessed by using group design chi-square test. Days of MTB-L culture positive by the two methods were recorded as mean±SD, and the differences of mean±SD were assessed by using Student's t-test.
④Differences among smear-positive and culture-negative rate of MTB-L culture method and TAS-L culture method, and differences among drug resistance rate of MTB、MTB-L were assessed by using group design chi-square test.
⑤SPSS13.0was used to define the differences.
Research results
⑥Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of our modified acid-fast staining method were similar to the modified IK acid-fast staining method. As in the modified IK acid-fast staining method, no heating is needed in our method. In addition, our method only needs about5min to complete, which is markedly shorter than the24h required to complete the modified IK acid-fast staining method. The results obtained using our methods have satisfactory concordance with those obtained by PCR. Moreover, the MTB and MTB-L positive detection rates of our method were significantly higher than those of the routine acid-fast staining method.
②Except1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis initial treated patients in Shenzhen Luohu district changed into negative after5month normalization treatment. The sputum negative conversion rates of MTB positive patients at the end of2,3months were higher than those of MTB and MTB-L positive patients and MTB-L positive patients sharply. Except2cases of MTB and MTB-L positive patients, and1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis retreated patients in Shenzhen Luohu district changed into negative after4month normalization treatment.
③MTB-L could grow more quickly on our modified mycobacterium tuberculosis solid medium than on modified TSA-L solid medium; the positive rates and required time of colonies growth of MTB-L using our modified solid medium were higher than those on modified TSA-L solid medium
④Initial treatment resistance rate of tuberculosis in Luohu district was lower than that of the fifth national tuberculosis epidemiological survey in2010; meanwhile, Retreated resistance rate of the former was than that of the latter.
Research conclusion
⑤A new, modified acid-fast staining method, which is convenient, specific, and sufficiently sensitive to examine sputum for the presence of MTB-L and MTB quickly and routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.
