Identification and characterization of nontuberculous mycobacteria isolated from suspected pulmonary tuberculosis patients in eastern china from 2009 to 2019 using an identification array system

Abstract Objective Nontuberculous mycobacteria (NTM) species are increasingly being isolated and have become a key factor affecting public health by causing pulmonary diseases. Most NTM species do not respond to conventional tuberculosis (TB) drugs. This study aimed to identify NTM isolated from suspected pulmonary TB patients from the Zhejiang province and analyze their distribution in the region. Methods A total of 1,113 NTM isolates from patients suspected to be suffering from acid-fast bacilli-positive tuberculosis were identified at the species level, using the CapitalBio Mycobacterium identification array and polymerase chain reaction amplification and sequencing of 16S-23S gene internal transcribed spacer (ITS), 16S rRNA, and hsp65. Results Of the 23,138 isolates, we identified 1,102 NTM (4.8%), mainly including Mycobacterium intracellulare (54.81%, 604/1,102), M. chelonae-M. abscessus (16.52%, 182/1,102), M. avium (13.16%, 145/1,102), M. kansasii (8.17%, 90/1,102), and M. gordonae (3.27%, 36/1,102). Conclusion The distribution of NTM species observed in patients with suspected pulmonary tuberculosis provides guidance for the diagnosis and treatment of NTM pulmonary diseases.

A prospective study of non-tuberculous mycobacterial disease among tuberculosis suspects at a tertiary care centre in north India

Background & objectives: The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide. The disease shares clinicoradiological features with tuberculosis (TB), Nocardia and several fungal diseases, and its diagnosis is frequently delayed. The present study was performed to determine the frequency of NTM disease among TB suspects in a tertiary care centre in north India. Methods: In this prospective study, mycobacterial culture isolates from pulmonary and extrapulmonary specimens among TB suspects were tested with immunochromatographic assay (ICA). All ICA-negative isolates were considered as NTM suspects and further subjected to 16S-23S rRNA internal transcribed spacer gene sequencing for confirmation and species identification. Patients with active disease were treated with drug regimen as per the identified NTM species. Follow up of patients was done to determine clinical, radiological and microbiological outcomes. Results: Of the 5409 TB suspects, 42 (0.77%) were diagnosed with NTM disease. Patients with active disease consenting for treatment were treated and followed up. Thirty four patients had NTM pulmonary disease (NTM-PD) and the remaining eight had extrapulmonary NTM (EP-NTM) disease. Mycobacterium intracellulare and M. abscessus, respectively, were most frequently isolated from NTM-PD and EP-NTM patients. Fifteen NTM-PD and seven EP-NTM patients successfully completed the treatment. Ten patients died due to unrelated causes, five were lost to follow up and another four declined the treatment. Interpretation & conclusions: Our study showed that the frequency of NTM disease was low among TB suspects at a large tertiary care centre in north India and this finding was similar to other Indian studies. More studies need to be done in other parts of the country to know the geographical variation in NTM disease, if any.

Characteristic profile of antibody responses to PPD, ESAT-6, and CFP-10 of Mycobacterium tuberculosis in pulmonary tuberculosis suspected cases in Surabaya, Indonesia

