Diagnostic Performance of the GENEDIA MTB/NTM Detection Kit for Detecting Mycobacterium tuberculosis and Nontuberculous Mycobacteria With Sputum Specimens

The GENEDIA MTB/NTM Detection Kit (GENEDIA MTB/NTM; Green Cross Medical Science Corp., Chungbuk, Korea) is a multiplex real-time PCR assay used for differential identification of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). While the importance of differential identification of MTB/NTM is recognized, there is limited data on the performance of GENEDIA MTB/NTM assay to date. A total of 687 consecutive sputum specimens were cultured and analyzed with the GENEDIA MTB/NTM and GENEDIA MTB assays. Nineteen specimens (2.8%) were MTBC-positive, and 69 (10.0%) were NTM-positive based on mycobacterial culture. All specimens showed concordant results for MTBC using both assays, with a kappa value of 1.00, overall sensitivity of 63.2% (12/19), and specificity of 100% (668/668). The overall NTM sensitivity and specificity were 23.2% (16/69) and 99.7% (616/618) for GENEDIA MTB/NTM. The association between NTM-positivity using GENEDIA MTB/NTM and the diagnosis of NTM pulmonary disease was not statistically significant. In conclusion, the two real-time PCR assays showed similar diagnostic performance for MTBC detection. However, the sensitivity for NTM detection was lower than that for MTBC detection.

Comparison of Histological, Microbiological, and Molecular Methods in Diagnosis of Patients with TBLN Having Different Anti-TB Treatment Background / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>The influence of anti-tuberculosis (TB) treatment history on tuberculous lymphadenitis (TBLN) diagnosis is unclear. Therefore, this study aims to evaluate the diagnostic methods, including histology, microbiology, and molecular tests, used for TBLN.</p><p><b>METHODS</b>In this study, suspected patients with TBLN and having different anti-TB treatment background were enrolled. All the samples were tested simultaneously by histology, Ziehl-Neelsen (ZN) staining, mycobacterial culture (culture), Xpert MTB/RIF (xpert), real-time PCR, and high-resolution melting curve PCR (HRM). Thereafter, the performance of these methods on samples with different anti-TB treatment background was assessed.</p><p><b>RESULTS</b>In our study, 89 patients were prospectively included 82 patients with TBLN and 7 with other diseases. The overall sensitivities of Xpert, real-time PCR, histology, ZN staining, and culture were 86.6%, 69.5%, 58.5%, 43.9%, and 22.0%, respectively. The anti-TB treatment history revealed dramatic influences on the sensitivity of culture (P < 0.0001). In fact, the treatment that lasted over 3 months also influenced the sensitivity of Xpert (P < 0.05). However, the treatment history did not affect the performance of remaining tests (P > 0.05). For rifampicin drug susceptibility test (DST), the anti-TB treatment showed only significant influence on the success rate of culture DST (P = 0.001), but not on those of Xpert and HRM tests (P > 0.05).</p><p><b>CONCLUSION</b>Other tests as well as culture should be considered for patients with TBLN having retreatment history or over 1-month treatment to avoid false negative results.</p>

Tuberculosis Verrucosa Cutis Confirmed by Culture / 대한피부과학회지

Tuberculosis verrucosa cutis is a warty form of cutaneous tuberculosis. It is a paucibacillary disorder caused by external reinfection of mycobacteria into the skin of previously sensitized individuals with moderate to strong cell-mediated immunity. Inoculation arises at sites of minor wounds, or rarely from the patient's sputum. Tuberculosis verrucosa cutis begins as a small papule and grows slowly by peripheral expansion, sometimes reaching a size of several centimeters or more in diameter. For definitive diagnosis of cutaneous tuberculosis, the demonstration of Mycobacterium tuberculosis is essential. However, the laboratory diagnosis of paucibacillary cutaneous tuberculosis is very difficult owing to the poor sensitivity of routine available methods. Herein, we report two cases of tuberculosis verrucosa cutis definitively confirmed by mycobacterial culture in Korean patients who had received bacille Calmette-Guerin vaccination earlier in life.

Does Polymerase Chain Reaction of Tissue Specimens Aid in the Diagnosis of Tuberculosis?

