Efficacy of Amplicor PCR for the diagnosis of tuberculosis in respiratory specimens other than sputum.

A total of 832 respiratory specimens not including the sputum (402 bronchial lavages, 241 bronchial brushing specimens, 136 pumping lavages, 41 pleural effusions, and 12 others) from 462 patients were assayed using the Roche Amplicor Mycobacterium tuberculosis test for amplification and identification of M. tuberculosis, M. avium and M. intracellulare (Amplicor PCR). The results were compared with those obtained using conventional microscopy and cultivation methods. Each patient had little or no sputum and showed an abnormal chest X-ray shadowing of unknown cause. No patients had previously undergone antituberculous therapy. Of the specimens obtained, 24 were both PCR and culture positive, 786 were both PCR and culture negative, 11 were PCR positive and culture negative, and 11 were PCR negative and culture positive. Based on these results, the sensitivity and specificity of Amplicor PCR were determined to be 68.67% and 98.6%, respectively, when compared with culture of respiratory specimens not including the sputum. After correcting for discrepancies due to differences in patient clinical data, the sensitivity of Amplicor PCR was found to be 68.6%, and the specificity to be 99.9%; the corresponding values for culture were 66.7% and 100%, and those for smear were 9.8% and 100%. Thus, Amplicor PCR was shown to possess a similar sensitivity to culture and to be a highly specific technique for the diagnosis of tuberculosis in the respiratory system using non-sputum specimens within hours in patients showing little or no sputum and abnormal chest X-ray shadowing of an indeterminant cause.     

Comparison of amplicor, in-house polymerase chain reaction, and conventional culture for the diagnosis of tuberculosis in children.

A total of 251 clinical specimens (235 gastric aspirates and 16 bronchoalveolar lavages) from 88 children were prospectively tested in a blinded manner for the presence of Mycobacterium tuberculosis complex, by use of the Amplicor M. tuberculosis test and by means of in-house polymerase chain reaction (PCR). The results were compared with those obtained by conventional culture and by direct microscopy. All of the children underwent extended follow-up to verify or exclude the clinical diagnosis of tuberculosis. The results of the different tests, when compared to the final clinical diagnosis, were a sensitivity of 60% and a specificity of 96.8% for in-house PCR, 44% and 93.7% respectively for the Amplicor test, 44% and 100% for mycobacterial culture and 12% and 100% for microscopy. Amplicor tests presented false-positive findings in children without tuberculous infection. We conclude that both in-house PCR and the Amplicor test are rapid methods that can be helpful for difficult or urgent diagnosis of tuberculosis in children. However, efforts should be aimed toward improvement of the sensitivity and specificity of an easy-to-use PCR kit.     

实时荧光PCR检测儿童肺结核粪便中Mtb-DNA的效果评价

目的 应用实时荧光PCR快速检测肺结核患儿粪便中Mtb-DNA,并初步评估其临床效果。 方法 收集肺结核住院儿童粪便标本76份,应用实时荧光PCR检测Mtb-DNA,检测结果与20例其他呼吸系统疾病患儿的粪便实时荧光PCR检测Mtb-DNA、76例粪便抗酸杆菌涂片检查以及41例患儿痰标本的涂片和(或)培养、实时荧光PCR检测结果进行比较。 结果 粪便实时荧光PCR检测敏感度达到23.68%(18/76),特异度为100.00％(20/20),肺结核患儿粪便PCR阳性率［23.68%(18/76)］明显高于涂片阳性率［6.58%(5/76)］,41例患儿中痰涂片和(或)培养阳性例数(15例)和痰PCR检测阳性例数(18例)高于粪便PCR检测阳性例数(11例)。 结论 实时荧光PCR检测儿童粪便Mtb-DNA,是一种特异度高、比较敏感、非侵入性且相对安全的儿童肺结核快速诊断方法。     

Direct detection of Mycobacterium tuberculosis in sputum samples from Guinea Bissau by an rRNA target-amplified test system

Setting: There is a need for more sensitive and rapid methods for laboratory confirmation in the diagnosis of tuberculosis.Objective: To investigate the applicability of a target rRNA amplified test system (AMTDT, Gen-Probe, CA) for rapid detection of Mycobacterium tuberculosis.Design: The rRNA amplified test system was compared to standard methods for acid fast microscopy and mycobacterial culture for the demonstration of M. tuberculosis in sputum samples from 247 patients in Guinea Bissau with suspected tuberculosis.Results: The highest incidence of positive samples was obtained with the AMTDT test. Out of 274 sputum samples 96 (35%) were positive by the AMTDT test, 82 (30%) were positive by culture and 38 (14%) by direct microscopy. Using culture as reference method the sensitivity of the test was 85% (after discrepancy analysis 87%), and the specificity was 86% (after discrepancy analysis 93%).Conclusion: The sensitivity and specificity of the AMTDT test used in this setting indicates that it may be a valuable complement for improving the laboratory diagnosis of tuberculosis.     