Abstract Accurate and rapid diagnostic tools are important aspects of managing tuberculosis (TB) cases appropriately. However, the sensitivity and specificity of diagnostic kits based on immune response such as the tuberculin skin test (TST) and interferon gamma release assay (IGRA) are still debated. Thus, the exploration and assessment of specific biomarker-targeted antibodies are needed for the development of an accurate and rapid diagnostic tool. The present study was conducted in patients with a respiratory problem suspected to be TB at Dr. Soetomo Hospital, Surabaya, Indonesia. Among 102 patients tested by GeneXpert and AFB, 59 serum samples were from cases retrospectively determined to have active TB. A total of 102 serum of healthy controls (HC) was also collected. The PPD antigen and the recombinant CFP-10 and ESAT-6 proteins were prepared. Antibody responses against these proteins were evaluated by ELISA. All samples were also screened for the possibility of Mycobacterium avium-intracellulare complex (MAC) infection using Capilla MaC kit. The results showed that TB patients had a significantly higher concentration of IgG antibody in response to PPD than the HC. In addition, the receiver operating characteristic (ROC) curve analysis showed that PPD was acceptable for diagnostic purposes with an AUC value of 0.835 (95% CI 0.770-0.900, p < 0.0001). However, ESAT-6 and CFP-10 had low AUCs, and 32 samples from both groups showed a low concentration of IgA antibody against all antigens. The MAC detection results also showed that the concentration of IgA in the HC group was the highest. The current results indicate that PPD is a better antigen for antibody-based detection of TB than ESAT-6 and CFP-10. Based on the MAC detection assay, 53 people in the HC group were probably infected with rapidly growing nontuberculous mycobacteria (NTM), although antibody response to PPD was low.

Survey of Drug Prescription Patterns Among Patients with Nontuberculous Mycobacterial Disease Using the Database of Health Insurance Claims / 薬剤疫学

In recent years, the number of patients with non-tuberculosis mycobacteria (NTM) has rapidly increasing.According to the nationwide survey conducted in 2014, the number of patients with NTM was reported to increase 9.7 times compared to the survey in 1980. Among them, the patients with Mycobacterium avium complex (MAC) account for about 88.8% of them. It is the main cause of the rapid increase of NTM patients mainly in middle-aged and elderly woman. To treat patients with MAC, it is common to do chemotherapy over one year after the bacteria becomes negative. Among experts of NTM, it is recommended to do chemotherapy preventing generation of resistant bacteria by using clarithromycin (CAM) and rifampicin and ethambutol (EB) in combination. Meanwhile, a monotherapy of CAM and high-dose EB administration over a long period are not currently recommended due to side effects. However, it has not been clarified so far how many such drug prescriptions had existed. Therefore, in this study, we investigated the actual drug prescription of 571 patients who were presumed to be NTM in health insurance data collected from 2015 to 2016. As a result, about 5.1% (29 cases) of CAM monotherapy and 4.4% (15 cases) of EB high-dose prescription over 3 months were observed. In general, because NTM is a case where a long-term antibiotic treatment is required, it increases the possibility of any disadvantages exerting on patients. Hence, we consider it is an important and urgent matter to inform the correct information widely to clinical workers and sites.

Use of green fluorescent protein labeled non-tuberculous mycobacteria to evaluate the activity quaternary ammonium compound disinfectants and antibiotics

Abstract Although infections with NonTuberculous Mycobacteria have become less common in AIDS patients, they are important opportunistic infections after surgical procedures, likely because they are ubiquitous and not efficiently killed by many commonly used disinfectants. In Venezuela there have recently been many non-tuberculous mycobacteria soft tissue infections after minor surgical procedures, some apparently related to the use of a commercial disinfectant based on a Quaternary Ammonium Compound. We studied the activity of this and other quaternary ammonium compounds on different non-tuberculous mycobacteria by transforming the mycobacteria with a dnaA-gfp fusion and then monitoring fluorescence to gauge the capacity of different quaternary ammonium compounds to inhibit bacterial growth. The minimum inhibitory concentration varied for the different quaternary ammonium compounds, but M. chelonae and M. abscessus were consistently more resistant than M. smegmatis, and M. terrae more resistant than M. bovis BCG.

Pulmonary Non-tuberculous Mycobacterial Infections in Category II Failures from National Tuberculosis Programme.

Pulmonary infections due to non-tuberculous mycobacteria (NTM) are increasingly being reported. These can mimic drugresitant tubercuolosis. A diagnosis of NTM infections needs a high degree of clinical suspicion and repeated isolation of the organism on culture. NTM infections occur commonly in immunocompromised individuals and in people with lung abnormalities. Currently there are no guidelines on drug combinations and the duration of treatment is not adequately defined. Two cases of pulmonary infection with NTM in immune-competent individuals are described in the present report. Although the bacteriological, radiological and clinical response to treatment was good; early discontinuation of treatment resulted in recurrence and change in drug susceptibility pattern, suggesting the need for prolonged treatment for achieving cure.

