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To evaluate COBAS TaqMan MAI test misidentification of Mycobacterium lentiflavum as Mycobacterium intracellulare.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.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.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.

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.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.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.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).

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.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.Chemotherapeutic drugs, including CAM, are clinically and bacteriologically effective for pulmonary M. szulgai disease.

We aimed to investigate the prevalence and possible transmission routes of rifampicin (RFP) mono-resistant Mycobacterium tuberculosis strains.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.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.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.

There appears to be some genetic diversity among Mycobacterium kansasii (M. kansasii) isolates recovered throughout the world. Restriction analysis of heat shock protein 65-polymerase chain reaction-restriction analysis (hsp65PRA) showed that M. kansasii contains seven subspecies genetically distinct from M. kansasii isolates. M. kansasii genotype I is predominant in Japan and shows a very tight clonal structure. Different molecular typing methods including the 16S-23S rRNA spacer (ITS) region, RFLP, and PFGE analysis have been applied to isolates worldwide, and M. kansasii genotype I, as defined by hsp65PRA, appears to be highly clonal and the most common genotype associated with human disease. However, the identification of M. kansasii at the subtype level may possibly be more than just an interesting epidemiological tool; it may be relevant to determining the infectious pathway and clinical management of individual cases, as it allows the differentiation of potentially pathogenic subtypes from nonpathogenic subtypes. This review has been followed by the first review of the epidemiology of M. kansasii, and summarizes the evidence of molecular epidemiology and establishes the validity and importance of studies of M. kansasii. Further, the more precise definition of various M. kansasii isolates herein should provide a significant contribution to the understanding of key aspects of its biology, genotype, and molecular epidemiology.

Mycobacterium kansasii, a slow-growing photochromogen, causes serious pulmonary diseases in humans. Since the 1980s the isolation rate of M. kansasii has been surpassed by that of Mycobacterium avium complex (MAC). Pulmonary disease caused by M. kansasii is known to be more common in urban areas than in rural areas, and its changing epidemiologic features and geographical distribution have been known for several decades. The disease had been found almost only in and around Tokyo areas until the 1970s, but after the 1990s, the disease spread to the rest of Japan. The incidence rate of the disease due to M. kansasii remained at the same level, while non-tuberculous mycobacterioses as a whole are rapidly increasing, especially because of the increase in Mycobacteriu avium complex diseases, and because of the improvement of identification techniques in recent years. This change in the epidemiologic picture of M. kansasii in Japan seems to be influenced by the international trends of this disease. The complicated environmental and other factors related to M. kansasii infection are discussed in this review.

To analyze the molecular epidemiology of Mycobacterium gordonae strains from patients and environments in the hospital.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.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.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.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 (the INNO-LiPA assay) for mycobacterial identification.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.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).The INNO-LiPA assay could provide rapid and correct identification results with clear-cut and easy interpretation.

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.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.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.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.

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.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.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 (p0.5) 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 (p0.5).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.

To clarify the genetic microheterogeneity of Mycobacterium lentiflavum and identify the predominant genotype.Clinical isolates of M. lentiflavum used in this study were obtained from sixteen patients of lung diseases. In order to assess their intraspecies variability, four gene fragments, from the 16S rDNA (1471 bp), 16S-23S ITS (282 bp), rpoB (306 bp), and hsp65 (401 bp), were sequenced.Intraspecies variabilities were found in all of the four targeting fragments. As multilocus sequence typing with these four targets, 16 clinical isolates were divided into 3 genotypes, i.e., MLST2, MLST3, and MLST4. Among them, MLST2 to which 12 clinical isolates belonged, was a predominant genotype. Three strains belonged to MLST3 and the remaining one strain belonged to MLST4. Drug susceptibility study indicated that there was no clear relation between sequence types and drug susceptibility.Multilocus sequence typing could aid in characterization and in better understanding of the epidemiology of M. lentiflavum.

Comparing multidrug-resistant M. tuberculosis strains with susceptible strains by molecular epidemiological methods.We examined 109 multidrug-resistant strains (MDR-TB) and 226 susceptible strains (S-TB) derived from National Hospital Organization Kinki-chuo Chest Medical Center by restriction fragment length polymorphism (RFLP) with IS6110, and Spacer oligonucleotide typing (Spoligotyping).In the case of MDR-TB, 47 strains (43.1%) belonged to 12 descriptions of clusters and the number of IS 6110 copies per isolate ranged from 9 to 25. Similarly, 99 strains (43.8%) belonged to 20 descriptions of clusters in S-TB and the distribution of IS 6110 copies were from 1 to 20. On the other hand, 84 strains of MDR-TB (77.1%) and 191 strains of S-TB (84.5%) belonged to Beijing family by Spoligotyping.MDR and susceptible M. tuberculosis strains were characterized similarities in ratio of clusters by RFLP patterns and high proportion of Beijing family by Spoligotyping. These finding supported the possibility that infectiousness of MDR-TB might be similar to that of S-TB.

To make molecular epidemiological analysis of Mycobacterium kansasii (M. kansasii) isolates.We examined 174 M. kansasii isolates from clinical samples of patients at National Hospital Organization Kinki-chuo Chest Medical Center from June 1, 2002 to August 31, 2005 by polymerase chain reaction (PCR) -restriction analysis (PRA) of the heat shock protein (hsp) 65 gene (hsp65-PRA), sequencing (ITS, 16S-23S internal transcribed spacer, and hsp65 for discrepant case between hsp65-PRA and ITS sequence), pulsed-field gel electrophoresis (PFGE), and restriction fragment length polymorphism (RFLP) with the major polymorphic tandem repeat (MPTR) probe and the IS1652 probe of genomic DNA.Of the 174 M. kansasii isolates, 170 strains were classified as M. kansasii type I using hsp65-PRA, while two isolates belonged to type II and one each isolate to type IIb and VI, respectively. Although the ITS sequence of these isolates also identified the same region of polymorphism by hsp 65-PRA, only type II b might be revealed atypical type II, a transitional type from typical type II to intermediate type I by hsp65 sequence. The polymorphic patterns by RFLPs with MPTR and IS1652 probe were shown specific for each homogeneous cluster by hsp 65-PRA. In addition, 159 isolates were recognized the same common pattern A by PFGE analysis. In contrast, the rest 15 isolates revealed significant polymorphism within 11 isolates of type I, and 4 isolates among type II, IIb, and VI.We verified the M. kansasii genotype I was predominant, with the same pattern of major worldwide type regions, and reflected a very tight clonal structure. Type I was furthermore indicated recognition of subtypes by PFGE analysis.

To detect rifampicin-resistant mutations in Mycobacterium kansasii (M. kansasii).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.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.We verified the association between rpoB gene mutations and rifampicin resistance in M. kansasii.