Detection of Mycobacterium tuberculosis by a polymerase chain reaction colorimetric dot-blot assay.

SETTING: A public health laboratory in a tuberculosis-endemic region in Brazil. OBJECTIVE: To evaluate the accuracy of a combined polymerase chain reaction (PCR) colorimetric dot-blot protocol for Mycobacterium tuberculosis detection in clinical samples in a public health laboratory. DESIGN: Eighty clinical samples (13 cerebrospinal fluid, 31 induced sputum, 17 expectorated sputum, eight bronchoalveolar lavage and 11 pleural fluid) were assayed with the developed protocol. The accuracy of polymerase chain reaction (PCR) dot-blot methodology was compared to PCR agarose gel electrophoresis (PCR-AG) using as a gold standard the bacteriological result (culture and biochemical identification) combined with clinical follow-up. One internal region of the IS6110 repetitive element of the M. tuberculosis complex was selected for amplification and the amplified product transferred to nylon membranes to be detected by biotinylated DNA probe. RESULTS: Overall sensitivity and specificity obtained were respectively 90% and 97% for PCR-AG and 95% and 97% for the PCR dot-blot. Among the 56 respiratory specimens, the sensitivity and specificity results for PCR-AG were respectively 88% and 95%, and for PCR dot-blot they were 94% and 95%. Among the 24 non-respiratory specimens the sensitivity and specificity results were respectively 83% and 100% for PCR-AG, and 100% and 100% for the PCR dot-blot protocol. CONCLUSION: The results demonstrated that the PCR dot-blot assay may be helpful in the diagnosis of tuberculosis, and feasible even in resource-poor countries.     

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％。     

应用磁纳米捕获-PCR技术检测痰液结核分枝杆菌的研究

目的采用磁纳米捕获技术富集痰液中的结核分枝杆菌,以提高PCR检测的灵敏度,快速诊断结核病。方法以普通PCR为对照,采用磁纳米捕获技术富集187份结核和非结核呼吸系统疾病患者痰标本中的结核分枝杆菌,进行PCR检测。结果 152份肺结核患者痰标本41份(27.0%)涂片抗酸染色阳性,72份(47.4%)普通PCR检测阳性,126份(82.9%)磁纳米捕获-PCR检测阳性。35份非结核呼吸系统疾病患者,痰标本中抗酸染色均为阴性,1份普通PCR和磁纳米捕获-PCR检测均阳性,该患者临床诊断肺部感染合并陈旧性肺结核。结论采用磁纳米捕获技术富集痰液中的结核分枝杆菌,可显著提高PCR检测的灵敏度。     

四种结核分枝杆菌检测方法的临床应用评价

目的评估涂片、培养、PCR和增菌PCR检测结核分枝杆菌临床应用价值。方法对124例临床确诊的肺结核、可疑结核患者和非结核病人痰标本的涂片、培养、PCR和增菌PCR四种方法的检测结果进行比较。结果涂片、培养、PCR和4及7d的增菌PCR检测31例临床确诊的肺结核病人痰标本阳性率分别为22.5%、32.2%、54.8%、64.5%和87.1%;检测59例临床可疑肺结核病人痰标本阳性率分别为13.6%、18.6%、28.8%、37.3%和52.5%。比较四种方法的阳性检测率有显著性差异(P<0.05)。检测34例非结核病人痰标本,涂片、培养均为阴性,而PCR和增菌PCR均有1例假阳性,假阳性率2.9%。比较PCR与增菌PCR对菌阳和菌阴病人的痰标本阳性检测率,有显著性差异(P<0.05),而两种方法的假阳性率相同。结论增菌PCR检测结核分枝杆菌具有很高的敏感性和特异性,可作为结核病的有效辅助诊断方法之一。     

Evaluation of the Idaho Technology LightCycler PCR for the direct detection of Mycobacterium tuberculosis in respiratory specimens.

