[Genetic analysis reveals misidentification of Mycobacterium lentiflavum as Mycobacterium intracellulare by the COBAS TaqMan MAI test].

OBJECTIVE: To evaluate COBAS TaqMan MAI test misidentification of Mycobacterium lentiflavum as Mycobacterium intracellulare. MATERIALS AND METHODS: Preliminary comparative analysis identified 13 clinical isolates used in this study as COBAS Amplicor MAV and MIN-negative but COBAS TaqMan MAI-positive. The COBAS TaqMan MAI test limit of detection and reproducibility were evaluated by tenfold dilution series from 3 x 10(8) CFU/mL. Isolate 16S rDNA nucleotide sequences were compared with Mycobacterium avium and M. intracellulare. RESULTS: Discrepancies were observed between isolates identified as M. lentiflavum by 16S rDNA sequencing and as M. intracellulare by the COBAS TaqMan MAI test. The false-positive results were verified by sequence comparison of a randomly sampled clinical isolate and the M. intracellulare reference strain. Sequence analysis of M. lentiflavum and M. intracellulare 16S rDNA amplification products showed at least 3 mismatches between species. The high identity in the sequence was found for M. intracellulare by COBAS TaqMan MAI. CONCLUSION: In Japan, commercially available nucleic acid probe- and amplification-based tests cannot identify M. lentiflavum. Correct identification, though challenging, is possible using standard cultivation procedures for colony growth. Misleading results using the COBAS TaqMan MAI kit may lead to erroneous diagnoses.

[Comparative evaluation of acid-fast staining for the detection of Mycobacterium fortuitum--clinical performance of fluorescent and Ziehl-Neelsen staining].

OBJECTIVE: Fluorescent staining is of paramount importance, not only for confirming the presence of mycobacteria in a given specimen but also for providing an estimated growth quantification. In this study, for rapidly growing Mycobacterium fortuitum, we evaluated the effectiveness of a rapid fluorescent staining method employing auramine-rhodamine (AR) fluorescent stain and acridine-orange (AO) fluorescent stain compared to that of the standard Ziehl-Neelsen (ZN) stain currently in use in our laboratory. METHOD: We evaluated the acid-fast nature of M. fortuitum strain ATCC6841 and 42 clinical isolates from each patient diagnosed at NHO Kinki-chuo Chest Medical Center. These isolates were preliminarily identified as M. fortuitum using DNA-DNA hybridization (DDH Mycobacteria; Kyokuto Pharmaceutical, Tokyo, Japan). These isolates were further identified by comparative sequence analysis of the ITS regions and the partial 16S rRNA gene. RESULTS: A total of 26 M. fortuitum strains (61.9%) demonstrated the lack of an acid-fast nature by AR staining, and slightly fewer demonstrated the same by AO staining. Sequence analysis of these 42 clinical isolates led to the identification of 35 M. fortuitum subsp. acetamidolyticum isolates (83.3%) and 7 closely M. fortuitum isolates. DISCUSSION: This work reported the loss of the acid-fast nature of specific M. fortuitum strains. It is likely that both the specific cell envelope of M. fortuitum and the staining mechanics could have been responsible for the loss of the acid-fast nature since the 2 different fluorescent stains yielded the same results. M. fortuitum is a mycobacterium species that does not stain with the commonly used fluorescence microscopy technique. Therefore, we suggested the use of an identification scheme for these organisms that employs ZN staining and the study of cultural characteristics (growth rate, temperature, and pigment production).

[Rapid mycobacterium identification and rapid susceptibility testing by the nucleic amplification method].