Characterization of mycobacterium isolates from pulmomary tuberculosis suspected cases visiting Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, Addis Ababa Ethiopia: a cross sectional study

OBJECTIVE@#To characterize mycobacterium isolates from pulmomary tuberculosis suspected cases visiting National Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, for diagnosis of pulmonary tuberculosis from January 4 to February 22, 2010 with total samples of 263.@*METHODS@#Sputum specimens were collected and processed; the deposits were cultured. For culturing Lowenstein Jensen medium (LJ) and Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) were used. Capilia Neo was used for detecting NTM isolates from isolates of BACTEC MGIT 960. In Armauer Hansen Research Institute, Addis Ababa Ethiopia, Deletion typing PCR method for species identification (from confirmed Mycobacterium tuberculosis complex (MTBC) isolates by Capilia Neo) was done.@*RESULTS@#Out of 263 enrolled in the study, 124 and 117 of them were positive for mycobacterium growth by BACTEC MGIT 960 and LJ culture method, respectively. From BACTEC MGIT 960 positive media of 124 isolates, 117 were randomly taken to perform Capilia TB Neo test. From these 7 (6%) of them were found to be NTM and 110 (94%) were MTBC. From these 110 MTBC isolates, 81 of them were randomly taken and run by the deletion typing RD9 PCR method of molecular technique. Out of these 78 (96.3%) were found to be species of Mycobacterium tuberculosis and 3 (3.7%) were found to be not in the MTBC. Regarding the types of methods of culture media, Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) method was found to have excellent agreement (with kappa value of 0.78) with the routine method of LJ.@*CONCLUSIONS@#Pulmonary tuberculosis suspected cases visiting the National Tuberculosis Reference Laboratory at EHNRI that were confirmed to be pulmonary tuberculosis are caused by the species of Mycobacterium tuberculosis, hence treatment regimen including pyrazinamide can be applied to the patients as the first choice in the study area in Addis Ababa, Ethiopia. There is indication of the presence of NTM in patients visiting the tuberculosis reference laboratory and this is important because NTM is known to cause pulmonary disease similar with sign and symptom of pulmonary tuberculosis but different in treatment. BACTEC MGIT 960 has excellent agreement with LJ media but it has high tendency of having high contamination rate unless a better decontamination method is designed.

Characterization of mycobacterium isolates from pulmomary tuberculosis suspected cases visiting Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, Addis Ababa Ethiopia: A cross sectional study

Objective: To characterize mycobacterium isolates from pulmomary tuberculosis suspected cases visiting National Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, for diagnosis of pulmonary tuberculosis from January 4 to February 22, 2010 with total samples of 263. Methods: Sputum specimens were collected and processed; the deposits were cultured. For culturing Lowenstein Jensen medium (LJ) and Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) were used. Capilia Neo was used for detecting NTM isolates from isolates of BACTEC MGIT 960. In Armauer Hansen Research Institute, Addis Ababa Ethiopia, Deletion typing PCR method for species identification (from confirmed Mycobacterium tuberculosis complex (MTBC) isolates by Capilia Neo) was done. Results: Out of 263 enrolled in the study, 124 and 117 of them were positive for mycobacterium growth by BACTEC MGIT 960 and LJ culture method, respectively. From BACTEC MGIT 960 positive media of 124 isolates, 117 were randomly taken to perform Capilia TB Neo test. From these 7 (6%) of them were found to be NTM and 110 (94%) were MTBC. From these 110 MTBC isolates, 81 of them were randomly taken and run by the deletion typing RD9 PCR method of molecular technique. Out of these 78 (96.3%) were found to be species of Mycobacterium tuberculosis and 3 (3.7%) were found to be not in the MTBC. Regarding the types of methods of culture media, Mycobacteria Growth Indicator Tube (BACTEC MGIT 960) method was found to have excellent agreement (with kappa value of 0.78) with the routine method of LJ. Conclusions: Pulmonary tuberculosis suspected cases visiting the National Tuberculosis Reference Laboratory at EHNRI that were confirmed to be pulmonary tuberculosis are caused by the species of Mycobacterium tuberculosis, hence treatment regimen including pyrazinamide can be applied to the patients as the first choice in the study area in Addis Ababa, Ethiopia. There is indication of the presence of NTM in patients visiting the tuberculosis reference laboratory and this is important because NTM is known to cause pulmonary disease similar with sign and symptom of pulmonary tuberculosis but different in treatment. BACTEC MGIT 960 has excellent agreement with LJ media but it has high tendency of having high contamination rate unless a better decontamination method is designed.