②A new, modified cultural method, which is convenient, specific, and sufficiently sensitive to drug sensitivity test of MTB-L routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.
③The sputum negative conversion rates of MTB positive patients in tuberculosis initial treated and retreated patients were higher than those of MTB+MTB-L or MTB-L positive patients. The sputum negative conversion rates between MTB+MTB-L positive patients and MTB-L positive patients changed little. So, MTB-L is an important reason for the lower sputum negative conversion rates of tuberculosis patients.
④The rates of initial resistance and retreated resistance of MTB-L were both46.7%, which indicated that the status of MTB-L drug resistance is still quite serious.
⑤The rates of initial resistance and retreated resistance of MTB in Luohu district were lower than those of national epidemiological sampling survey of tuberculosis in2010(22.5%vs.42.7%,33.3%vs.38.5%), which indicated that satisfactory progress is made in the control and prevention of MTB in Luohu district.
⑥The rates of initial resistance and retreated resistance of MTB-L were higher than those of MTB respectively, which indicated that MTB-L and the severity of drug fast is closely related.
进入21世纪后,我国肺结核防治局面依然十分严峻。结核病疫情呈现感染率高、患病率高、发病率高、耐药率高、死亡率高,结核病控制进程慢等“五高一慢”的特点。结核病疫情农村高于城市、青壮年患病率和死亡比例高,严重危害着人民群众的生命安全。结核分枝杆菌(Mycobacterium Tuberculosis, MTB) L型(L form, L)是结核分枝杆菌抵抗外界环境和赖以生存的重要形式,可在体内长期存活、生长、繁殖,导致结核病缓慢地进展、恶化、复发和耐药。近年来国内外的多项研究结果显示,菌阴肺结核约占活动性肺结核病的70%,而在菌阴肺结核标本中,MTB-L阳性率约为30%；在耐多药结核病(MDR-TB)患者中MTB-L阳性率达50%。因此,探索MTB-L检测方法对确定结核感染的性质和进展,选择治疗策略及其效果评价和判定预后、以及修正预防措施具有非常重要的意义。
研究目的
针对当前结核病防控的严峻形势,以及MTB-L生物学特性和致病性特点,本研究拟系统分析深圳市罗湖辖区结核病感染及耐药状况,建立快速简便、灵敏度高、特异度好的MTB-L的涂片染色和培养药敏的检测方法,并探讨其对结核病诊疗的临床价值。
研究内容
本研究拟开展以下工作：①建立快速简便、灵敏度高、特异度好的MTB-L的涂片染色和培养药敏的检测方法,并进行方法学评价。②采用改进的MTB-L涂片染色方法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间结核病门诊收治的960例结核病确诊患者及其治疗期间的痰标本同时进行MTB和MTB-L涂片染色检查。③采用改进的MTB-L药敏培养方法对上述痰标本进行MTB-L培养及药敏分析。④采用罗氏药敏培养基对上述痰标本进行MTB培养及药敏分析。⑤分析深圳市罗湖辖区结核病流行现状,MTB和MTB-L的耐药情况,以及其对结核病临床诊断和治疗预后的影响。
研究方法
1MTB-L涂片染色方法的改进及其方法学评价
1.1Ziehl-Neelsen(Z-N)抗酸染色法
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：玻片经火焰固定后,滴加石碳酸复红染液盖满痰膜,在火焰高处徐徐加热,切勿沸腾,出现蒸汽即暂时离开,若染液蒸发减少,应再加染液,以免干涸,加热3～5min,待标本冷却后流水自玻片一端轻缓冲洗,洗去染色液,沥去玻片上剩余的水。自痰膜上端外缘滴加3%盐酸酒精,流过痰膜,需脱至痰膜无可视红色为止,脱色应单片进行。流水自玻片一端轻缓冲洗,冲去脱色液,沥去玻片上剩余的水。滴加亚甲蓝复染液,染色30s。流水自玻片一端轻缓冲洗,冲去复染液,沥去标本上剩余的水。
③镜检：待玻片干燥后镜检,记录结果。
1.2改良IK抗酸染色法