SETTING: The rapid detection of Mycobacterium tuberculosis (TB) in clinical samples is an important goal. The LightCycler heralds an advance in thermal cycle technology combining rapid cycle DNA amplification with fluorimetry, eliminating the need to perform amplification and product analysis separately. OBJECTIVES: To evaluate the LightCycler for direct detection of M. tuberculosis complex in respiratory specimens. To evaluate a DNA extraction method based on Chelex 100 resin, heating and ultrasonication for the prevention of endogenous inhibitions in respiratory samples. DESIGN: DNA was extracted from sputum samples using the Chelex method and polymerase chain reaction (PCR) for TB performed with the LightCycler. RESULTS: For 88 sputum samples positive by microscopy and culture for M. tuberculosis, 95% were PCR-positive. None of the five sputum samples that were smear-negative but culture-positive for M. tuberculosis, the 79 culture-negative sputum samples and the 29 sputum samples that were culture-positive for mycobacteria other than TB yielded positive PCR results. PCR inhibitors were not detected in any of the samples. CONCLUSION: The LightCycler proved a simple, reproducible and rapid system, reducing the time to result from weeks (culture) or days (conventional PCR) to hours. The Chelex 100 resin method produced good results for the smear-positive specimens. However, a larger study is required to determine the efficacy of the method with smear-negative specimens and for specimens known to contain endogenous inhibitors.     

Effectiveness of the BDProbeTec ET system for detection of Mycobacterium tuberculosis complex in sputum and bronchoalveolar lavage specimens

ObjectiveThe diagnostic efficacy of the BDProbeTEC ET Mycobacterium tuberculosis (MTB) complex direct detection assay (DTB) performed on bronchoalveolar lavage (BAL) specimens and sputum smears was compared with acid-fast bacilli (AFB) smear microscopy.MethodAFB smear microscopy, DTB and culture results of 286 patients with pulmonary tuberculosis were retrospectively reviewed. A total of 120 patients provided expectorated sputum samples, and 166 patients provided BAL specimens. Culture results and clinical diagnosis were used as gold standards.ResultsThe sensitivity and specificity of the DTB assay in detecting MTB in sputum specimens was significantly higher compared to AFB smear microscopy (83.7% and 82.4%, vs. 75.6%, and 41.2%, respectively). The sensitivity and specificity of the DTB assay in detecting MTB in sputum samples was 77.2% and 100% compared to clinical diagnosis, while AFB smear had a sensitivity and specificity of 70.3% and 26.3%, respectively. Compared to culture, DTB had a sensitivity and specificity of 82.8% and 93.2%, respectively, in detecting MTB from BAL specimens; AFB smear had a sensitivity and specificity of 41.9% and 87.7%, respectively. Compared to clinical diagnosis, DTB had a sensitivity and specificity of 67.2% and 100%, respectively, in detecting MTB from BAL specimens; AFB smear had a sensitivity and specificity of 34.8% and 79.5%, respectively.ConclusionsThe superior performance of the DTB assay relative to AFB smear microscopy makes it a valuable tool to enable early diagnosis of MTB, thereby improving patient care and reducing transmission.     

Evaluation of the FASTPlaqueTB assay for direct detection of Mycobacterium tuberculosis in sputum specimens.

SETTING: Sputum samples were collected from suspected tuberculosis patients attending out-patient clinics at the Ojha Institute of Chest Diseases, Karachi, Pakistan. OBJECTIVE: To evaluate the performance of the FASTPlaqueTB assay for rapid diagnosis of pulmonary tuberculosis. DESIGN: A comparative study of 584 sputum samples using acid-fast smear microscopy, Lowenstein-Jensen culture and FASTPlaqueTB. RESULTS: A total of 514 samples yielded complete results. Seventy specimens were lost to analysis due to the overgrowth of contaminants. The addition of antimicrobials inhibited growth of gram-positive contaminants, and reduced the overall contamination rate from 18.2% to 7.2%. Mycobacterium tuberculosis was isolated from 175 smear-positive and 70 smear-negative specimens. FASTPlaqueTB detected M. tuberculosis in 81.6% of specimens, with a specificity of 97.7%. The sensitivity and specificity of the assay for smear-positive specimens were respectively 87.4% and 88.2%. For smear-negative specimens, the sensitivity of the assay was 67.1% and the specificity was 98.4%. The combined sensitivity of smear and FASTPlaqueTB for M. tuberculosis was 90%. Test results were available in 48 hours. CONCLUSION: FASTPlaqueTB is a sensitive and specific test for rapid diagnosis of pulmonary tuberculosis in high prevalence areas. The test is sensitive enough to confirm a large number of clinically suspected smear-negative cases and improve case finding.     