Characterization of mycobacterium isolates from pulmomary tuberculosis suspected cases visiting Tuberculosis Reference Laboratory at Ethiopian Health and Nutrition Research Institute, Addis Ababa Ethiopia：a cross sectional study

Objective:To characterize mycobacterium isolates from pulmomary tuberculosis suspected cases visitingNationalTuberculosisReferenceLaboratory atEthiopianHealth andNutritionResearch Institute, for diagnosis of pulmonary tuberculosis fromJanuary4 toFebruary22,2010 with total samples of263.Methods:Sputum specimens were collected and processed; the deposits were cultured.For culturingLowensteinJensen medium(LJ) andMycobacteriaGrowthIndicatorTube (BACTECMGIT960) were used.CapiliaNeo was used for detectingNTM isolates from isolates of BACTECMGIT960.InArmauerHansenResearchInstitute,AddisAbabaEthiopia,Deletion typing PCR method for species identification(from confirmedMycobacterium tuberculosis complex (MTBC) isolates byCapiliaNeo) was done.Results:Out of263 enrolled in the study,124 and117 of them were positive for mycobacterium growth byBACTECMGIT960 andLJ culture method, respectively.FromBACTECMGIT960 positive media of124 isolates,117 were randomly taken to performCapiliaTBNeo test.From these7(6%) of them were found to beNTM and110(94%) were MTBC.From these110MTBC isolates,81 of them were randomly taken and run by the deletion typingRD9PCR method of molecular technique.Out of these78(96.3%) were found to be species ofMycobacterium tuberculosis and3(3.7%) were found to be not in theMTBC.Regarding the types of methods of culture media,MycobacteriaGrowthIndicatorTube(BACTECMGIT960) method was found to have excellent agreement(with kappa value of0.78) with the routine method of LJ.Conclusions:Pulmonary tuberculosis suspected cases visiting theNationalTuberculosis ReferenceLaboratory atEHNRI that were confirmed to be pulmonary tuberculosis are caused by the species ofMycobacterium tuberculosis, hence treatment regimen including pyrazinamide can be applied to the patients as the first choice in the study area inAddisAbaba,Ethiopia.There is indication of the presence ofNTM in patients visiting the tuberculosis reference laboratory and this is important becauseNTM is known to cause pulmonary disease similar with sign and symptom of pulmonary tuberculosis but different in treatment.BACTECMGIT960 has excellent agreement withLJ media but it has high tendency of having high contamination rate unless a better decontamination method is designed.

Comparison of AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR for Detection of Mycobacterium tuberculosis Complex in Routine Clinical Practice