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：涂片于空气中自然干燥后,丙酮固定；玻片倾斜,蒸馏水自一端流下清洗。加苯酚品红液后置湿盒内,于室温24h后细流水洗。自痰膜上端外缘滴加0.5%盐酸乙醇脱色剂,脱至无红色为止。加复染剂亚甲蓝,复染0.5～2min,水洗后干燥。
③镜检：待玻片干燥后镜检,镜检方法、结果判断和分级报告标准同Ziehl-Neelsen抗酸染色法。
1.3本研究改进的MTB-L抗酸染色法(以下简称‘'MTB-L抗酸染色法”)
①涂片：收集病例,按《结核病诊断细菌学检验规程》要求,挑取痰标本中干酪样、脓样或可疑部分约0.05～0.1ml,于玻片正面右侧2/3处,均匀涂抹成10mm×20mm的卵圆形痰膜,痰膜朝上静置自然干燥,每位病人涂片2张。
②染色：玻片经火焰固定后,滴加石碳酸复红染液盖满痰膜,然后加滴2～3滴双氧水,混匀,保持染色1～2min。流水自玻片一端轻缓冲洗,洗去染色液,沥去玻片上剩余的水。自痰膜上端外缘滴加3%盐酸酒精,流过痰膜,需脱至痰膜无可视红色为止,脱色应单片进行。流水自玻片一端轻缓冲洗,冲去脱色液,沥去玻片上剩余的水。滴加亚甲蓝复染液,染色30s。流水自玻片一端轻缓冲洗,冲去复染液,沥去标本上剩余的水。
③镜检：待玻片干燥后镜检,镜检方法、结果判断和分级报告标准同Ziehl-Neelsen抗酸染色法。本改良方法染色合格的玻片呈亮蓝色,且放置于报纸上可透过痰膜分辨报纸上的文字。
1.4荧光定量PCR法
①DNA提取：收集样本,按试剂说明书要求处理样本,提取DNA；
②PCR扩增：按试剂说明书要求进行荧光定量PCR扩增检测；
③结果分析：分析扩增曲线,判定结果。
1.5方法学评价
取经我院肺科门诊确诊的活动性肺结核患者100例和健康体检者20例的痰标本,分别采用荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法,以及改良IK抗酸染色法进行检测,计算并评估荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法和改良IK抗酸染色法的特异度、灵敏度、试验总有效率等指标；分别比较荧光定量PCR法MTB-L阳性检出率与后三种方法MTB-L阳性检出率的差异。
2改进的MTB-L涂片染色法的临床应用
采用MTB-L抗酸染色法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间结核病门诊收治的960例结核病确诊患者及其治疗期间的痰标本同时进行MTB和MTB-L涂片染色检查,分析深圳市罗湖辖区结核病流行现状,MTB和MTB-L的感染情况,以及其对结核病临床诊断和治疗预后(阴转率)的影响。
3MTB-L培养基的改进及其方法学评价
3.1罗氏培养基常规培养法
收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15-20min,其间振荡2～3次,使痰液化。取前处理消化后痰液0.1ml,无菌操作接种于罗氏培养基斜面上,每份标本同时接种两支培养基,放37℃孵育。接种后3～7d观察,此后每周观察一次菌落生长情况,有菌落生长需经抗酸染色确认是否为结核分支扦菌。若至第8周仍无菌落生长可报告阴性结果。
3.2改良胰胨大豆蛋白胨L型菌培养基(tryptone soybean tryptone ager, TSA-L)培养法[6]
收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15-20min,其间振荡2～3次,使痰液化,再加无菌生理盐水至约40ml,离心10min,去上清,再用无菌生理盐水洗一次,余约0.5ml,取此前处理液0.1ml,无菌操作接种于改良TSA-L上,均匀涂布后置5%CO2恒温培养箱中37℃培养6周。每周肉眼观察1次,若有可疑菌落,显微镜下观察菌落形成情况。平板上一般先出现颗粒状(G型)幼小菌落,由十几个到数十个球状体组成。随时间延长,典型“油煎蛋”状(L型)菌落逐渐增多。培养时有时亦可见丝状菌落(F型菌落)。出现菌落时即可涂片抗酸染色镜检。无菌落生长者盲刮检查可提高阳性率。
3.3本研究改进的MTB-L培养基培养方法(以下简称"MTB-L培养基培养法”)
对TSA-L培养基配方进行改进,加入有利于分枝杆菌生长的胆固醇和卵磷脂等特殊营养成分。收集肺结核确诊病人痰样本,采用碱处理法进行前处理,痰液中加4%NaOH约2～4倍量,振荡器振荡1min后,室温放置15～20min,其间振荡2～3次,使痰液化,再加无菌生理盐水至约40ml,离心10min,去上清,再用无菌生理盐水洗一次,余约0.5ml,取此前处理消化后痰液0.1ml,无菌操作接种于改进的MTB-L培养基上,均匀涂布后置5%CO2培养箱中37℃培养6周。接种后第3和7日观察培养情况,此后每周肉眼观察1次,若有可疑菌落,显微镜下观察菌落形成情况。平板上一般先出现颗粒状(G型)幼小菌落,由十几个到数十个球状体组成。随时间延长,典型“油煎蛋”状(L型)菌落逐渐增多。培养时有时亦可见丝状菌落(F型菌落)。出现菌落时即可涂片抗酸染色镜检。无菌落生长者盲刮检查可提高阳性率。
3.4方法学评价
取50例经荧光定量PCR法确诊的MTB-L标本,分别采用罗氏培养基、改良TSA-L培养基和MTB-L培养基进行培养,比较其培养阳性检出率及阳性菌落形成所需时间。
4改进的MTB-L培养基的临床应用
采用MTB-L培养法对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间收治的960例结核病确诊患者及其治疗期间MTB-L培养阳性样本进行4个抗结核病一线药的药物敏感试验,分析MTB-L对4个一线药品的耐药情况。
5MTB药物敏感试验