BACKGROUND: Mycobacterial culture is the gold standard test for diagnosing tuberculosis (TB), but it is time-consuming. Polymerase chain reaction (PCR) is a highly sensitive and specific method that can reduce the time required for diagnosis. The diagnostic efficacy of PCR differs, so this study determined the actual sensitivity of TB-PCR in tissue specimens. METHODS: We retrospectively reviewed 574 cases. The results of the nested PCR of the IS6110 gene, mycobacterial culture, TB-specific antigen-induced interferon-γ release assay (IGRA), acid-fast bacilli (AFB) staining, and histological findings were evaluated. RESULTS: The positivity rates were 17.6% for PCR, 3.3% for the AFB stain, 22.2% for mycobacterial culture, and 55.4% for IGRA. PCR had a low sensitivity (51.1%) and a high specificity (86.3%) based on the culture results of other studies. The sensitivity was higher (65.5%) in cases with necrotizing granuloma but showed the highest sensitivity (66.7%) in those with necrosis only. The concordance rate between the methods indicated that PCR was the best method compared to mycobacterial culture, and the concordance rate increased for the methods using positive result for PCR or histologic features. CONCLUSIONS: PCR of tissue specimens is a good alternative to detect tuberculosis, but it may not be as sensitive as previously suggested. Its reliability may also be influenced by some histological features. Our data showed a higher sensitivity when specimens contained necrosis, which indicated that only specimens with necrosis should be used for PCR to detect tuberculosis.

Annual Report on External Quality Assessment Scheme for Clinical Microbiology in Korea (2014)

Annual proficiency surveys were performed in March, June and September 2014 by clinical microbiology division of The Korean Association of Quality Assurance for Clinical Laboratory. Parasitology part has been newly incorporated in this survey. For each trial, three sets which were composed of different combinations of five bacteria and yeast were distributed for gram stain, culture, identification, and antimicrobial susceptibility tests of general bacteriology and five fixed sputum smear on slides were distributed for acid fast bacilli stain. Two advanced bacteriology survey materials for culture and identification of anaerobic bacteria and mold were distributed to the voluntary participants in every trial and five mycobacterial culture and identification specimens, five anti-tuberculosis susceptibility testing specimens, and two Mycobacterium tuberculosis strains for rapid detection of rifampin and isoniazid resistance were distributed to the voluntary participants in March and June trials. Five virtual microscopic slides for stool parasite examination were open for the registered participants in June trial. A total of 340 laboratories were enrolled and 330 (97.0%), 331 (97.4%), and 331 (97.4%) returned the results on trial I, II, and III, respectively. For bacterial identification, the percent acceptable identification of Burkholderia cepacia, Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pneumoniae, Streptococcus agalactiae, Plesiomonas shigelloides, and Enterococcus faecalis were greater than 95%. Group C and group D Salmonella species challenged as the different sets of M1422 resulted in the acceptable rate lower than 95% because nine participants reported the identification of different sets. Surveillance cultures for methicillin-resistant S. aureus and vancomycin-resistant enterococci were correctly determined by 89.6% and 69.0% of the respondents, respectively. Correct identification to species level of Candida albicans, Candida auris, Candida glabrata, and Candida parapsilosis were 86.1%, 1.6%, 48.1%, and 83.8%. Vancomycin disk diffusion test in S. aureus, missing oxacillin screen or penicillin susceptibility test in S. pneumoniae and lack of reliable methods of quinolone resistance detection in Salmonella species caused unacceptable results in antimicrobial susceptibility testing. Advanced bacteriology trials revealed low performance in species identification of mold. Mycobacterial culture, identification and susceptibility test performance was kept in excellence. The performance of identification of stool parasites was acceptable >90% for detection of helminth eggs and amebic cysts but 28.6% false positive responses resulted from negative specimens. In conclusion, species-level identification of fungi of both candida species and mold were challenging to clinical microbiology laboratories. Vancomycin disk diffusion method for S. aureus and lack of proper penicillin susceptibility test for S. pneumoniae were still common cause of inaccurate results. Virtual microscopic survey has been successfully introduced in parasitology.