The AdvanSure tuberculosis/non-tuberculous mycobacterium (TB/NTM) PCR (LG Life Science, Korea) and COBAS TaqMan Mycobacterium tuberculosis (MTB) PCR (Roche Diagnostics, USA) are commonly used in clinical microbiology laboratories. We aimed to evaluate these two commercial real-time PCR assays for detection of MTB in a large set of clinical samples over a two-year period. AdvanSure TB/NTM PCR and COBAS TaqMan MTB PCR were performed on 9,119 (75.2%) and 3,010 (24.8%) of 12,129 (9,728 respiratory and 2,401 non-respiratory) MTB specimens, with 361 (4.0%) and 102 (3.4%) acid-fast bacilli (AFB)-positive results, respectively. In MTB culture, 788 (6.5%) MTB and 514 (4.2%) NTM were identified. The total sensitivity and specificity of the AdvanSure assay were 67.8% (95% confidence interval [CI], 63.9-71.6) and 98.3% (95% CI, 98.0-98.6), while those of the COBAS TaqMan assay were 67.2% (95% CI, 60.0-73.8) and 98.4% (95% CI, 97.9-98.9), respectively. The sensitivities and specificities of the AdvanSure and COBAS TaqMan assays for AFB-positive and AFB-negative samples were comparable. Furthermore, the AdvanSure assay showed fewer invalid results compared with the COBAS TaqMan assay (5.0 vs. 20.4 invalid results/1,000 tests, P<0.001). AdvanSure assay represents a comparable yet more reliable method than COBAS TaqMan for the identification of mycobacteria in routine clinical microbiology.

Multidrug resistant tuberculosis versus non-tuberculous mycobacterial infections: a CT-scan challenge

INTRODUCTION: Clinical, laboratory and imaging findings in patients with multidrug resistanttuberculosis (MDR-TB) and non-tuberculosis mycobacterium (NTM) are similar, and the majority of these patients present with positive smear for Acid Fast Bacilli (ADB) and no response to first line anti-TB treatment, so sputum culture and PCR are necessary, especially in NTM. OBJECTIVE: In this study we evaluate more details of imaging findings to help earlier diagnosis of pathogens. MATERIALS AND METHODS: 66 patients with positive smear for AFB and no response to first line anti-TB drugs were divided into two groups by PCR and culture: MDR-TB (43 patients) and NTM (23 patients). Age, sex, history of anti-TB treatment, smoking and CT-scan findings (parenchymal, pleural and mediastinal variables) by details and lobar distribution were analyzed. RESULTS: Mean age of NTM patients was slightly higher (52 versus 45) and there is no significant difference in sex and smoking. In MDR-TB group, history of anti-TB treatment and evidence of chronic pulmonary disease such as calcified and fibrodestructed parenchyma, volume loss and pleural thickening were higher significantly. Cavities in MDR-TB were thickwall in the background of consolidation, while NTM cavities were more thin-walled with adjacent satellite nodules in same segment or lobe. Prevalence of bronchiectasis was similar in both groups, while bronchiectasis in MDR-TB group was in fibrobronchiectatic background in upper lobes, and in NTM group the distribution was more uniform with slightly middle lobes predominance. Prevalence and distribution of nodular infiltrations were similar more in Tree in Buds and scattered pattern. Calcified or non-calcified lymph nodes and also pleural changes were more frequent in MDR-TB but prevalence of lymphadenopathy was mildly higher in NTM. CONCLUSION: A check-list with multiple variables is helpful for differentiation between the two groups.

Evaluation of Nucleic Acid Probe for Rapid Identification of Mycobacterium tuberculosis Complex in Extra-Pulmonary Culture Isolates.

Patients infected with Non-tuberculous mycobacteria (NTM) usually do not respond to conventional anti-tubercular treatment and are misdiagnosed as infection with multi-drug resistant strains of mycobacterium tuberculosis (M.tb) due to lack of correct species identification, particularly in the developing countries like India. One of the challenges faced by clinicians in the treatment of tuberculosis is the absence of an easy, reliable and rapid identification tool that can accurately differentiate disease caused by M.tb complex from NTM. Keeping this in consideration, the performance of species specific nucleic acid probe i.e. Accuprobe was assessed and compared with conventional niacin production, nitrate reductase assay techniques for identification of M.tb complex in 80 mycobacterial isolates obtained from different extra - pulmonary sites. Accuprobe identified 62 isolates (77.5%) as M. tuberculosis complex and remaining 18 isolates (22.5%) as NTM whereas 64 isolates (80%) were identified as M.tb and rest 16 (20%) were interpreted as NTM by conventional biochemical techniques. The overall agreement between both techniques was 96.9% The sensitivity, specificity, positive predictive value(PPV) and negative predictive value (NPV) shown by accuprobe were 96.9%, 100%, 96.9%, and 88.9% respectively. Thus, accuprobe has showed impressive sensitivity and specificity giving results in <3 hrs from culture-positive isolates and have sure edge over conventional biochemical methods which are, nonetheless, labour intensive and cumbersome to perform thus delaying prompt mycobacterial identification.