采用罗氏药敏培养基对深圳市罗湖区慢性病防治院2010年1月至2011年12月期间收治的960例结核病确诊患者及其治疗期间MTB培养阳性样本进行4个抗结核病一线药的药物敏感试验,分析MTB对4个一线药品的耐药情况。
统计学处理
1MTB-L抗酸染色法、Z-N抗酸染色法及改良IK抗酸染色法与荧光定量PCR法MTB-L阳性检出率比较,分别采用配对设计的两样本率的卡方检验(McNemar配对法),校正检验水准a'=0.05/3=0.017。统计学软件为SPSS13.0, P<校正检验水准a’为差异有统计学意义。
2MTB-L抗酸染色法分别与Z-N抗酸染色法、改良IK抗酸染色法的MTB、 MTB-L阳性检出率结果比较采用配对设计的非参数检验(McNemar配对法),校正检验水准a'=0.05/2=0.025。初治及复治结核病患者治疗期间MTB、MTB-L阴转率比较,均采用成组设计的两样本率的卡方检验,校正检验水准a'=0.05/3=0.017。统计学软件为SPSS13.0,P<校正检验水准a’为差异有统计学意义。
3MTB-L培养基和TAS-L培养基MTB-L培养阳性率检测结果比较采用配对设计的非参数检验(McNemar配对法)；两种培养法培养MTB-L阳性所需天数采用x士s表示,两者比较采用成组设计t检验。统计学软件为SPSS13.0, P<0.05为差异有统计学意义。
4MTB-L培养基培养法和TAS-L培养基培养法的涂阳培阴率比较采用配对设计的非参数检验(McNemar配对法),初治及复治结核病患者治疗期间MTB、MTB-L耐药率比较,均采用成组设计的两样本率的卡方检验。统计学软件为SPSS13.0, P<0.05为差异有统计学意义。
研究结果
1荧光定量PCR法、MTB-L抗酸染色法、改良IK抗酸染色法和Z-N抗酸染色法特异度均为100%,荧光定量PCR法、MTB-L抗酸染色法和改良IK抗酸染色法的灵敏度依次为30%、26%和28%,均高于Z-N抗酸染色法(14%)。荧光定量PCR法MTB-L抗酸染色法和改良IK抗酸染色法的总有效率依次为41.7%、38.3%和40.0%,均高于Z-N抗酸染色法(28.3%)。
2100例痰标本经荧光定量PCR法、MTB-L抗酸染色法、Z-N抗酸染色法和改良IK法分析,MTB-L阳性检出率依次为30%、26%、14%和28%。MTB-L抗酸染色法MTB-L阳性检出率与荧光定量PCR法比较差异无统计学意义(Exact P=0.344>0.017), Z-N抗酸染色法MTB-L阳性检出率显著低于荧光定量PCR法(Exact P=0.000<0.017),改良IK法MTB-L阳性检出率与荧光定量PCR法比较差异无统计学意义(Exact P=0.688>0.017)
32010年度468例结核患者经检测,MTB-L抗酸染色法MTB、MTB-L阳性检出率分别为59.0%、26.9%,改良IK抗酸染色法MTB、MTB-L阳性检出率分别为57.5%、27.6%,Z-N抗酸染色法MTB、MTB-L阳性检出率分别为38.5%、16.0。MTB-L抗酸染色法MTB、MTB-L阳性检出率与改良IK抗酸染色法比较差异均无统计学意义(P=0.092>0.025; P=0.549>0.025); MTB-L抗酸染色法MTB、MTB-L阳性检出率均显著高于Z-N抗酸染色法,差异均有统计学意义(χ2=85.142, P=0.000<0.025;χ2=40.984, P=0.000<0.025)。
42011年度492例结核患者经检测,MTB-L抗酸染色法MTB、MTB-L阳性检出率分别为60.8%、27.8%,Z-N抗酸染色法MTB、MTB-L阳性检出率分别为39.2%、15.4%。MTB-L抗酸染色法MTB、MTB-L阳性检出率均显著高于Z-N抗酸染色法,差异均有统计学意义(χ2=96.711, P=0.000<0.025;χ2=50.704, P=0.000<0.025)。
52010年1月至2011年12月间初治结核病患者痰液经涂片抗酸染色检查,MTB阳性患者411例,MTB+MTB-L阳性患者130例,MTB-L阳性患者129例。经规范化治疗5个月后,除1例MTB-L阳性患者外,其余全部转阴。按《中国结核病防治规划实施工作指南》(2008年版)规范要求计算结核病涂阳患者2、3月末痰菌阴转率(痰菌阴转率=某月末累计痰菌阴转患者数/涂阳患者登记数×100%)。其中MTB阳性患者2、3月末阴转率均分别高于MTB+MTB-L阳性患者(χ2=115.330,P=0.000<0.017；校正χ2=32.889,P=0.000<0.017)及MTB-L阳性患者(χ2=120.031,P=0.000<0.017；校正χ2=33.178P=0.000<0.017)；而MTB+MTB-L阳性患者2、3月末阴转率与MTB-L阳性患者无显著性差异(χ2=0.037,P=0.848>0.017；χ2=0.000,P=0.983>0.017)。
62010年1月至2011年12月间复治结核病患者痰液经涂片抗酸染色检查,MTB阳性患者12例,MTB+MTB-L阳性患者19例,MTB-L阳性患者13例。经规范化治疗4个月后,除2例MTB+MTB-L阳性患者和1例MTB-L阳性患者外,其余全部转阴。按《中国结核病防治规划实施工作指南》(2008年版)规范要求计算结核病涂阳患者2、3月末痰菌阴转率(痰菌阴转率=某月末累计痰菌阴转患者数/涂阳患者登记数×100%)。其中MTB阳性患者2月末阴转率分别略高于MTB+MTB-L阳性患者及MTB-L阳性患者,但无统计学差异(Exact P=0.032>0.017;Exact P=0.041>0.017);MTB阳性患者3月末阴转率分别略高于MTB+MTB-L阳性患者及MTB-L阳性患者,但无统计学差异(Exact P=0.363>0.017;Exact P=0.593>0.017);MTB+MTB-L阳性患者2、3月末阴转率与MTB-L阳性患者均无显著差异(均exact P=1.000>0.017)。