环介导等温扩增法快速检测结核分枝杆菌的临床应用评估

目的 进行环介导等温扩增法（LAMP法）快速检测结核分枝杆菌的临床验证与应用,评估该方法在结核病临床诊断中的效能。方法 选取广东6地市专业结核病防治机构（简称“结防机构”）的肺部不同疾病及健康人员的痰标本2129份,分别采用直接涂片法、罗氏固体培养法及LAMP法检测痰标本中结核分枝杆菌;另广州、汕头两市337例结核病患者的痰标本浓缩处理后进行实时荧光定量（qPCR）及LAMP两种方法检测。 结果 2129例痰标本中,直接涂片法、罗氏固体培养法和LAMP法的阳性检出率分别为13.2%（282/2129）、21.7%（463/2129）和20.3%(432/2129);同罗氏培养法相比,LAMP法灵敏度、特异度、阳性和阴性预测值分别为89.1%(432/485)、95.5%(1570/1644)、85.4%(432/506)和96.7%(1570/1623);337例浓缩处理后的痰标本qPCR阳性率为35.0% (118/337),LAMP阳性率为35.9% (121/337);LAMP法同直接涂片法检测、罗氏固体培养法和qPCR法比较,检测结果实际一致率分别为87.9%（Kappa值＝0.612）、96.1%（Kappa值＝0.89）和91.4%（Kappa值＝0.812）。 结论 LAMP法用于痰标本中结核分枝杆菌检测,其效能同罗氏培养法及qPCR法相当,同直接涂片法相比,LAMP法能够大幅提高阳性检出率,具有很好的临床应用和推广前景。     

Comparative study of PCR, smear examination and culture for diagnosis of cutaneous tuberculosis

Diagnosis of tuberculosis (TB), especially cutaneous TB by conventional laboratory method is unreliable and time consuming. We assessed the utility of Polymerase Chain Reaction (PCR) test vis a vis other laboratory tests in 37 clinical samples of skin biopsy from equal number of patients with different variants of cutaneous TB. The PCR test amplifying 165bp region of 65kDa antigen coding gene specific for M. tuberculosis was performed on skin biopsy samples obtained from cases with a strong clinical evidence of cutaneous TB. The samples were also subjected to other laboratory tests e.g. smear examination, conventional (LJ based culture) and rapid BACTEC culture and histopathological examination for mycobacteria. Significant difference (p<0.05) was observed in the sensitivity of PCR test vis-a-vis other tests e.g. smear examination, LJ and BACTEC culture. PCR test showed a higher sensitivity than histopathological examination but the difference was not found to be statistically significant (p>0.05). PCR test showed the maximum positivity of 79.4% followed by histopathology (73.5%), BACTEC culture (47.5%), LJ media culture (29.4%) and smear examination (5.8%). The sensitivity and specificity of PCR test employing culture as the "gold standard" were 95.2% and 100%. The mean time taken for a positive result in different tests were less than 24 hours for smear examination, 1 day for PCR test, 23.42 days for BACTEC culture and 38.02 days for LJ culture. These results show that PCR amplification of 165bp region of 65kDa antigen coding gene of M. tuberculosis is a rapid and sensitive test for diagnosis of cutaneous TB using skin biopsy samples.     

A comparative study of the polymerase chain reaction and conventional procedures for the diagnosis of tuberculous pleural effusion.