The analysis of identification for non-tuberculous Mycobacterium with molecular assay genotype Mvcobacterium kit / 中华微生物学和免疫学杂志

Objective To identify non-tuberculous Mycobacterium(NTM) rapidly with HAIN molecular assay genotype Mycobacterium kit,and investigate the advantages and disadvantages of this method.Methods Seventy-four clinical NTM isolates were collected from hospitals in Zhejiang and Anhui province.Clinical strains were identified with HAIN molecular assay genotype Mycobacterium kit.16S rRNA gene sequencing was used to estimate and compare with this method.Results The results of kit showed that there were thirty-one M.intracellulare strains,twelve M.chelonae strains,eight M.fortuitum strains,six M.kansasii strains,five M.avium strains,three M.smegmatis strains,two M.phlei strains,two M.scrofulaceum strains and one M.gordon strain.Four strains were identified as Mycobacterium without further identification.Eight M.tuberculosis strains were identified correctly too.Compared with 16S rRNA gene sequencing,except for four strains identified as Mycobacterium,others 70 strains got the same results as 16S rRNA gene sequencing,the coincidence was 94.59％,and it could further identify thirteen Mycobacterium chelonae complex and eight Mycobacterium kansasii complex to subspecies M.abscessus and M.kansasii,respectively.If only to identify strains under the identification range of this kit,the coincidence reach to 100％,Conclusion The method of HAIN molecular assay genotype Mycobacterium kit is simple and accurate,the time is shorter and should widely be applied clinically.

Isolation Trend of Nontuberculosis Mycobacteria at a Tertiary-care Hospital in 2003-2011 / 고신대학교의과대학학술지

OBJECTIVES: This study was performed to investigate the prevalence of nontuberculous mycobacteria (NTM) species and to determine the clinical significance of NTM isolates. METHODS: From January 2003 to July 2011, NTMs were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or reverse blot hybridization assay (REBA). And pulmonary NTM infection was diagnosed by clinical history, underlying disease, radiological and microbiological findings according to the diagnostic criteria of 2007 American Thoracic Society (ATS). RESULTS: Of the 697 AFB culture-positive specimens, NTM was detected in 149 (21.4%) specimens. Among 154 NTM isolates from 149 specimens, M. avium-intracellulare complex (MAC) (48.1%) was the most frequently isolated organisms followed by M. abscessus (13.6%), M. gordonae (9.1%), M. kansasii (8.4%), M. szulgai (3.9%), M. fortuitum complex (3.3%), M. scrofulaceum (2.0%), M. malmoense (1.3%), M. chelonae (1.3%), M. marinum (1.3%), M. genavense (1.3%), M. lentiflavum (1.3%) and M. mucogenicum (0.6%). Among 147NTM isolates from 142 respiratory specimens, 54 NTM isolates (36.7%) were causative organisms in NTM pulmonary infection. CONCLUSIONS: The isolation rate of NTM was 21.4% in clinical specimen, and in some cases NTM species results in pulmonary NTM infection. Because the treatment of pulmonary NTM infection depends on the infecting species, accurate identification and clinical significance of NTM are required for adequate treatment.

