• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.745-752
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• 2015

Tuberculosis (TB) is the most common mycobacterial infection in developing countries, requiring a rapid, accurate, and well-differentiated detection/diagnosis. For the rapid detection and discrimination of Mycobacterium tuberculosis complex (MTC) from non-tuberculous mycobacteria (NTM), a novel, simple, and primer-combined single-step multiplex PCR using three primer pairs (6110F-6110R, 1081F-1081R, and 23SF-23SR; annealing on each of IS6110, IS1081, and 23S rDNA targets), hereafter referred to as a triplex PCR, has been developed and evaluated. The expected product for IS6110 is 416 bp, for IS1081 is 300 bp, and for 23S rDNA is 206 bp by single PCR, which was used to verify the specificity of primers and the identity of MTC using DNA extracted from the M. tuberculosis H37Rv reference strain (ATCC, USA) and other mycobacteria other than tuberculosis (MOTT) templates. The triplex PCR assay showed 100% specificity and 96% sensitivity; the limit of detection for mycobacteria was ~100 fg; and it failed to amplify any target from DNA of MOTT (50 samples tested). Of 307 blinded clinical samples, overall 205 positive M. tuberculosis samples were detected by single PCR, 142 by conventional culture, and 90 by AFB smear methods. Remarkably, the triplex PCR could subsequently detect 55 positive M. tuberculosis from 165 culture-negative and 115 from 217 AFB smear-negative samples. The triplex PCR, targeting three regions in the M. tuberculosis genome, has proved to be an efficient tool for increasing positive detection/discrimination of this bacterium from clinical samples.

• Tuberculosis and Respiratory Diseases
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• v.59 no.6
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• pp.606-612
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• 2005

Background : It has been reported that nontuberculosis mycobacterium(NTM) isolates account for approximately 10% of patients with a positive Acid-Fast Bacilli(AFB) smear. Therefore, it is necessary to consider NTM pulmonary disease when such a positive test is encountered. The aim of this study was to evaluate the etiologies and clinical characteristics of patients with NTM pulmonary disease who had been treated at a national tuberculosis hospital. Methods : The NTM isolates were recovered from the sputum or bronchial washing specimens submitted to a clinical laboratory of National Masan TB Hospital from August 2002 to July 2003. All samples were identified using a polymerase chain reaction-restriction fragment length polymorphism analysis method, which amplifies the rpoB gene. The patients were diagnosed with NTM disease according to the American Thoracic Society diagnostic criteria. Results : One hundred NTM isolates were recovered from 57 patients. Of the 100 isolates, M. avium complex(MAC) was the most common species, which was found 55%(n=55) of patients, followed by M. abscessus(n=25), and M. fortuitum( n=9). 26(45.6%) patients had NTM disease. Twenty-six (45.6%) patients had NTM disease according to The American Thoracic Society classification. The main organisms involved in NTM disease were MAC(n=19, 73.1%) and M. abscessus(n=5, 19.2%). The pathogenic potential was 67.9% in M. intracellulare and 41.7% in M. abscessus. The predictive factors related to NTM disease were a positive sputum smear (OR 6.4, p=0.02) and the isolation of either MAC or M. abscessus(OR 6.9, p=0.007). Fifteen patients(57.7%) were cured. There were no significant factors associated with the treatment success. Conclusion : There was a relatively high proportion of NTM disease in NTM isolates and the common species were MAC and M. abscessus. The predictive factors for NTM disease were a positive sputum smear and the isolation of either MAC or M. abscessus.

• 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.47 no.6
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• pp.747-756
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• 1999

Background: Acid-fast stain and cultures for diagnosis of pulmonary tuberculosis are primary and essential method, but have their limitation : low sensitivity and time consuming. The objective of this study is comparison of amplified Mycobacterium tuberculosis direct test(MTD) by the conventional AFB smears and cultures in the detection of Mycobacterium tuberculosis in respiratory specimens. Methods: During the period between November, 1997 and May, 1998 a total of 267 respiratory specimens (sputum 173, bronchial washing 94) from 187 patients suspected pulmonary tuberculosis were subjected to AFB smears, cultures and MID test. MID is based on nucleic acid amplification. We compared the MID with 3% Ogawa culture method. In positive AFB smear and negative MID specimen, positive culture identification between nontuberculous mycobacterium and M.tuberculosis was assesed by using Accuprobe M.tuberculosis complex probe. In negative AFB smear and negative AFB culture, MTD results are assessed by clinical follow-up. Results : 1) Compared with culture in sputum and bronchial fluid specimens, sensitivity and specificity of MTD in positive AFB smear is 79.7% and 20.0%, sensitivity and specificity of MTD in negative AFB smear specimens is 75.0% and 79.7%. 2) Discrepant analysis is assessed by clinical follow-up and other specimen results beyond study. Culture negative but MTD positive specimens were proved to be true positive and gave MTD sensitivity 79.2%, specificity of 84.4%, positive predictive value 80.5% and negative predictive value 83.2%. 3) 14 out of 31 specimens in negative AFB smear, negative AFB culture and positive MTD showed pulmonary tuberculosis diagnosed on clinical follow-up and sensitivity is 45.2%. 4) 2 out of 13 specimens in positive AFB smear, positive AFB culture and negative MID diagnosed as non tuberculous mycobacterium by Accuprobe culture. Conclusion: This study suggested that MID in respiratory specimens is simple and rapid diagnostic method, but considered adjuvant method rather than replace the conventional AFB smear and culture.