This review was designed to review mycobacterial infections from the viewpoint of clinical practices. We showed the usefulness of the rapid mycobacterium identification system for the detection of various genes by the nucleic amplification method. However, most PCR-based identifications required NALC-NaOH preprocessing, a special technique, or lengthy, hard work. Although 16S rRNA gene sequences may be provided successfully to identify many mycobacterial species, they lack sufficient discrimination to differentiate certain isolates from some species. We also explained the current methodologies of rapid susceptibility testing for M. tuberculosis and clarified these abilities. Therefore, molecular detection and identification should be considered to isolate these organisms, in settings where bacteria were microscopically visible in clinical samples, involving culturing and performing drug susceptibility testing for mycobacteria. Finally, we should emphasize the importance of collaboration between clinical microbiologists and basic bacterial researchers to promote current and future clinical strategies against mycobacteria.

[Molecular epidemiology of rifampicin mono-resistant Mycobacterium tuberculosis].

PURPOSE: We aimed to investigate the prevalence and possible transmission routes of rifampicin (RFP) mono-resistant Mycobacterium tuberculosis strains. METHODS: Drug susceptibility testing was used to identify 15 RFP-resistant strains out of 4633 M. tuberculosis isolates. Sequencing of the rpoB gene and VNTR analysis were performed to further confirm the genetic classification. RESULTS: Resistance-conferring mutations in the RFP resistance-determining region (RRDR) of the rpoB gene were found in 14 of the 15 strains with phenotypic RFP mono-resistance. VNTR analysis revealed 2 clusters of 5 identical strains each. CONCLUSIONS: Although the community prevalence of RFP mono-resistant M. tuberculosis is low, the results of VNTR analysis suggested that rather than being recently transmitted, these strains may have been widely transmitted as latent infections in the population.

[Analysis of the clinical features of pulmonary disease caused by Mycobacterium szulgai].

SUBJECTS & METHODS: We reviewed the patient characteristics, radiological findings, treatments, and clinical outcomes in 12 cases of pulmonary Mycobacterium szulgai disease diagnosed at our hospital from April 1998 to March 2008. In addition, drug susceptibility testing of the causative isolates was performed with several antibiotics, including clarithromycin (CAM) and rifampicin (RFP), using BrothMIC NTM. RESULTS: The patients included 10 men and 2 women, with a mean age of 57.2 years. Among them, 10 were smokers, 5 previously had pulmonary tuberculosis, 3 had a history of gastric ulcers, and 1 had a history of esophageal cancer surgery. All patients had been previously treated with various chemotherapeutic regimens. Six of them were treated with chemotherapy, including CAM, and they improved both radiologically and bacteriologically. The minimal inhibitory concentration of CAM for all the strains tested was less than 0.25 microg/mL, which is the likely critical concentration for clinical efficacy of CAM. The present study suggested that, in addition to smoking and a history of pulmonary tuberculosis, gastroesophageal disorders were relevant underlying conditions in patients with pulmonary M. szulgai disease. CONCLUSION: Chemotherapeutic drugs, including CAM, are clinically and bacteriologically effective for pulmonary M. szulgai disease.

Comparison of rifabutin susceptibility and rpoB mutations in multi-drug-resistant Mycobacterium tuberculosis strains by DNA sequencing and the line probe assay.

We compared rifabutin susceptibility and rpoB mutations in 98 multi-drug-resistant strains of Mycobacterium tuberculosis (MDR-TB) by DNA sequencing and with a line probe assay using the commercially available INNO-LiPA Rif. TB kit (the LiPA). Our results indicated that rifabutin continues to remain active against MDR-TB strains harboring certain genetic alterations and also that the LiPA might be useful in identifying MDR-TB strains susceptible to rifabutin.

[Detection of molecular epidemiology of Mycobacterium gordonae isolates].

OBJECTS: To analyze the molecular epidemiology of Mycobacterium gordonae strains from patients and environments in the hospital. SUBJECTS: A total of 46 clinical strains were obtained from patients registered at the NHO Kinki-chuo Chest Medical Center and 3 strains from hospital environments. METHODS: By using genetic data from the 16S rRNA gene and hsp65PRA, pulsed-field gel electrophoresis (PFGE) assessment of their intraspecies variability and epidemiology was carried out. RESULTS: Strains from six patients and environmental cultures exhibited the different genotypes of 16S rRNA gene sequencing and the hsp65PRA type. The PFGE analysis suggested no pseudo-outbreak and showed a polyclonal infection in one patient. CONCLUSION: These findings suggest that we should maintain effective surveillance of environments in the hospital and continuously perform molecular epidemiological investigations for infection control of M. gordonae.