Nontuberculous Mycobacterial Infection after Breast Reconstruction with an Implant: A Case Report

PURPOSE: Breast implant surgery is increasing in Korea. NTM (non tuberculous mycobacteria) infection after breast implant surgery is rare, but it has been there reported in several foreign countries. However, no report has been issued on NTM infection after breast reconstruction surgery with an implant in Korea. The purpose of this article is to report a case of NTM infection after breast reconstruction surgery with an implant. METHODS: A female patient who underwent total mastectomy and immediate breast reconstruction with a latissimus dorsi myocutaneous flap and an implant exhibited signs of inflammation after the surgery. Fluid cultures taken at the time of wound exploration were initially negative, but NTM was isolated by culture 10 days later. RESULTS: The implant was removed. M. fortuitum was identified by acid-fast culture and NTM-PCR. The patient was treated with combined antibiotic therapy. CONCLUSION: Although it is difficult to diagnose NTM infection after breast surgery, it is important that surgeons include NTM infection in the differential diagnosis of a post mammoplasty infection after breast implant surgery.

Detection of Rifampin Resistant Mycobacterium tuberculosis complex using Denaturing HPLC / 대한진단검사의학회지

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Mycobacterium tuberculosis and Nontuberculous Mycobacteria by PCR Assay / 대한임상미생물학회지

BACKGROUND: The purpose of this study was to evaluate the usefulness of direct PCR for a rapid detection of Mycobacterium tuberculosis (MTB), the differentiation of MTB from nontuberculous mycobacteria (NTM), and the identification of NTM species in acid-fast bacilli (AFB) smear-positive specimens. METHODS: A total of 255 AFB smear-positive respiratory specimens were studied. For the differentiation of MTB from NTM, the bands of the 360 bp, which exists both in MTB and NTM, and the 190 bp, which exists only in MTB, were amplified using the onetube nested PCR targeting regions of the rpoB gene. The two-tube nested PCR targeting 16S-23S rRNA spacer gene was done with 34 specimens that were negative by one-tube nested PCR. The specimens that were tested positive for NTMs were subsequently subjected to PCR-restriction fragment length polymorphism (PCR-RFLP) analysis based on the rpoB gene for mycobacterial species identification. RESULTS: Detection rate of MTB and NTM was 87% after the one-tube nested PCR. The detection range of MTB and NTM increased up to 93% after the two-tube nested PCR. The results of PCR-RFLP analysis identified those NTMs as M. avium and M. intracellulare. CONCLUSION: This result seems to suggest strongly that the PCR testing especially aiming for differentiation of MTB from NTM, and identificatin of mycobacterial species using AFB smear-positive specimens would be highly importnat in clinical settings for effective treatment of patients.

Clinical Utility of Polymerase Chain Reaction for the Differentiation of Nontuberculous Mycobacteria in Patients with Acid-fast Bacilli Smear-positive Specimens / 결핵및호흡기질환

BACKGROUND: In Korea, polymerase chain reaction (PCR) test for M. tuberculosis has been used for the diagnosis of acid-fast bacilli (AFB) smear-negative tuberculosis in order to increase diagnostic sensitivity. However, there have been no data dealing with the clinical utility of PCR in AFB smear-positive patients to differentiate between M. tuberculosis and nontuberculous mycobacteria. METHOD: We retrospectively analyzed the PCR test results which have been performed in patients who had AFB smear-positive sputum but had ambiguous clinical manifestations of active tuberculosis. PCR test was done using AMPLICORa M. tuberculosis kit. The sensitivity, specificity, and positive and negative predictive values of the PCR test were calculated based on culture and final clinical diagnosis result. RESULTS: Fifty-six consecutive patients (62 PCR tests) were included in the study. Active tuberculosis was diagnosed in 23 patients (41.0%), while 9 patients had NTM infection (16.0%). The sensitivity, specificity, positive- and negative-predictive value of PCR test were 88.8%, 86.8%, 76.1% and 94.3%, respectively, according to the culture result. In comparison, they were 91.3%, 100%, 100%, 94.3%, respectively, according to the final clinical diagnosis. All 15 patients with NTM isolates, including 6 patients who had other lung diseases but expectorated NTM isolate, were negative for PCR test. CONCLUSION: Even though tuberculosis is still prevalent in Korea, PCR test is useful to differentiate between M. tuberculosis and NTM in patients with AFB-smear positive sputum but with ambiguous clinical manifestations of active tuberculosis.