750例MTB-L阳性标本在含不同卵磷脂和胆固醇浓度的MTB-L培养基上生长情况不一致,以卵磷脂加入量≥0.6g和胆固醇加入量≥0.6g时生长状况最佳,因此,卵磷脂和胆固醇的最小加入量均为0.6g。
8MTB-L培养法MTB-L阳性检出率为30.6%,改良TAS-L培养法MTB-L阳性检出率为23.9%,MTB-L培养法MTB-L阳性检出率显著高于改良TAS-L培养法(χ2=23.077,P=0.000<0.05),同时其培养MTB-L阳性所需时间显著缩短(t=14.851,P=0.000<0.05)。
9分别采用改良TAS-L培养法和MTB-L培养法对281例涂片染色MTB-L阳性标本进行分离培养,两种方法(?)培阴率[涂阳培阴率=(涂阳培阴例数/涂阳总例数)×100%]分别为9.3%和1.8(?)MTB-L培养法涂阳培阴率显著低于改良TAS-L培养法(P=0.000<0.05)
10深圳市罗湖辖区MTB初治耐药率为22.4%,其中单耐药率为：INH4.7%、 RFP3.3%、EB1.7%、SM5.8%,多耐率为3.4%,耐多药率为3.7%；复治耐药率为33.3%,其中单耐药率为：INH5.6%、RFP5.6%、SM5.6%,多耐率为5.6%,耐多药率为11.1%。初治结核病患者治疗2月末结核分枝杆菌耐药率为61.5%,其中单耐药率为：：INH23.1%、RFP5.1%、EB12.8%、SM10.2%,多耐率为5.1%,耐多药率为5.1%；复治结核病患者治疗2月末结核分枝杆菌耐药率为40%,其中单耐药率为：INH20%,多耐率为20%,耐多药率为13.6%。
11深圳市罗湖辖区MTB-L初治耐药率为46.7%,其中单耐药率为：INH11.9%、 RFP1.2%、EB11.0%、SM2.0%,多耐率为16.3%,耐多药率为4.5%；复治耐药率为46.7%,其中单耐药率为:INH10.0%、EB10.0%、SM3.3%,多耐率为16.7%,耐多药率为6.7%。初治结核病患者治疗2月末结核分枝杆菌耐药率为46.6%,其中单耐药率为INH11.6%、RFP1.0%、SM1.9%,多耐率为17.5%,耐多药率为3.9%；复治结核病患者治疗2月末结核分枝杆菌耐药率为50%,其中单耐药率为：INH16.7%, EB16.7%,多耐率为8.3%,耐多药率为8.3%。
12深圳市罗湖辖区MTB-L初治始耐药率(46.7%)显著高于MTB初治始耐药率(22.5%)(χ2=47.148,P=G.000<0.05), MTB-L复治耐药率(46.7%)略高于MTB复治耐药率(33.3%),但无统计学差异(χ2=0.823,P=0.364>0.05)。结果提示MTB-L的存在与耐药严重程度密切相关。
研究结论
1本研究改进的MTB-L抗酸染色方法具有良好的灵敏度、特异度,操作简便易行,适合于痰MTB-L常规检测,能够在基层结核病实验室推广应用。
2本研究改进的MTB-L培养基配方简单、成本低廉,培养MTB-L所需时间短且细菌生长良好,适合MTB-L培养鉴定及其药物敏感试验,能够在基层结核病实验室推广应用。
3本辖区初治和复治结核病患者中痰MTB阳性者转阴率高于MTB+MTB-L阳性者及MTB-L阳性者,MTB+MTB-L阳性者和MTB-L阳性者转阴率无明显差异。结果表明MTB-L是结核病患者阴转率低的重要原因,因此加强结核病患者痰MTB-L的检测是当前结核病防控的重要策略。
4深圳市罗湖辖区MTB-L初治耐药率和复治耐药率均为46.7%,结果表明本辖区MTB-L耐药形势依然严峻,因此有必要加强结核病患者MTB-L的耐药性监测。
5深圳市罗湖辖区MTB初治耐药率为22.5%,复治耐药率为33.3%,较2010年第5次全国结核病流行病学抽样调查初始耐药率42.7%,复治耐药率38.5%的水平低,结果表明深圳市罗湖辖区结核病防控维持在一个良好的水平。
6深圳市罗湖辖区MTB-L初治耐药率和复治耐药率均分别高于MTB初治耐药率和复治耐药率,结果表明MTB-L的存在与耐药严重程度密切相关,因此有必要强结核病患者MTB-L感染及其耐药性监测。
Research background
The prevention and cure of pulmonary tuberculosis remain severe extremely in21century in our country. The epidemic situation of tuberculosis shows5characters: high infection rate, high prevalence, high incidence, high resistance rate, high death rate and slow tuberculosis control process. The epidemic situation of tuberculosis in countryside is higher than that in city. The prevalence and death rate of young adults are higher, which harm the life safety of the people severely. Mycobacterium tuberculosis (MTB) can form cell wall-deficient mutants (L forms) easily, and M. tuberculosis L