Preliminary reports by ourselves and others suggest that amplification of mycobacterial DNA by the polymerase chain reaction (PCR) is a sensitive and rapid diagnostic test for tuberculosis. We recently described a PCR assay with a 336 bp repetitive sequence specific for Mycobacterium tuberculosis as the DNA target, which gave encouraging results in culture-positive smear-negative clinical specimens. In the present prospective study of patients with pleural effusions we compared PCR of the pleural fluid with conventional procedures. 84 adult patients with pleural effusions were divided into 4 groups. In group A (44 patients), M. tuberculosis was detected by culture of pleural fluid, pleural biopsy or extrapleural source. In group B (6 patients), tuberculous infection was confirmed by histology (group A excluded). Group C (3 patients) had clinical evidence of tuberculosis. Group D (31 patients) had no evidence of active M. tuberculosis infection. Analysis of the pleural fluid confirmed a sensitivity for PCR of 81%. The sensitivity of pleural fluid culture, culture of pleural biopsy, and histology of biopsy was 52.8%, 69.8% and 77.3% respectively. There were however 7 PCR positive results within group D; 6 of these were in patients with malignant effusions. We conclude that for the diagnosis of M. tuberculosis PCR is more sensitive than laboratory culture as determined by the analysis of pleural fluids. Positive PCR results among patients with malignant effusions may be false-positives or the result of latent tuberculous infections. PCR should remain an investigational procedure until prospective studies in high and low prevalence areas have critically evaluated the specificity of the assay.     

Detection of Mycobacterium tuberculosis from sputum specimens using one-tube nested PCR

One-tube nested PCR was developed for diagnosis of pulmonary tuberculosis using sequences based on thel6SrRNA gene. The usage of primers 16SOL, 16SOR, 16SIL and 16SIR with optimized conditions could detect 555 bp DNA band from 21 species, 41 strains of mycobacteria and one isolate of Nocardia asteroides. It also revealed a specific 306 bp DNA band from 59 strains of M. tuberculosis complex. Cross amplification was observed in M. marinum, M. ulcerans and a few isolates of M. fortuitum complex. The developed method could detect as little as 100 fg of M. tuberculosis DNA. The PCR mixtures could be stored at 0 degrees C for 2 months or at -20 degrees C for at least 20 months without decrease in sensitivity. Using one-tube nested PCR for detection of M. tuberculosis compared with acid fast staining and culture results from 153 sputum specimens revealed 88.6% sensitivity and 89.2% specificity in smear positive specimens and 93.2% sensitivity and 85.0% specificity in culture positive specimens.     

结核抗体lgG/lgM检测在肺结核和肺外结核中的诊断价值

目的 探讨异种血清抗体检测技术在结核病诊断中的应用价值。方法 采用IgG/IgM抗体试剂盒分别检测102例结核病患者(包括73例肺结核和29例肺外结核)、223例其他肺部疾病患者和100例对照者结核感染情况,以临床诊断为标准评价该方法的敏感度和特异性,同时分别与痰菌培养及痰涂片平行检验的结果作比较,统计学分析采用χ²检验。结果 结核抗体IgG/IgM检测结核病患者的敏感度为74.51%、特异度为91.64%。结核抗体IgG/IgM检测肺结核和肺外结核的敏感度分别为82.19%、55.17%,肺内和肺外结核的敏感度差异有统计学意义(P＜0.05),结核抗体lgG/IgM检测结核患者阳性检出率明显高于痰培养法和痰涂片法(P<0.05)。102例结核病患者年龄段分组分析,少年组和老年组检出率分别为58.33%、36%,远低于青年组和中年组的96.15%和89.74%。不同年龄组间进行卡方比较分析显示,P＜0.05,差异有统计学意义。425例标本中,共发现8例非结核分枝杆菌,其中6例胞内分枝杆菌, 2例脓肿分枝杆菌,lgG/lgM抗体检测均为阴性。结论 IgG/IgM血清抗体检测肺内、外结核具有快速方便、经济和较高的敏感度,适合用于临床结核筛查。     

实时荧光定量PCR法分析结核分枝杆菌对异烟肼耐药的分析

目的利用实时荧光定量PCR技术快速检测异烟肼耐药结核分枝杆菌。方法收集到医院就诊的结核病疑似患者痰液样本,提取痰液样本的总DNA,利用实时荧光定量PCR(Real-time PCR)技术对结核分枝杆菌感染进行快速筛查,并与传统药敏试验进行比较,对两者的灵敏度、特异性、一致性进行比较分析。结果检测346例结核病人临床分离培养样本,药敏试验检出257例异烟肼敏感标本,101例异烟肼耐药标本;实时荧光定量PCR法共检测出异烟肼敏感和耐药标本225例98例,灵敏度为86.64%,特异性为93.92%,一致率为93.12%。结论跟传统药物敏感性实验相比,实时荧光定量PCR法检测速度快速、特异性强、灵敏度较高,可用于结核分枝杆菌耐异烟肼突变的快速检测,适于耐多药结核病的快速筛查。     