Isolation of Nontuberculous Mycobacteria by DNA Probe and Clinical Characteristics of Patients with NTM Pulmonary Disease / 결핵및호흡기질환

BACKGROUND: Nontuberculous mycobacterial (NTM) infections are increasingly being recognized as a cause of chronic pulmonary disease. This study describes the prevalence of NTM species from clinical specimens and the clinical characteristics of NTM pulmonary disease. MATERIAL AND METHODS: The NTM isolated from March 2003 to December 2003 at the Kosin Medical Center were identified using an oligonucleotide chip containing the internal transcribed space (ITS) sequence. The medical records of the patients with the NTM isolates, who fulfilled the 1997 ATS diagnostic criteria for NTM pulmonary disease, were analyzed, retrospectively. RESULTS: Twenty four species (24.2%) of NTM were isolated from 99 cultured AFB specimens. M. avium complex (MAC) (13 isolates), M. szulgai (3), M. kansasii (2), M. malmoense (2), M. abscessus (1), M. chelonae (1), M. scrofulaceum (1), and unclassified (1). Of the 23 patients with isolated NTM, 11 patients were found to be compatible with a NTM pulmonary infection according to the ATS criteria; MAC was found in 6 cases (54.5%), M. szulgai in 2 cases (18.2%), and M. abscessus, M. szulgai, M. kansasii and M. malmoense in 1 case each (9.1%). Ten patients (91%) were male and the median age at diagnosis was 61 years. In the pre-existing diseases, malignant disease was found in 6 cases including 5 patients with lung cancer, and history of old pulmonary tuberculosis was identified in 4 cases. The radiological patterns showed lung destruction lung in 3 cases, a cavitary mass in 3 cases, a nodular pattern in 2 cases, and reticulonodular, consolidation and a bronchiectasis pattern were in 1 case each. CONCLUSION: Various types of NTM pulmonary diseases were`found in a tertiary hospital at Busan, Korea. The NTM pulmonary diseases were caused by MAC, M. szugai, M. kansasii, M. malmoense, M. abscessus, M. chelonae, and M. scrofulaceum in the order of frequency.

Clinical Significance of Low-colony Count Scotochromogen Nontuberculous Mycobacteria / 결핵및호흡기질환

BACKGROUND: Even though it has been suggested that low-colony, scotochromogen nontuberculous mycobacteria (NTM) are usually contaminants and not true pathogens, evidence for this hypothesis has not been provided. This study investigated the colony characteristics, organism identification, and clinical significance of low-colony scotochromogen. METHODS: The laboratory cultured 6,898 respiratory clinical specimens for an examination of mycobacteria over a three-month period. A low-colony count was arbitrarily defined as < or = 20 colonies. This study analyzed the recovery rate of the mycobacteria, the number of colonies and their gross characteristics, and their clinical significance. PCR- restriction fragment length polymorphism analysis was carried out to identify the NTM species. NTM pulmonary disease was defined according to the American Thoracic Society. RESULTS: A total of 6,898 respiratory specimens for mycobacterium were cultured. Of these, 263 (3.8%) grew NTM, and 382 (5.5%) grew M. tuberculosis. Of the 263 cultured NTM specimens, 124 (47.1%) were scotochromogens. The smear-positive rate was significantly lower in these scotochromogens (4.8%) than in the non-scotochromogens (23.7%) (p<0.05). The most common isolates were M. gordonae (83/102, 81.4%) in the scotochromogens, and MAC (52/121, 43.0%) in the non-scotochromogens. Even though three out of 113 patients with a low-colony scotochromogen has been diagnosed with NTM pulmonary disease, the isolated scotochromogen was not considered to be the cause of the NTM disease but was just a contaminant. CONCLUSION: In this study, the most common isolate of a low-colony count scotochromogen was M. gordonae, which appeared to be contaminants and not true pathogens. Greater efforts in the quality control of a mycobacterium laboratory are needed in cases where there is a high recovery rate of low-colony count scotochromogen.