[Evaluation of the INNO-LiPA MYCOBACTERIA v2 for mycobacterial identification].

PURPOSE: Evaluation of the INNO-LiPA MYCOBACTERIA v2 (the INNO-LiPA assay) for mycobacterial identification. MATERIALS AND METHODS: The laboratory identifications consisting of Cobas Amplicor systems, AccuProbe, and DDH, are commonly used to identify mycobacterial isolates in Japan. We compared the results between the INNO-LiPA assay and the common methods. A total of 122 clinical isolates from NHO Kinki-chuo Chest Medical Center from 1 February to 30 June 2006 were tested. RESULTS: There was agreement between the INNO-LiPA assay and the common methods for 112 mycobacterium isolates. The six discordant isolates have showed same results between sequencings and the INNO-LiPA assay. The one M. fortuitum isolates was indicated correctness by DDH and the one M. intracellulare isolates was recognized by Cobas Amplicor systems and as MAC by AccuProbe MAC. Moreover, discrepant results between sequencings and mycobacterial identifications including the INNO-LiPA assay were 2 isolates (M. paraffinicum, M. mucogenicum variant type). CONCLUSION: The INNO-LiPA assay could provide rapid and correct identification results with clear-cut and easy interpretation.

[Comparison of BBL Mycoprep and 2%NaOH decontamination procedures for MGIT].

OBJECTIVES: We compared the BBL Mycoprep (Becton Dickinson Japan) and home-made 2%NaOH decontamination procedures by using an equal amount of expectorated sputum in the aerosol-free 30 ml KT centrifuge tube with the rugged inner surface. METHOD: A total of 113 sputum specimens obtained in NHO Kinki-Chuo Chest Medical Center in November 2004 were subjected to two decontamination methods. All specimens were divided into two equal portions after concentrating the sediments processed by semi-kaline protease (SAP), then decontaminated, and inoculated into MGIT. The tubes were incubated at 37 degrees C and monitored for up to forty-second days. RESULTS: Comparing these decontamination procedures, the time of the recovery of mycobacteria strains in the 2%NaOH (mean 8 days) was significantly faster than in the BBL Mycoprep (mean 11 days). Of these, 19 specimens (16.9%) processed by the BBL Mycoprep were positive for growth of mycobacteria, and similarly 18 specimens (16.0%) processed by the 2%NaOH (p>0.5) were positive. The 19 mycobacteria recovered by the BBL Mycoprep were identified as 14 M. tuberculosis strains and 5 NTM strains. The decontamination rate was 0.9% in 2%NaOH and 6.2% in Mycoprep, however the difference was statistically not significant (p>0.5). DISCUSSION: We verified that the 2%NaOH was an alternative method suitable for the digestion and decontamination procedure, and 2%NaOH was useful for the isolation and detection of mycobacteria as well.

[Evaluation of the discrepant Mycobacterium tuberculosis strains between any ordinary susceptibility testing and rpoB gene analysis by the line probe assay].

PURPOSE: Evaluation of rifampicin-resistance by the line probe assay, for rifampicin-susceptible Mycobacterium tuberculosis strains which were classified as rifampicin-resistant by the phenotypic drug susceptibility testings. MATERIALS AND METHODS: A total of 15 clinical isolates from NHO Kinki-chuo Chest Medical Center consisting of 6 rifampicin-resistant strains by the line probe assay despite susceptible result by the drug susceptibility testings, and 9 clinical isolates which showed the fluctuating results on repeated drug susceptibility testings. After we conducted 3 drug susceptibility testings and the line probe assay, we have examined the sequence analysis for confirming mutations in the rpoB gene. RESULTS: All strains were determined rifampicin-susceptible or intermediate by the drug susceptibility testings with Minimum Inhibitory Concentration (MIC) which ranged from 0.25 to 4 microg/ml by BrothMIC MTB-1, whereas these isolates indicated rifampicin-resistance by the line probe assay and revealed mutations in the hot-spot region (69 bp) by the sequence analysis. CONCLUSION: We verified that the line probe assay might be useful for the correct determination of drug susceptibility, especially about the low-level rifampicin-resistant M. tuberculosis strains.

