Diagnostic value of gastric aspirate smear and polymerase chain reaction in smear-negative pulmonary tuberculosis

OBJECTIVE: The aim of this study was to determine the validity of acid-fast bacilli (AFB) smear and polymerase chain reaction (PCR) from gastric aspirates for the diagnosis of smear-negative pulmonary tuberculosis. METHODOLOGY: A cross-sectional study was conducted in a university hospital. One hundred and nine patients with suspected pulmonary tuberculosis in whom either sputum smears were negative or who were not producing sputum were recruited to the study. All patients underwent gastric aspiration after an overnight fast followed by standard fibreoptic bronchoscopy. Specimens were subjected to AFB smear, culture, and pathological examination. PCR was performed on culture filtrate after 1 week of incubation. RESULTS: Eight patients did not complete the follow-up schedule. Of the 101 patients with final outcomes, a diagnosis of pulmonary tuberculosis from microbiological evidence was established in 54 patients. The gastric aspirate smear, PCR, or either one of them was positive in 34, 30, and 39 tuberculosis patients, respectively. There were 13 false positive smears from 47 non-tuberculosis patients, with five resulting from non-tuberculous mycobacteria (NTM). The PCR was falsely positive in eight patients, five of whom had previous histories of tuberculosis. The overall sensitivity, specificity, positive predictive value, and negative predictive value of gastric aspirate examination by combined smear and PCR were 72, 58, 66, and 64%, respectively. CONCLUSIONS: Gastric aspiration is a useful tool for the diagnosis of smear-negative pulmonary tuberculosis warranting institution of antituberculosis treatment. Interpretation of the results should be cautious in those who have had tuberculosis in the past or who have been at risk for acquisition of NTM.     

聚合酶链反应检测肺结核患者外周血单个核细胞中结核分 …

探讨外周血单个核细胞中结核分支杆菌ＤＮＡ检测在肺结核诊断中的价值。方法采用改良Ｔｒｉｔｏｎ－ｘ－１００法分离，制备单个核细胞中模板，ＤＮＡ，聚合酶链反应扩增结核分支杆菌２４０ｂｐ基因片段，同时分析了影响ＰＣＲ结果的有关因素。结果８９例肺结核患者的血标本，８４例肺结核患者的痰标本中，结核分支杆菌ＤＮＡ阳性率分别为７３％和５７％；８４例肺结核患者外周血，痰标本配对检测总阳性率可达８７％。     

实时荧光定量聚合酶链反应检测支气管肺泡灌洗液中结核分枝杆菌-DNA对肺结核的诊断价值

目的探讨实时荧光定量聚合酶链反应法（FQ-PCR）检测支气管肺泡灌洗液（BALF）中结核分枝杆菌-DNA（TB-DNA）对肺结核的诊断价值。方法肺结核52例（菌阳20例,菌阴32例）,肺炎35例,采用FQ—PCR法检测其支气管肺泡灌洗液中TB—DNA水平。结果52例肺结核组的阳性率为65．4％（菌阳19例,菌阴15例）,35例非肺结核组的阳性率为5．7％,经统计学比较,FQ-PCR法检出结核杆菌阳性率显著高于痰涂片抗酸染色和培养法（P均〈0．05）,FQ-PCR法特异性高。结论FQ-PCR法检测BALF中TB-DNA为痰涂片阴性及无痰患者提供良好的诊断依据。     