Role of Gene Xpert in smear negative pulmonary tuberculosis

BackgroundTuberculosis is a major health problem contributing to significant morbidity and mortality. Early diagnosis and treatment is the key for TB control. Sputum microscopy is a rapid and inexpensive test but due to low and variable sensitivity, many cases can be missed. Culture is considered to be the gold standard but is time consuming. Gene Xpert is a novel and rapid cartridge based nucleic acid amplification test (CBNAAT) that can be used for prompt diagnosis.AimTo compare the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of Gene Xpert with culture in diagnosing tuberculosis in sputum smear negative patients.MethodsThe study is a prospective observational study conducted from December 2017 to January 2019 on 189 patients, who were sputum smear negative but had signs and symptoms suggestive of tuberculosis. Their respiratory samples were taken (either sputum or bronchoalveolar lavage) and sent for Gene Xpert. The results were compared with culture, which was taken as the gold standard, and diagnostic accuracy was assessed.ResultA total of 189 patients were included in the study. In 25 patients sputum was taken and in 164 patients BAL was taken (which included 22 patients in whom sputum Gene Xpert was negative but there was high clinical suspicion of tuberculosis). The sensitivity, specificity, PPV and NPV of Gene Xpert in diagnosing smear negative pulmonary tuberculosis was found to be 96.3%, 81.3%, 87.5% and 94.2% respectively.ConclusionGene Xpert can be used as a rapid diagnostic tool in patients who are sputum smear negative but have clinical features highly suggestive of tuberculosis. It additionally helps in detecting rifampicin resistance. But every Gene Xpert positive case does not necessarily mean an active disease, therefore, past history of tuberculosis along with radiological signs of disease activity are to be considered. In case of negative Gene Xpert but high clinico-radiological suspicion of TB, patients should be followed up on regular intervals, while awaiting their culture.     

TaqMan聚合酶链反应技术检测结核分支杆菌DNA及其临？…

目的 探讨ＴａｑＭａｎ聚合酶链反应（ＴａｑＭａｎ－ＰＣＲ）技术在肺结构诊断中的价值。方法 对１６８例活动性肺结核、５７例肺癌患者的痰和外周血及３４－例健康对照外周血,同时应用ＴａｑＭａｎ－ＰＣＲ、ＰＣＲ检测,并与痰涂片法、ＢＡＣＴＥＣ法及改良罗氏培养法结果进行比较。结果 ＴａｑＭａｎ－ＰＣＲ检测痰和外周血总的阳性率分别为５３．０％和６１．３％,显著高于ＰＣＲ、痰涂片法、ＢＡＴＣＴＥＣ法及改良罗氏培     

Evaluation of culture and PCR-based assay for detection of Mycobacterium tuberculosis from sputum collected and stored on filter paper

We evaluated the use of culture and PCR-based assay for the direct detection of Mycobacterium tuberculosis (MTB) from sputum collected and stored on filter paper at room temperature for 5 days; the results were compared with those of staining and conventional culture of fresh sputum before storage (the 'gold standard'). Out of 231 sputum specimens examined, MTB was recovered from 124 samples by culture before storage. The culture positivity rate was significantly decreased to 70% after 5 days storage. For PCR assay, a fragment of 377 bp of the IS6110 sequence was amplified and detected using three methods: first PCR product combined with agarose gel electrophoresis (AGE); first PCR product with dot blot hybridization (DBH); nested PCR with AGE. Compared with culture, the sensitivity, specificity, and efficiency for first PCR with AGE were 71.8, 100 and 84.9% respectively; PCR with DBH gave results of 89.5, 96.3 and 92.6% respectively; the same values for nested PCR were 96.0, 97.2, and 96.5% respectively. Of these three methods, nested PCR gave excellent sensitivity and specificity with no significant difference (p = 0.727) from conventional culture. The storage of sputum on filter paper and storage at room temperature for 5 days had no apparent effect on the performance of nested PCR. We propose that this collection and storage method be considered for transporting sputum specimens from peripheral health centers or from the field; specimens may be sent by post to a central point for both culture and PCR analysis by trained technicians supervised in accordance with a well-established quality control system.