Association between sequevar and antibiotic treatment outcome in patients with Mycobacterium abscessus complex infections in Japan.

PURPOSE: Macrolide susceptibility differs between subspecies in the Mycobacterium abscessus complex, likely due to differences in erm(41) sequevars. Patients with M. abscessus complex infection generally show poor clinical outcomes in response to antibiotic treatment. Here, the association between genotype and treatment outcome was investigated. METHODOLOGY: We collected 69 isolates from 35 patients with non-cystic fibrosis bronchiectasis: 24 had M. abscessus complex lung disease and non-cystic fibrosis bronchiectasis, and 11 were colonized. Outcome analysis was performed in the 24 infected patients. Molecular analyses, including erm(41) and rrl sequencing, and variable-number tandem-repeat (VNTR) analysis of 69 isolates, from 24 infected and 11 colonized patients, were performed to elucidate the influence of genotype on antibiotic susceptibility. RESULTS: Among the 24 patients, 18 (14 infected with M. abscessus subsp. abscessus and 4 with M. abscessus subsp. massiliense) showed unfavourable outcomes; six (three infected with M. abscessus subsp. abscessus and three with M. abscessus subsp. massiliense) exhibited favourable outcomes. Patients with unfavourable outcomes showed acquired clarithromycin resistance (33.3 vs 0 %), mixed sequevars (38.9 vs 16.7 %) and differing VNTR patterns between initial and serial isolates (33.3 vs 16.7 %). In contrast, in the 11 colonized patients, M. abscessus subsp. abscessus C28 (sequevar 02) and M. abscessus subsp. massiliense were the most prevalent subspecies. CONCLUSION: Patients infected with multiple sequevars and genotypes were more likely to exhibit treatment failure and/or recurrence. The precise identification of subspecies and analyses of mycobacterial characteristics may help to predict treatment outcomes in patients with M. abscessus complex lung disease.

Hypervariable loci that enhance the discriminatory ability of newly proposed 15-loci and 24-loci variable-number tandem repeat typing method on Mycobacterium tuberculosis strains predominated by the Beijing family.

The newly proposed 15- and 24-loci mycobacterial interspersed repetitive unit (MIRU)-variable-number tandem repeat (VNTR) typing method was evaluated for its ability to differentiate 181 Mycobacterium tuberculosis Beijing family strains. Compared with the original 12-loci MIRU-VNTR typing method, the 15-loci system dramatically improved the discriminatory power for Beijing strains; however, large clusters that could be further differentiated by IS6110 restriction fragment length polymorphism (RFLP) were still obtained. The clonal stability and allelic diversity of a total of 31 VNTR loci were evaluated. VNTRs 3232, 3820, and 4120 were identified as the effective hypervariable VNTR set for the second-line typing of clustered strains following the 15-loci based scheme. Consequently, the discriminatory power of the new scheme (18 loci) equaled that of IS6110 RFLP.

[Detection of rpoB mutations in rifampicin-resistant Mycobacterium kansasii].

PURPOSE: To detect rifampicin-resistant mutations in Mycobacterium kansasii (M. kansasii). METHODS: We examined the M. kansasii isolates from sputum of patients at National Hospital Organization Kinki-chuo Chest Medical Center from January 1, 2001 to November 30, 2005 using drug-susceptibility testing, and analyzed 69-bp fragment of rpoB gene in rifampicin-resistant strains. RESULTS: Three strains from 314 isolates were determined as rifampicin resistant using drug-susceptibility testing. Those strains showed a rise in minimum inhibitory concentration (MIC), and had the mutations in rpoB gene. These point mutations in codons 513 and 516 were common mutations found in rifampicin-resistant clinical isolates of M. tuberculosis. DISCUSSION: We verified the association between rpoB gene mutations and rifampicin resistance in M. kansasii.