forms (MTB-L) are able to survive under harsh environmental conditions. MTB-L can survive over long periods in vivo and can be isolated and cultivated from blood samples of patients with active sarcoidosis. The biological characteristics, pathogenicity, and drug susceptibility of MTB-L differ from those of MTB, and these differences might be important factors underlying tardy progression, aggravation, recurrence, and drug resistance in tuberculosis. Approximately70%of active pulmonary tuberculosis cases are reported to be smear and culture negative. However, the MTB-L positive rate exceeds30%for sputum specimens from smear- and culture-negative pulmonary tuberculosis cases, and the MTB-L positive rate exceeds50%for sputum specimens from multidrug-resistant tuberculosis cases. Thus, a fast and convenient method to detect MTB and MTB-L would be useful in determining the nature of a tuberculosis infection, selecting a treatment strategy, and identifying preventive measures.
Research purpose
To aim directly at severe situation of the prevention and cure of pulmonary tuberculosis, and the bionomics and pathogenic characters of Mycobacterium tuberculosis L forms; we planed:①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms;②to analyze the infection and drug fast of tuberculosis in Luohu district of Shenzhen city systematically and to evaluate the diagnosis and treatment value of the modified method.
Research contents
①to establish a novel, high sensibility, high specificity method for smear dyeing and drug sensitivity test of Mycobacterium tuberculosis L forms, and to evaluate the diagnosis and treatment value of the modified method.
②the sputum specimens of dubious tuberculosis patients and directly observed therapy, short-course tuberculosis patients from Luohu Chronic Disease Prevention and Cure Hospital during January,2010to December,2011were detected for Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms by the modified acid-fast staining.
③the drug sensitivity test of MTB-L of the above samples were carried out by the modified MTB-L culture medium.
④the drug sensitivity test of MTB of the above samples were carried out by the modified Roche medium.
⑤the epidemic situation of tuberculosis in Luohu district was analyzed, the affection of the drug fast to Mycobacterium tuberculosis and Mycobacterium tuberculosis L forms on the clinical diagnosis and treatment prognosis of tuberculosis was evaluated.