Early Detection of Mycobacteria Using a Novel Hydrogel Culture Method

BACKGROUND: Early laboratory detection of Mycobacterium tuberculosis is crucial for controlling tuberculosis. We developed a hydrogel mycobacterial culture method that retains the advantages of both solid and liquid methods in terms of speed, cost, and efficiency. METHODS: Mycobacterium bovis bacillus Calmette-Guerin (BCG) suspensions and 200 acid-fast bacilli (AFB)-positive clinical specimens were inoculated in Middlebrook 7H9 liquid media (Becton-Dickinson and Company, USA) and mixed with 75 microL of 9-fluorenylmethoxycarbonyl (Fmoc)-Phe-Phe-OH hydrogel stock solution in an Eppendorf tube just before culture incubation. The mixtures were cultured at 37degrees C for as long as 14 days to monitor culture status. RESULTS: The number of M. bovis BCG increased with time. For 200 AFB smear-positive specimens, 155 of 158 conventional culture-positive specimens and 4 culture-negative or contaminated specimens yielded positive cultures within 14 days. For 128 specimens positive with the liquid culture method, the time to positive culture using the hydrogel method (mean, 12.6 days; range, 7 to 14 days) was significantly shorter than that for conventional liquid culture (mean, 16.2 days; range, 6 to 31 days; P<0.0001). CONCLUSIONS: The hydrogel scaffold culture system is useful for timely, economical, and efficient detection of mycobacteria in clinical specimens.

Survey on Changes in Mycobacterial Testing Practicies in Korean Laboratories / 대한임상미생물학회지

BACKGROUND: Since 1997 in which first survey for mycobacterial practices of hospitals in Korea were carried on, changes of practice and concept in mycobacterial testing have been expected because advanced testing methods have been surged for last five-year-period. We purposed to follow-up survey to monitor practices changes, and in addition, situation of quality control. METHODS: Questionnaires was composed of items including mycobacterial test methods, test volume, turnaround time (TAT), and quality control measure. It was sent to 90 laboratories of general hospitals, tuberculosis specialty hospitals and commercial laboratories in April 2001. RESULTS: Sixty-seven (74%) of 90 laboratories replied to this survey. Five of 67 laboratories (7%) were using fluorochrome method for AFB stains. In five among 58 laboratories that performed AFB cultures (8%), liquid media have been used. Mycobacterial species was identified by molecular biologic methods or high-performance liquid chromatography in 18 laboratories (34%). Average TAT of culture and identification for Mycobacterium tuberculosis was 11 days at the laboratory using Mycobacteria Growth Indicator Tube (MGIT) 9600 system and PCR method, while that at the laboratory using Ogawa media and biochemical method was 35.8 days. TATs of susceptibility tests undertaken at three laboratories were 28-60 days. Only six laboratories were joining external quality control program, even though most laboratories wanted to participate in. CONCLUSIONS: TATs for mycobacterial culture and susceptibility tests were too long to have the laboratories take a pivotal role to control tuberculosis. To improve Korean mycobacterial laboratories, it is necessary that the national health insurance system supports the newer rapid methods for mycobacterial tests and nationwide external quality control program is introduced.

A Prospective Study of Ziehl-Neelsen Stain and Mycobacterial Culture of Pleural Fluid and Closed Pleural Biopsy Specimen in Tuberculous Pleural Effusion / 감염

BACKGROUND: Tuberculous pleural effusion is the most common extrapulmonary site of all disease due to Mycobacterium tuberculosis. The diagnosis of tuberculous pleural effusion is most often established by histologic examination of pleural biopsy specimens. This study documents the utility of smear and culture of pleural fluid and pleural biopsy specimens for tubercle bacilli. METHODS: Between March 1998 and August 1999, we performed thoracentesis with or without pleural bio-psies on 148 patients with pleural effusion according to protocol with Abrams needle. Before the pleural biopsy, a diagnostic thoracentesis was performed. Aliquots of pleural fluid (30 mL) were submitted for biochemical, cytologic, and bacteriologic studies, Ziehl-Neelsen staining and culture in Lowenstein-Jensen medium. At least five samples of parietal pleural tissue were obtained, one for mycobacterial study and another for histologic study. RESULTS: Thirty-seven of the 148 patients were proved to have tuberculosis (24 men and 13 women) with a median age of 32 years (range, 21~91). Pleural biopsy was performed on 35 of the 37 patients with tuberculous pleural effusion. Granuloma was present in 33 of the 35 patients investigated with acid-fast bacilli in 9 patients. The smear for acid-fast bacilli of pleural fluid was positive in 1 patient and the culture for M. tuberculosis was positive in 5 of 37 patients. Pleural biopsy culture was positive in 3 of 35 patients. The 2 patients who could not carry out the pleural biopsy were positive in pleural fluid and pleural tissue mycobacterial culture, respectively. CONCLUSION: In our test, Ziehl-Neelsen staining and culture for M. tuberculosis of pleural fluid and pleural specimen gave a higher yield (5.4%) than the histologic methods alone in establishing the diagnosis of tuberculous pleural effusion.