[A study on inoculum density and reproducibility of drug susceptibility testing by BACTEC MGIT 960].

OBJECTIVE: The BACTEC MGIT 960 drug susceptibility system (MGIT AST) has been recently introduced in Japan. The issue of discordant MGIT results compared with the conventionally used Ogawa method has been raised. It has been speculated that discordant results might be due to MGIT inoculum density since there is no standardization step other than dilution of growth for tubes beyond 2 days after MGIT turns out to be positive. In this study, we examined the reproducibility of the MGIT AST system. MATERIALS AND METHODS: Nineteen sputum specimens from drug-resistant and susceptible pulmonary tuberculosis patients were processed with CCE pretreatment reagent (Japan BCG), inoculated into 3 MGIT tubes, and loaded into the MGIT 960. Inocula for MGIT AST were prepared 1, 3, and 5 days after MGIT tubes became positive. Cultures on day 3 and 5 were diluted 1: 5 with saline. Ten-fold dilutions from each positive culture were plated on Middlebrook 7H11 agar plates for CFU determination. MGIT AST results were compared with those of the conventional proportion method on Ogawa egg and Vite-spectrum (Kyokuto), or Pyrazinamidase (Pzase) assay and Kyokuto PZA test. RESULTS AND CONCLUSION: A total of 15 specimens were culture positive in all 3 tubes. Four of 19 cases were removed from the analysis because of negative cultures in one or more tubes. Three of 4 culture negative cases were MDR-TB. Colony counting showed the mean CFU/ml of inocula prepared from tubes 1, 3, and 5 days after MGIT tube became positive were 3.6 x 10(6), 1.6 x 10(6), 3.1 x 10(6), respectively. There was no significant difference although the CFU range was wide (8 x 10(4)-2 x 10(7)). MGIT AST results were consistent among 3 inocula. Moreover, overall concordance rates between MGIT AST and the conventional methods were over 90% for 5 first-line antituberculosis drugs. These results indicate that the BACTEC MGIT 960 system is very useful for rapid diagnosis of drug resistant tuberculosis.

[Present trends of drug-resistant tuberculosis and how to manage it by mycobacterial laboratories].

Global, domestic, and local trends of drug-resistant tuberculosis and how to manage increase in the resistance by mycobacterial laboratories were discussed based on literatures and our own data. At first, how to make drug-resistant tuberculosis was explained. Genetic drug-resistant bacteria were emerged spontaneously by mutation of the genome and were selected by inadequate treatment(mono-therapy or functional mono-therapy): acquired drug resistance(single, and then multi-drug resistance). In a mean time, some people were infected with the drug-resistant bacteria from the beginning and a part of them developed active disease: primary drug resistance (single or multi-drug resistance). Estonia, Latvia, Iran, and some part of Russia, China, and India were reported to be the most endemic region of drug resistant and multi-drug resistant tuberculosis in the world. The rate of primary resistance in Japan was as high as the median of the world, but the rate of acquired resistance was almost twice of the median. Since delays in reporting of the drug resistance from the laboratory seemed one of reasons for the inadequate treatment, drug susceptibility testing should be more rapid than usual, by using liquid media such as BACTEC MGIT 960 system, or gene analysis.

Rapid identification of strains belonging to the Mycobacterium abscessus group through erm(41) gene pyrosequencing.