支气管肺泡灌洗液行Xpert MTB/RIF检测对涂阴肺结核的诊断价值

目的评价支气管肺泡灌洗液行Xpert MTB/RIF检测对涂阴肺结核的诊断价值。方法选取从2014年11月至2015年12月徐州市传染病医院收治的3次痰涂片阴性的可疑肺结核患者110例,对所有患者行支气管镜检查进行刷检及收集BALF,进行涂片镜检找抗酸杆菌、结核分枝杆菌培养及Xpert MTB/RIF检测,同时进行诊断。分别以BALF的罗氏培养结果及临床诊断标准作为肺结核诊断的阳性标准,计算Xpert MTB/RIF诊断涂阴肺结核的敏感度、特异度、阳性预测值及阴性预测值。结果以BALF培养阳性结果作为判断肺结核的阳性标准,BALF行Xpert MTB/RIF检测对诊断涂阴肺结核的敏感度、特异度、阳性预测值、阴性预测值分别为100%、97.4%、97.8%、100%。以临床诊断标准为诊断肺结核的阳性标准,BALF行Xpert MTB/RIF检测对诊断涂阴肺结核的敏感度、特异度、阳性预测值、阴性预测值分别为81.9%、97.4%、98.3%、74.0%。以DST结果为金标准,Xpert MTB/RIF检测利福平耐药的敏感度、特异度分别为75.0%、96.0%。结论BALF行Xpert MTB/RIF检测在涂阴肺结核中的敏感度、特异度均较高,且检测快速并能判断是否利福平耐药,对涂阴肺结核的快速诊断及治疗具有较大的应用价值。     

荧光定量PCR测定支气管肺泡灌洗液中TbDNA对涂阴肺结核的诊断价值

目的探讨纤支镜肺泡灌洗液中结核分枝杆菌DNA在涂阴肺结核诊断中的价值。方法应用荧光定量聚合酶链反应（FQ-PCR）技术对300例涂阴肺结核,178例菌阳肺结核及119例肺炎或肺癌患者的纤支镜肺泡灌洗液标本进行结核分枝杆菌DNA检测。结果3种病因肺泡灌洗液结核分枝杆菌DNA阳性检出率分别为：74.3%（223/300）、94.9%（169/178）、17.6%（21/119）。结论荧光定量聚合酶链反应检测肺泡灌洗液结核分枝杆菌DNA,用于肺结核诊断可提高肺结核诊断的敏感性和准确性,明显优于痰抗酸染色,亦较痰结核菌培养诊断灵敏、快速,可作为肺结核诊断较可靠指标。     

肺结核患者支气管肺泡灌洗液细胞因子的水平及其动态变化

目的研究肺结核患者支气管肺泡灌洗液(BALF)中γ干扰素(IFN-γ)、白细胞介素12(IL-12)、白细胞介素4(IL-4)、白细胞介素10(IL-10)水平的变化及其临床意义。方法采用双抗体夹心ELISA法检测60例活动性肺结核患者、33例非活动性肺结核患者BALF中IFN-γ、IL-12、IL-4、IL-10的水平,对其中32例活动性肺结核患者抗结核治疗后的上述细胞因子水平进行随访。组间比较采用t检验。结果活动性肺结核病例BALF中IFN-γ、IL-12的水平要显著高于非活动性肺结核病例(P0.01),而IL-4、IL-10的水平两组间无明显差异(P0.05)。菌阳及重症肺结核的IFN-γ、IL-4、IL-12水平要明显高于菌阴及轻症(P0.05),IL-10值无明显差异(P0.05)。32例活动性肺结核的病例在抗痨治疗一个月后复查支气管镜,BALF中IFN-γ、IL-4、IL-12的水平均较治疗前有明显的下降(P0.01)。而IL-10值亦有所下降,但差异无统计学意义(P0.05)。结论肺结核患者BALF中IFN-γ、IL-4、IL-12水平的检测可作为了解病灶活动性、严重程度、监测疗效的参考。     

