• Tuberculosis and Respiratory Diseases
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• v.53 no.2
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• pp.161-172
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• 2002

Background : To Investigate the association between bronchial anthracofibrosis (AF) and tuberculosis (TB), and the clinical utility of a polymerase chain reaction (PCR) on bronchial specimens for rapid diagno-sis of active pulmonary TB in patients with bronchial AF. Method : Thirty patients (25 women and 5 men ranging in age from 53 to 88), who were diagnosed with bronchial AF by a bronchoscopic exami-nation, were enrolled in this study. PCR targeting the IS6110 segment of Mycobacterium tuberculosis was performed on the bronchial wash fluid and anthracofibrotic bronchial tissue. The PCR results were compared with the bacteriological, histological, and clinical findings. Results : Eighteen of the 30 patients (60%) were associated with TB, nine of whom were confirmed as having active TB. The remaining 9 had a past history of TB. The sputum or bronchial aspirate AFB smear, culture, and histological findings were positive in 4 (13%), 9 (30%), and 5 (17%) patients, respectively. PCR of the AF tissue and bronchial wash fluid was positive in 5 (17%) and 11 (37%) of the 30 patients, respectively. PCR was more sensitive than the AFB smears for diagnosing pulmonary TB (22 % us 89 %, respectively, p<0.05). All 5 patients with positive AF tissue PCR results also had both histological findings and positive bronchial wash fluid PCR results. Of the 3 patients with positive PCR but negative bacteriological or histological results, 2 of these patients appeared to have active tuberculosis on a clinical basis. Conclusion: Although TB-PCR did not reveal an increased association between bronchial AF and TB compared with traditional methods, PCR on the bronchial wash fluid appears to be useful for the rapid diagnosis of pulmonary TB in patients with bronchial AF. TB-PCR on AF bronchial tissue itself did not yield additional benefits for diagnosing TB, which suggests that an AF lesion itself may not be an active or original site of the infection, but a secondary change of TB.

• Korean Journal of Veterinary Service
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• v.34 no.4
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• pp.333-339
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• 2011

Loop-mediated isothermal amplification (LAMP) was developed to detect Mycobacterium tuberculosis complex (MTC) and non-tuberculous mycobacterium (NTM) genomic DNA in blood samples of Korea native cattle. A set of four primers, two outer and two inner, were designed from M. bovis and M. avium genomic DNA targeting the IS6110 and 16S rRNA gene, respectively. Based on 85 Intradermal Tuberculin Test (ITT) positive blood sample and using conventional PCR and LAMP, the agreement quotient (kappa), which measures agreement beyond chance were 0.93 (conventional PCR) and 0.97 (LAMP), respectively. The detection limit of the LAMP method was $2.0{\times}10^2$ copy/ml M. bovis and M. avium cells, compared to $2.0{\times}10^3$ copy/ml M. bovis and M. avium cells for conventional PCR. These results suggest that the LAMP is a powerful tool for rapid, sensitive, and practical detection of MTC and NTM in blood samples of Korea native cattle.

• Korean Journal of Clinical Laboratory Science
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• v.47 no.2
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• pp.83-89
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• 2015

Although Mycobacterium tuberculosis complex strains remain responsible for the majority of diseases caused by mycobacterial infections worldwide, the increase in HIV (human immuno deficiency virus) infections has allowed for the emergence of other non-tuberculous mycobacteria as clinically significant pathogens. M. tuberculosis was detected by two-tube nested polymerase chain reaction (PCR) and non-tuberculous mycobacteria was detected by PCR-restriction fragment length polymorphism (RFLP) with Msp I. Result of niacin test is equal to result of two-tube nested PCR after culture for M. tuberculosis. In this study, acid fast bacilli stain (AFB. stain) >2+ case, Detection of Mycobacteria is similar to result before culture and after culture. AFB. stain <1+ case, result of mycobacteria is distinguished. Conclusionly, these results suggest that identification of mycobacteria must go side by side both culture and PCR for more fast and accuracy.

• Tuberculosis and Respiratory Diseases
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• v.42 no.1
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• pp.35-41
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• 1995

Background: Tuberculous cervical lymphadenitis can be diagnosed by clinical findings, chest X-ray, Mantoux test, but confirmed only by excisional biopsy. The polymerase chain reaction(PCR) is now widely applied to test very small amount of pathogen and would be used to detect Mycobacterium tuberculosis in biopsied tissues and fine needle aspirates. Method: We carried out the PCR using IS-1 and IS-2 primers in 16 samples from tuberculous cervical lymphadenitis patients, and 13 samples from non-tuberculous cervical lymphadenopathy patients. Acid fast staining and culture for Mycobacterium were all negative. Results: All of 8 pathologically confirmed tuberculous cervical lymphadenitis samples showed positive PCR results, and of 5/8 clinically diagnosed samples were positive. None of 6 pathologically excluded samples were positive, and among 7 clinically undiagnosed samples 2 showed positive PCR results. Conclusion: In patients with suspected tuberculous cervical lymphadenitis, PCR could be used to detect Mycobacterium tuberculosis using biopsied tissues and even fine needle aspirates with good sensitivity and specificity.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1001-1010
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• 1997