Mycobacterium abscessus and Mycobacterium massiliense lung infections have different clarithromycin susceptibilities, making proper identification important; however, standard multi-gene sequencing in clinical laboratories is laborious and time consuming. We developed a pyrosequencing-based method for rapid identification of strains belonging to the M. abscessus group by targeting erm(41). We examined 55 isolates from new pulmonary M. abscessus infections and identified 28 M. abscessus, 25 M. massiliense, and 2 Mycobacterium bolletii isolates. Multi-gene sequencing of 16S rRNA, hsp65, rpoB, and the 16S-23S ITS region was concordant with the results of erm(41) pyrosequencing; thus, the M. abscessus group can be identified by single-nucleotide polymorphisms in erm(41). The method also enables rapid identification of polymorphic, inducible clarithromycin-resistant sequevars (T28 or C28). Pyrosequencing of erm(41) is a rapid, reliable, high-throughput alternative method for identifying and characterizing M. abscessus species. Further testing of a diverse collection of isolates is necessary to demonstrate the discriminatory power of erm(41) sequencing to differentiating species with this highly divergent group.

Further isolation of Mycobacterium abscessus subsp. abscessus and subsp. bolletii in different regions of Japan and susceptibility of these isolates to antimicrobial agents.

The aim of this study was to genetically analyse Mycobacterium abscessus subsp. abscessus (hereafter M. abscessus) and M. abscessus subsp. bolletii (hereafter M. bolletii) isolates from six different regions of Japan and to determine the antimicrobial susceptibility of these isolates. Subspeciation of 143 clinical isolates of M. abscessus group was done by comparative sequence analysis of the rpoB and hsp65 genes and the internal transcribed spacer (ITS) region. Genetic analysis led to the identification of 90 M. abscessus (62.9%) and 53 M. bolletii (37.1%; comprising 50 'M. massiliense' and 3 'M. bolletii' in the old nomenclature). No significant differences were found between the M. abscessus and M. bolletii isolates in any characteristics. Susceptibility to clarithromycin and linezolid for M. bolletii isolates was significantly higher than that for M. abscessus (P<0.05). Moreover, the results demonstrated that 82 M. abscessus isolates with T28 sequevar were resistant to clarithromycin owing to the expression of erm(41), which was induced by clarithromycin, whilst 8 isolates with C28 sequevar were susceptible. Acquired clarithromycin resistance in 'M. bolletii' isolates was significantly associated with previous Mycobacterium avium complex (MAC) treatment compared with that of M. abscessus isolates; however, intrinsic inducible susceptibility of M. abscessus isolates was not associated with MAC treatment. However, acquired resistance to clarithromycin by mutation in the rrl gene encoding 23S rRNA did not occur in 14 of 18 resistant isolates. Strains with acquired resistance to clarithromycin and mutation in rrl consisted of two M. bolletii (one 'M. massiliense' and one 'M. bolletii') and two M. abscessus T28 sequevar.

Rapid detection of Mycobacterium tuberculosis in respiratory samples by transcription-reverse transcription concerted reaction with an automated system.

The aim of this study was to evaluate the performance of the transcription-reverse transcription concerted (TRC) method for the detection of Mycobacterium tuberculosis complex (MTC) 16S rRNA in clinical respiratory samples for the diagnosis of pulmonary tuberculosis. TRC is a novel method that enables the rapid and the completely homogeneous real-time monitoring of isothermal sequence RNA amplification without any postamplification procedure. The detection limit of the TRC method for MTC was one organism per 100 mul of sputum. The specificity of the method was confirmed by the absence of positive signals for sputum containing 10(6) M. avium or M. kansasii organisms per 100 microl. A total of 201 respiratory samples from patients diagnosed with or suspected of having tuberculosis were tested. Of the 72 MTC culture-positive samples, the TRC method was positive for 52 (sensitivity, 72.2%), whereas the Roche COBAS AMPLICOR PCR was positive for 58 (sensitivity, 80.6%). Both the TRC method and the COBAS AMPLICOR PCR showed no positive identification for any of the 129 culture-negative samples. The percent agreement between the two methods was 95% (191 of 201 samples). The high sensitivity and specificity together with shorter detection time (within 1 h) of the TRC method allow it to be proposed as a useful method for the rapid detection of MTC in respiratory samples.