• Tuberculosis and Respiratory Diseases
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• v.86 no.1
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• pp.47-56
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• 2023

Background: There is a global increase in isolation of nontuberculous mycobacteria (NTM). The aim of the study was to analyze longitudinal trends of NTM identification and pattern of antimicrobial susceptibility testing. Methods: NTM recovery rates, distribution of NTM species identification, and antimicrobial susceptibility pattern of NTM at Pusan National University Yangsan Hospital between January 2016 and December 2020 were retrospectively analyzed. Results: A total of 52,456 specimens from 21,264 patients were submitted for mycobacterial culture, of which 2,521 from 1,410 patients were NTM positive over five years (January 2016 to December 2020). NTM isolation showed an increasing trend from 2016 to 2020 (p<0.001, test for trend) mainly caused by Mycobacterium avium complex. The vast majority of M. avium complex were susceptible to key agents clarithromycin and amikacin. For Mycobacterium kansasii, resistance to rifampin and clarithromycin is rare. Amikacin was the most effective drug against Mycobacterium abscessus subspecies abscessus and Mycobacterium subspecies massiliense. Most of M. subspecies massiliense were susceptible to clarithromycin, while the majority of M. abscessus subspecies abscessus were resistant to clarithromycin (p<0.001). Conclusion: There was an increasing trend of NTM isolation in our hospital. Resistance to key drugs was uncommon for most NTM species except for M. abscessus subspecies abscessus against clarithromycin.

• Tuberculosis and Respiratory Diseases
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• v.57 no.2
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• pp.125-131
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• 2004

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate BCG vaccine is urgently needed. We constructed three recombinant Mycobacterium bovis BCG (rBCG) strains over-expressing antigen (Ag) 85A, Ag85B, or both of M. tuberculosis using their own promoter and secretory sequence, or hsp60 promoter. SDS-PAGE analysis of rBCG proteins showed overexpression of Ag85A and Ag85B proteins in higher level than of those in their parental strain of BCG. In addition, rBCG(rBCG/B.FA) over-expressing Ag85A and Ag85B induced strong IFN-${\gamma}$ production in splenocytes. However, there was no significant difference in protective efficacy between rBCG and their parental BCG strain. In this study, therefore, rBCG over-expressing Ag85A, Ag85B, or both failed to show enhanced protection against M. tuberculosis infection in a mouse model.

• 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.

• The Journal of the Korean Society for Microbiology
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• v.34 no.5
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• pp.491-500
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• 1999

A recent study showed that comparative sequence analysis of rpoB DNAs could reveal natural relationships in genus Mycobacterium [J Clin Microbial. 37 (6). 1999]. rpoB DNAs showed interspecies variation and intraspecies conservation. Based on these data, we developed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) protocols which enable species differentiation in genus Mycobacterium. When this assay was applied to 24 clinical isolates identified as M. avium complex (MAC) by biochemical test, these were successfully differentiated into M. avium and M. intracellulare. These results were concordant with those obtained by 16s rDNA analysis. It is the first report that PCR-SSCP analysis of rpoB DNA could be used for species differentiation of MAC strains.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1259-1264
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• 2009

We assessed the capacity of two liquid-medium culture methods with automated incubation and reading systems (MB/BacT ALERT 3D System and BACTEC MGIT 960 System) and one solid-medium culture method ($L\ddot{o}wenstein$-Jensen) to detect mycobacteria in different types of clinical samples. Out of 1,770 cultured clinical samples (1,519 of respiratory origin and 251 of non respiratory origin), mycobacteria were isolated in 156 samples (135 M. tuberculosis complex, 8 M. chelonae, 6 M. kansasii, 4 M. fortuitum, 2 M. gordonae, and 1 M. marinum) by at least one of the methods used. The BACTEC MGIT 960 System proved to be the most sensitive method (86.5%), especially in the detection of M. tuberculosis complex (89.1%). However, $L\ddot{o}wenstein$-Jensen culture was the most sensitive (76.2%) to detect nontuberculous mycobacteria. The BACTEC MGIT 960 System showed the lowest mean detection time for mycobacterial growth (15.3 days), significantly shorter than the other two methods. Highest sensitivity (95.5%) and specificity (99.6%) values were obtained using the BACTEC MGIT 960 System with the $L\ddot{o}wenstein$-Jensen culture method, which was also the only combination capable of detecting 100% of the nontuberculous mycobacteria.