Background : PCR technique is useful in diagnosis of pulmonary tuberculosis. But, its sensitivity and specificity is some different among several studies. Our aim is compare our PCR results with other's previous PCR results in AFB smear negative patients. Methods : PCR were performed in patients that their disease were suspected as active pulmonary tuberculosis and that their initial serial sputum AFB smear results were negative. Total number of patients studied by PCR technique was 177. Also, we analyzed the data only in patients whose bronchial washing fluid AFB smear was negative. And the primer had been used was IS 6110. Results : In our retrograde study, the number of patients who are diagnosed as having active pulmonary tuberculosis, inactive pulmonary tuberculosis and nontuberculous pulmonary disease was 99, 28, 50, respectively. In the sputum study, the sensitivity of PCR is 41.5% (27 PCR positive cases/65 active TBc cases). And the sensitivity of TB culture is 53.8% (35 TB culture positive cases/65 active TBc cases). In the bronchial washing specimen study, the sensitivity of PCR is 53.8% (21 PCR positive cases/39 active TBc cases). And the sensitivity of TB culture is 43.6% (17 TB culture Positive cases/39 active TBc cases). The specificity of PCR in our study is 94.9%. (74 PCR negative cases/78 inactive TBc or nontubereulous cases) In the cases of patients who were never takened anti-TBc medication, the sensitivity of PCR (45.6%--25 positive cases/55 cases) is some lower than culture (58.2%--32 positive cases/55 cases). In the cases of patients who had been takened anti-TBC medication. the sensitivity of PCR (60%--18 positive cases/30 cases) is some superior than culture (50%--15 positive cases/30 cases). Conclusion : We think that PCR results in cases of sputum AFB smear negative patients is nearly same as culture. And PCR is especially useful in patients who had been takened anti-TBc medication on admission.

• Tuberculosis and Respiratory Diseases
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• v.40 no.1
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• pp.43-51
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• 1993

Background: The polymerase chain reaction (PCR) is a very sensitive method for the detecting of mycobacterial DNA. There are many reports revealing the efficacy of PCR for the diagnosis of M. tuberculosis, but there are many different methods for DNA extraction from Mycobacterium tuberculosis. Bead beater method is a very useful method for DNA extraction from clinical spectimens, but its procedures are relatively complicated and time-consuming. So we studied other methods for the DNA extraction from Mycobacterium tuberculosis $H_{37}Rv$ and some clinical specimens (5 smear positive sputa and 5 smear negative CSF). Method: We extracted the mycobacterial DNA with 6 different methods from H37Rv strain and clinical specimens. The methods included SDS-microwave oven method, NaOH lysis method, Triton X-100-Proteinase K method, Lysis buffer method, SDS-proteinase K method and bead beater method. The target DNA was 123bp of IS6110 and was detected by examination of ethidium bromide-stained agarose gels. Results: Among 6 methods, SDS-proteinase K method, bead beater method, lysis buffer method and triton X-100-proteinase K method were excellent, but SDS-proteinase K method was the best method in the aspect of simplicity and cost-effectiveness. Conclusion: We suggest that SDS-porteinase K method is a simple and convinient method and might be the best method for the extraction of mycobacterial DNA.

• Tuberculosis and Respiratory Diseases
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• v.43 no.1
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• pp.30-37
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• 1996

Background: The extraction methods of DNA from clinical samples are the major obstacle to use the PCR(polymerase Chain Reaction) in routine labortary for early detection of M. tuberculosis. We tried to improve the extraction method of DNA from sputum for establishment of the PCR in routine labortary by reducing the possibility of cross contamination and performing it easily and safely. Methods: We used the $InstaGene^{TM}$ DNA extraction kit(BioRad Co.) using Chelex 100 ion exchange resin for preparation of DNA. We compared InstaGene method in 100 cases of sputum from proteinase K method which is known as the most commonly used method for DNA purification(Experiment 1). And we compared InstaGene method in 98 cases of sputum from Microwave method developed by a company in Korea(Experiment 2). In experiment 1,245bps of IS6110 were amplified and then 188bps were amplified by nested PCR. In experiment 2,536bps in primary PCR and 276bps in nested PCR were amplified and analysed by agarose gel electrophoresis and EtBr staining. Results: When we chose AFB smear, culture, or AFB smear and culture as a standard test, PCR had low specificity and positive predictive value in both experiments. The InstaGene method has higher value in sensitivity and negative predictive value significantly than proteinase K method. The InstaGene method and the Microwave methods were similar in sensitivity, specificity, positive predictive value and negative predictive value. Conclusion: Even though both methods had lower possibility of cross contamination, shorter time requirement, simplicity, and economic advantages than Proteinase K method, the InstaGene method was a little simpler than the Microwave method. Therefore, in terms of usefulness in clinical application, the Instagene method seems to be the most useful method in DNA extraction for detection of M. tuberculosis using PCR. The reliability of this method will be clarified by further studies with enough clinical samples.

• Tuberculosis and Respiratory Diseases
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• v.50 no.2
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• pp.222-228
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• 2001

Backgrounds : Recent technological developments have introduced a new method to identifying M. tuberculosis complex DNA in clinical samples directly. The direct amplification test (DAT) is approved for identifying M. tuberculosis complex in respiratory specimens that are smear-positive for acid-fast bacilli (AFB). When there is a discrepancy between the AFB smear and DAT, no information on their clinical utility is currently available. In this study, the diagnostic reliability of DAT was investigated in suspected pulmonary tuberculosis patients whose sputum AFB smear was negative. Methods : From June 1, 1998 through May 30, 1999, 909 patients with presumed active pulmonary tuberculosis were enrolled. A sputum AFB stain, culture, DAT and/or biopsy were performed. Using the criteria of clinical tuberculosis or confirmed tuberculosis, the positive predictive value of DAT in diagnosing pulmonary tuberculosis was investigated. Results : The positive predictive value of DAT was 82.1% by the clinically active tuberculosis criteria. However, it decreased to 61.5% when diagnosis was restricted to only to culture positive or biopsy proven cases. The false positive rate of DAT was 18.0%. Conclusion : The DAT is a valuable diagnostic method in suspected patients whose sputum AFB is was negative.