一种直接检测结核病痰标本的PCR技术的建立与评价

目的建立并评价聚合酶链式反应(polymerase chain reaction,PCR)在结核病痰标本检测中的应用价值。方法根据结核分枝杆菌复合体IS6110序列设计引物INS1和INS2,并建立PCR反应体系和反应条件。运用PCR方法分别检测标准菌株、结核分枝杆菌PCR检测标准品和拟诊结核病患者痰标本,采用痰涂片和细菌培养为对照。结果比较的统计学分析采用卡方检验。结果PCR方法对结核分枝杆菌、牛分枝杆菌、卡介苗标准株的最小检出浓度分别达到102,103,103个细菌/毫升,能够特异地检出结核分枝杆菌复合体。在PCR检测的574例拟诊病例中,PCR检测阳性病例241例,42%;痰涂片和细菌培养的阳性率分别为19.69%和26.31%,PCR检测阳性率高于传统细菌学检验方法,经χ2检验,差异具有显著统计学意义(χ2=103.67,P<0.01)。以痰培养结果为标准,计算PCR检测方法的敏感度为67.53%。结论PCR检测方法与传统的细菌学检测方法相比可以提高阳性标本检出率,而且具有快速、特异、简便的特点,有望成为结核病大规模筛查和临床快速检测方法。     

Clinical evaluation of a polymerase chain reaction assay to detect Aspergillus species in bronchoalveolar lavage samples of neutropenic patients

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. Using a recently developed two-step polymerase chain reaction (PCR) assay to detect 10 fg of Aspergillus DNA, corresponding to 1-5 colony-forming units (CFU)/ml of spiked samples in vitro, we prospectively examined 197 bronchoalveolar lavage (BAL) samples from 176 subjects, including 141 neutropenic, febrile patients with lung infiltrates, at risk for invasive fungal disease. Underlying diseases of these patients were haematological malignancies; 93 patients suffered from acute leukaemias. Thirty-one of these immunocompromised patients (17.6%) were PCR positive, correlating with positive BAL culture, positive histology from lung surgery or from autopsy, positive computerized tomography scans or positive galactomannan enzyme-linked immunosorbent assay. Six patients (4.3%) of this group had positive PCR results without any correlation to clinical or other diagnostic data, probably owing to contamination of the samples by ubiquitous Aspergillus spores. The samples of two patients (1.4%) with a subsequent histologically proven mould infection were PCR negative. All 102 immunocompromised patients (72.3%) with a negative PCR showed no evidence of invasive fungal disease. From 35 patients without immunodeficiency, four (11.4%) showed positive results, without evidence of invasive or non-invasive pulmonary aspergillosis. In this haematological population, the sensitivity and specificity values of the test reached 93.9% and 94.4%, the positive predictive value 83.8%, the negative predictive value 98.1%. Our data support the considerable clinical value of this PCR assay for confirming and improving diagnosis of pulmonary aspergillosis in high-risk patients.     

Role of bronchoalveolar lavage fluid and serum interleukin-27 in the diagnosis of smear-negative pulmonary tuberculosis

Background:To evaluate the value of interleukin (IL)-27 measured in serum and bronchoalveolar lavage fluid (BALF) for the diagnosis of smear-negative pulmonary tuberculosis (TB).Methods:This was a prospective study of patients planned to undergo bronchoscopy at Wuxi No.5 People''s Hospital between January 2017 and September 2018. The patients were grouped as the TB and control groups. BALF and serum IL-27 were measured by ELISA. Receiver operating characteristic (ROC) curves were used to assess the diagnostic value and calculate the optimal cutoff values.Results:There were 40 patients in the control group and 87 in the TB group. In the TB group, 20 had positive sputum smear results and 67 were negative. The area under the ROC curve (AUC) of BALF IL-27 for pulmonary TB was 0.897 (95% CI: 0.830–0.944) (P < .001). The AUC of serum IL-27 for pulmonary TB was 0.703 (95% CI: 0.616–0.781) (P < .001). In patients with negative sputum smear results, the AUCs of BALF IL-27 and serum IL-27 for pulmonary TB was 0.882 (95% confidence interval [CI]: 0.805–0.936) (P < .001) and 0.679 (95% CI: 0.601–0.782) (P < .001), respectively.Conclusions:BALF IL-27 can be used for the diagnosis of pulmonary TB, particularly in those with a negative sputum smear result. Serum IL-27 could be an auxiliary method for TB screening.     