Research methods
1The improvement and methodological evaluation of MTB-L acid-fast staining method
1.1Procedures of our modified acid-fast staining method (modified MTB-L acid-fast staining method), modified IK acid-fast staining method and traditional acid-fast staining method
Modified IK acid-fast staining method and traditional Ziehl-Neelsen acid-fast staining method were executed as previously reported. Colorants should be added onto the slide repeatedly and heating was needed in the process of traditional acid-fast staining method. Heating was not needed in the process of modified IK acid-fast staining method; but the process should be sustained for24hours. However, heating was not needed in the process of our modified acid-fast staining method either and the process should be sustained for about5minutes only. The procedures of the three methods were presented in the table below.
1.2Fluorescent quantitation PCR method
MTB DNA was extracted from50sputum samples from patients with definite tuberculosis by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da'an Gene) according to the manufacturer's instructions. Extracted DNA was stored at-20℃in extract buffer until analysis.
MTB DNA was analyzed by using a previously reported real-time PCR method. Briefly, real-time PCR was carried out by using the commercially available Mycobacterium tuberculosis (TB) fluorescent polymerase chain reaction diagnostic kit (Da'an Gene) in a40-μl reaction mixture containing PCR buffer, deoxynucleoside triphosphates (i.e., dATP, dTTP, dCTP, and dGTP), forward and reverse primers (5'-TCGCCCGTCTACTTGGTGTT-3';5'-TGATGTGGTCGTAGTAGGTC-3'), and the fluorescence probe (5'-ACAACGCCGAATTGCGAAGGGC-3'), along with3μl Taq DNA polymerase and2μl of the extracted DNA sample. The thermal cycler was programmed as follows:one cycle of93℃×2min,10cycles of93℃×45s and55℃×1min, and30cycles of93℃×30s and55℃×45s. The fluorescence signals of FAM were acquired at the end of the third program (i.e., after55℃×45s). Fluorescence data analysis was conducted using SDS software (Version1.4; Applied Biosystems). The threshold fluorescence level, used to derive threshold cycle (Ct) values, was automatically determined by the SDS software.
1.3Evaluation of our modified acid-fast staining method
The sensitivity, specificity, positive predictive value, negative predictive value, positive rate, and diagnostic efficiency of the modified MTB-L acid-fast staining method were evaluated by using the PCR method as the comparison's gold standard. Briefly, fifty sputum samples from patients with definite tuberculosis were analyzed by comparing results from our modified acid-fast staining method and the PCR method.
2Clinical application of the modified MTB-L acid-fast staining method
Nine hundred and sixty sputum samples from patients with definite tuberculosis were smeared onto individual glass slides to form egg-shaped sputum membranes of size10×20mm (6smears per sample). Two of the six smears were analyzed using the traditional acid-fast staining method; two, using the modified IK acid-fast staining method; and two, using our modified acid-fast staining method. The positive detection rates for MTB and MTB-L in the smears of the960samples treated with each of the three methods were compared. The epidemic situation of tuberculosis in Luohu district, the infection status of MTB and MTB-L, and the affection of MTB and MTB-L on clinical diagnosis and treatment prognosis to tuberculosis were analyzed.
3The improvement and methodological evaluation of MTB-L medium
3.1The routine cultivation using Roche medium
The sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the Roche medium at37℃. After3～7days, the colonies were observed once a week and defined using acid-fast staining method. Negative results were reported when no colonies were found till8th week.
3.2The cultivation using modified tryptone soybean tryptone ager for MTB-L (TSA-L medium)
The sputum samples were collected from clinically diagnosed patients with tuberculosis. Then the samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the TSA-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.
3.3The cultivation using our modified medium for MTB-L (MTB-L medium)
The prescription of TSA-L were improved, that is to say, cholesterol and lecithin in favor of mycobacteria growth were added into TSA-L. Then the sputum samples were collected from clinically diagnosed patients with tuberculosis. The samples were liquefied through pre-treatment by4%sodium hydroxide. About0.1ml liquefied samples were inoculated onto the MTB-L at37℃with5%～10%CO2. The colonies were observed once a week. The doubtful colonies were defined using microscope.
3.4Evaluation of our modified acid-fast staining method
Fifty MTB-L positive samples defined by Fluorescent quantitation PCR method were cultivated using Roche medium, TSA-L medium and MTB-L medium respectively. The positive rates and required time of colonies growth were analyzed.