支气管肺泡灌洗液多指标联合检测对不典型肺结核的诊断价值探讨

目的探讨支气管肺泡灌洗液（BALF）多指标联合检测对不典型肺结核的诊断价值。方法56例不典型肺结核患者治疗前均进行支气管镜检查,行支气管刷检涂片法、镜检后痰涂片法、BALF涂片法以及联合进行BALF涂片、BALF聚合酶链反应（BALF-PCR）、BALF结核抗原（BALF-TBAG）和BALF腺苷脱氨酶（BALF-ADA）四项指标检测方法,分析不同的检测方法对不典型肺结核诊断的阳性率。结果56例患者行支气管刷检涂片法、镜检后痰涂片法、BALF涂片法的阳性率分别为39.2%,46.4%,75.0%,联合进行BALF涂片、BALF-PCR、BALF-TBAG和BALF-ADA四项指标检测的阳性率为92.8%,与前三种检测方法相比,差异有显著性（P〈0.05）。结论BALF多指标联合检测对不典型肺结核具有较高的诊断价值,特异性高,快速,大大缩短诊断时间,可以明显提高诊断阳性率,是一项较好的组合方式。     

Rapid diagnosis of invasive pulmonary aspergillosis by quantitative polymerase chain reaction using bronchial lavage fluid

Polymerase chain reaction (PCR) is a sensitive method for detection of Aspergillus DNA in bronchoalveolar lavage fluid, but it has not yet been able to distinguish infection from contamination. We have established a technique to quantify Aspergillus DNA using a real-time PCR method to resolve this problem, and we report herein a successful application of real-time PCR to diagnose invasive pulmonary aspergillosis by comparing the amount of Aspergillus DNA in bronchial lavage fluid from an affected area to that from an unaffected area. This novel tool will provide rapid, sensitive, and specific diagnosis of pulmonary aspergillosis.     

Combined bronchoalveolar lavage and polymerase chain reaction in the diagnosis of pulmonary tuberculosis in smear-negative patients.

SETTING: The polymerase chain reaction (PCR) may be sensitive and specific for the diagnosis of tuberculosis, but most reports are of studies conducted in well-controlled laboratories. A study to evaluate the clinical value of bronchoalveolar lavage (BAL) combined with PCR was necessary. OBJECTIVE: One hundred and thirty one patients were recruited into the study from March 1994 to February 1997. DESIGN: Patients with a positive acid-fast stain on sputum smear were recruited into group A as positive controls, patients with lung cancer and a negative acid-fast stain on sputum smear were put into group B as negative controls, and patients who had clinical symptoms of pulmonary TB without sputum or with negative smear results were the investigating group. PCR was performed on the sputum samples from group A and B patients and on the BAL fluid from those in group C. RESULTS: The sensitivity of PCR was 96% in group A, and the specificity was 100% in group B. The sensitivity of PCR in the BAL fluid from the group C patients was 36% and the specificity was 96%; the positive predictive value was 94% and the negative predictive value was 45%. CONCLUSION: BAL plus PCR is useful in the rapid diagnosis of pulmonary TB in non-productive or smear-negative patients.     

涂阴老年不典型肺结核的多种CT影像表现

目的探讨涂阴老年不典型肺结核的多种CT表现。方法收集我院住院的经临床治疗、痰检及纤支镜证实的涂阴老年不典型肺结核30例,就其CT表现回顾性分析。结果 1大片状肺实变影3例(占10%);2单发球形或肿块2例(占6.7%):1例结核球;1例肿块;3多发结节及肿块:2例(占6.7%);4间质改变11例(占36.7%);5空洞征3例:薄壁空洞1例(占3.3%),厚壁空洞2例(占6.7%);6树芽征6例(占20%);7磨玻璃影3例(占10%)。结论胸部CT扫描可识别涂阴老年不典型肺结核的多种征象,判断病变的性质和程度等方面具有较高的敏感度,有助于评价和监测不典型继发性肺结核的诊断、征象的转归等演变过程,为临床提供不可或缺的支持。     

Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction

A polymerase chain reaction (PCR) assay for the rapid detection of Mycobacterium tuberculosis in sputum samples is described. The target DNA is a 123-base pair (bp) segment of IS6110, which is repeated in the M. tuberculosis chromosome and is specific for the M. tuberculosis complex. Methodology used to lyse the mycobacteria, extract the DNA, and amplify the 123-bp target DNA is presented. The amplified PCR product is detected by examination of ethidium-bromide-stained acrylamide gels. An internal control using the same primers as the target DNA has been constructed to assess the efficacy of each individual reaction. Of 162 sputum samples tested, 82 were smear-positive for acid-fast bacilli. Of the 94 specimens from patients in whom pulmonary tuberculosis was diagnosed, 51 were culture-positive, smear-positive, or both. Fifty of these were PCR positive. Of the 42 specimens from patients with nontuberculous mycobacterial pulmonary disease, 41 were PCR negative. All 26 specimens from patients without mycobacterial infection were PCR negative. This assay provides a sensitive and specific means for the laboratory diagnosis of tuberculosis within 48 h that is relatively simple to perform.     

应用多重聚合酶链反应法检测并鉴别结核分支杆菌与非结核分支杆菌DNA

OBJECTIVE: To improve the specificity and sensitivity of polymerase chain reaction (PCR) technique for detecting and identification of DNA of M. tuberculosis and M. nontuberculosis. METHOD: Three pairs of oligonucleotide primer were used in triplex-PCR. A 383 bp DNA fragment encoding part of the 65,000 mycobacterial surface antigen, a 123 bp fragment corresponding to a specific M. tuberculosis complex sequence which was the insertion sequence 6110 (IS 6110) and a 268 bp fragment for human beta-globin were amplified by triplex-PCR respectively. RESULT: The sensitivity of the triplex-PCR-electrophoresis for the DNA of mycobacterium was 0.6 pg. The specific bands of 383 bp and 123 bp among the amplified DNA from M. hominis, M. bovis, BCG and M. simiae were present in the agarose gel. By contrast, only a band of 383 bp was found among the M. nontuberculosis which contained M. avium, M. chelonae, M. scrofulaceum, M. xenopi, M. kansasii, M. intracellulare and M. smegmatis. Compared with the standard strains, there was an additional 268 bp band in simulated clinical samples infected by Mycobacterium. The above 3 specific bands were found neither in other 15 bacterial species tested nor in Mycoplasma pneumoniae. 182 clinical samples were examined by culture, smear and triplex-PCR. 72 nontuberculous clinical samples were all negative. In 110 tuberculosis clinical samples, the positive rates were 2.7%, 13.6% and 32.7%, respectively. CONCLUSION: The triplex-PCR possesses a high specificity and sensitivity. This method could detect and identify the DNA of M. tuberculosis and M. nontuberculosis except M. simiae. It is a valuable tool for early diagnosis and differentiation for infection of M. tuberculosis and M. nontuberculosis.     

Use of polymerase chain reaction in the diagnosis of abdominal tuberculosis

BACKGROUND: Early diagnosis and prompt treatment of abdominal tuberculosis is vitally important as it greatly reduces disease and treatment related morbidity and even mortality in extreme cases. A polymerase chain reaction (PCR) test was evaluated for its feasibility as a diagnostic tool in abdominal tuberculosis (TB) in the Indian scenario. METHODS: PCR for the identification of M. tuberculosis amplified a 340 bp nucleotide sequence located within the 38 kDa protein gene of M. tuberculosis. Tissues for processing were obtained from patients suspected to have abdominal TB. These were from various sources such as abdominal lymph nodes, segments of intestine and bowel obtained at various times and in different ways such as laparoscopy, colectomy, bowel and lymph node resection. Fifty such patients had their tissues sent for PCR. RESULTS: PCR results were compared with histopathology (HP). Of the 50 samples, 31 were positive for abdominal TB by HP whereas 30 were positive by PCR. Twenty-four of these were positive for both HP and PCR while of the seven samples positive for HP, five were negative and two gave inhibition by PCR. Six samples negative by HP were positive by PCR. CONCLUSION: This study demonstrates that PCR can be used as an effective tool to diagnose abdominal TB.     

支气管镜肺泡灌洗治疗脑卒中合并肺部感染的临床观察

目的 探讨支气管镜肺泡灌洗治疗脑卒中合并肺部感染的临床疗效.方法 54例脑卒中合并肺部感染患者随机分为治疗组28例,对照组26例,对照组予常规抗感染,对症支持及原发病等综合治疗;治疗组在对照组基础上予支气管镜肺泡灌洗治疗.结果 治疗组总有效率明显高于对照组(P<0.01);感染控制时间及住院时间较对照组明显缩短(P<0.01);需机械通气率及死亡率明显减少(P<0.05).结论 支气管镜肺泡灌洗治疗脑卒中合并肺部感染疗效确切.     

Novel method for sputum induction using the Lung Flute in patients with suspected pulmonary tuberculosis

Background and objective:   The Lung Flute is a small self-powered audio device that generates sound waves, which vibrate in tracheobronchial secretions. This was a preliminary trial to evaluate the usefulness of the Lung Flute for sputum sampling in patients suspected of pulmonary tuberculosis (TB).
Methods:   Thirty-four patients who were not expectorating sputum, but for whom sputum examination was required for the differential diagnosis of TB or other diseases, were enrolled in the study. Patients were instructed to blow out fast and hard through the Lung Flute and to repeat this for a total 20 sets of two blows each.
Results:   Using the Lung Flute, sputum samples were collected within 10 or 20 min from 30 of 34 patients (88%). The device permitted a rapid diagnosis of TB in seven of 15 confirmed TB cases. In three patients acid-fast bacillus smears were positive. In four patients acid-fast bacillus smears were negative, but PCR tests for TB were positive. Hyperventilation-related symptoms occurred in three patients.
Conclusions:   The application of the Lung Flute may represent a promising technique for the rapid diagnosis of pulmonary TB.     

结核分支杆菌DNA的单管巢式聚合酶链反应检测

目的探讨单管巢式聚合酶链反应（ＳＮＰＣＲ）检测石蜡包埋组织结核分支杆菌ＤＮＡ的特异性和敏感性。方法应用普通ＰＣＲ（ＧＰＣＲ）、双管巢式ＰＣＲ（ＤＮＰＣＲ）和ＳＮＰＣＲ对结核分支杆菌ＢＣＧ和３０例结核性淋巴结炎石蜡包埋组织进行结核分支杆菌复合群ＩＳ６１１０特异插入序列片段ＤＮＡ检测。结果ＤＮＰＣＲ和ＳＮＰＣＲ检测ＢＣＧＤＮＡ均于１５ｆｇ以上呈现阳性结果，其敏感性明显优于ＧＰＣＲ（４８０ｆｇ）。ＧＰＣＲ、ＤＮＰＣＲ和ＳＮＰＣＲ检测３０例结核性淋巴结炎阳性率分别为４３％、１００％和１００％，抗酸染色阳性率（１０％）与３种ＰＣＲ法相比差异有非常显著意义（均Ｐ＜０．０１）。ＧＰＣＲ阳性率与ＤＮＰＣＲ和ＳＮＰＣＲ相比差异亦具非常显著意义（均Ｐ＜０．０１）。ＳＮＰＣＲ阳性率与ＤＮＰＣＲ相同。结论巢式ＰＣＲ检测淋巴结石蜡包埋组织结核分支杆菌的敏感性显著高于ＧＰＣＲ，其中ＳＮＰＣＲ具有与ＤＮＰＣＲ相同的特异性和敏感性，并具有更大的实用价值。