4Clinical application of the modified MTB-L medium
Drug sensitivity test for MTB-L positive samples were carried out using the modified MTB-L medium. The resistant states of MTB-L to first line drug were analyzed.
Drug sensitivity test for MTB positive samples were carried out using Roche medium. The resistant states of MTB to first line drug were analyzed.
Statistical treatment
①Differences between values of MTB-L acid-fast staining method, Z-N acid-fast staining method, modified IK acid-fast staining method and PCR method were assessed by using McNemar's chi square test.
②Differences among values of MTB-L acid-fast staining method, modified IK acid-fast staining method, and Z-N acid-fast staining method, and differences among values of negative conversion rate of MTB、MTB-L were assessed by using group design chi-square test.
③Differences among values of culture positive rate of MTB-L culture method and TAS-L culture method were assessed by using group design chi-square test. Days of MTB-L culture positive by the two methods were recorded as mean±SD, and the differences of mean±SD were assessed by using Student's t-test.
④Differences among smear-positive and culture-negative rate of MTB-L culture method and TAS-L culture method, and differences among drug resistance rate of MTB、MTB-L were assessed by using group design chi-square test.
⑤SPSS13.0was used to define the differences.
Research results
⑥Sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of our modified acid-fast staining method were similar to the modified IK acid-fast staining method. As in the modified IK acid-fast staining method, no heating is needed in our method. In addition, our method only needs about5min to complete, which is markedly shorter than the24h required to complete the modified IK acid-fast staining method. The results obtained using our methods have satisfactory concordance with those obtained by PCR. Moreover, the MTB and MTB-L positive detection rates of our method were significantly higher than those of the routine acid-fast staining method.
②Except1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis initial treated patients in Shenzhen Luohu district changed into negative after5month normalization treatment. The sputum negative conversion rates of MTB positive patients at the end of2,3months were higher than those of MTB and MTB-L positive patients and MTB-L positive patients sharply. Except2cases of MTB and MTB-L positive patients, and1case of MTB-L positive patient, the detection rate of MTB and MTB-L in tuberculosis retreated patients in Shenzhen Luohu district changed into negative after4month normalization treatment.
③MTB-L could grow more quickly on our modified mycobacterium tuberculosis solid medium than on modified TSA-L solid medium; the positive rates and required time of colonies growth of MTB-L using our modified solid medium were higher than those on modified TSA-L solid medium
④Initial treatment resistance rate of tuberculosis in Luohu district was lower than that of the fifth national tuberculosis epidemiological survey in2010; meanwhile, Retreated resistance rate of the former was than that of the latter.
Research conclusion
⑤A new, modified acid-fast staining method, which is convenient, specific, and sufficiently sensitive to examine sputum for the presence of MTB-L and MTB quickly and routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.
②A new, modified cultural method, which is convenient, specific, and sufficiently sensitive to drug sensitivity test of MTB-L routinely, had been established successfully. Our method can be easily utilized in a general tuberculosis laboratory.
③The sputum negative conversion rates of MTB positive patients in tuberculosis initial treated and retreated patients were higher than those of MTB+MTB-L or MTB-L positive patients. The sputum negative conversion rates between MTB+MTB-L positive patients and MTB-L positive patients changed little. So, MTB-L is an important reason for the lower sputum negative conversion rates of tuberculosis patients.
④The rates of initial resistance and retreated resistance of MTB-L were both46.7%, which indicated that the status of MTB-L drug resistance is still quite serious.
⑤The rates of initial resistance and retreated resistance of MTB in Luohu district were lower than those of national epidemiological sampling survey of tuberculosis in2010(22.5%vs.42.7%,33.3%vs.38.5%), which indicated that satisfactory progress is made in the control and prevention of MTB in Luohu district.
⑥The rates of initial resistance and retreated resistance of MTB-L were higher than those of MTB respectively, which indicated that MTB-L and the severity of drug fast is closely related.
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14.朱明利,张永乐,张利诚等.肺结核患者结核菌L型感染及其荧光定量PCR检测的意义.中国实验诊断学,2008,12(5)：632-635.
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19.胡荣盛,徐亚丽,荧光定量PCR法在结核杆菌检测中的价值,现代实用医学.2008：20(10),767-769.
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