Anti-IL-6 receptor antibody causes less promotion of tuberculosis infection than anti-TNF-α antibody in mice.

Objective. Our aim was to investigate the effects of IL-6 blockade on the progression of Mycobacterium tuberculosis (TB) and compare them with those of TNF-α blockade in mice. Methods. Mice were intravenously infected with TB and injected with antibodies. Survival was monitored and histological and immunological studies were carried out. Results. All anti-IL-6R Ab-treated mice and 8 of 10 control mice survived until sacrificed 224 days after TB challenge, whereas anti-TNF-α Ab-treated mice all died between 120 and 181 days. Anti-IL-6R Ab-treated mice exhibited no significant differences in TB CFU in organs, including the lungs, and no deterioration in histopathology compared to control mice at 4 weeks. In contrast, anti-TNF-α Ab-treated mice exhibited increased TB CFU and greater progression of histopathological findings in organs than control mice. Spleen cells from anti-TNF-α Ab-treated mice had decreased antigen-specific response in IFN-γ release and proliferation assays. The results in anti-IL-6R Ab-treated mice suggest that spleen cell responses were decreased to a lesser degree. Similar results were obtained in IL-6 knockout (KO) mice, compared with TNF receptor 1 (TNFR1) KO and TNFR1/IL-6 double KO (DKO) mice. Conclusion. IL-6R blockade promotes the progression of TB infection in mice far less than TNF-α blockade.

Novel prophylactic vaccine using a prime-boost method and hemagglutinating virus of Japan-envelope against tuberculosis.

OBJECTIVE: Mycobacterium tuberculosis infection is a major global threat to human health. The only tuberculosis (TB) vaccine currently available is bacillus Calmette-Guérin (BCG), although it has no efficacy in adults. Therefore, the development of a novel vaccine against TB for adults is desired. METHOD: A novel TB vaccine expressing mycobacterial heat shock protein 65 (HSP65) and interleukin-12 (IL-12) delivered by the hemagglutinating virus of Japan- (HVJ)- envelope was evaluated against TB infection in mice. Bacterial load reductions and histopathological assessments were used to determine efficacy. RESULTS: Vaccination by BCG prime with IgHSP65+murine IL-12/HVJ-envelope boost resulted in significant protective efficacy (>10, 000-fold versus BCG alone) against TB infection in the lungs of mice. In addition to bacterial loads, significant protective efficacy was demonstrated by histopathological analysis of the lungs. Furthermore, the vaccine increased the number of T cells secreting IFN-γ. CONCLUSION: This vaccine showed extremely significant protection against TB in a mouse model, consistent with results from a similar paper on cynomolgus monkeys. The results suggest that further development of the vaccine for eventual testing in clinical trials may be warranted.

Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.

PURPOSE: BCG is not efficacious against M. tuberculosis (TB) in adult. Therefore, novel TB vaccines were established by using three kinds of animal models (cynomolgus monkey model which is the best animal model of human TB, IL-2R knock out SCID mice as a human immune model, and granulysin transgenic mouse). METHODS AND RESULTS: DNA vaccine expressing TB Hsp65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. The BCG prime followed by Hsp65+IL-12/HVJ vaccine boost showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys were alive in BCG alone group. Furthermore, the prolongation of survival period of the monkey was observed by the combination of BCG and DNA vaccine even when the boost was performed after long-term period (4month) from prime. This combination also improved the erythrocyte sedimentation rate (ESR), increased the body weight, and augmented the proliferation of PBL and IL-12 production at higher levels than BCG alone or saline. Furthermore, this vaccine exerted therapeutic efficacy in IL-2R knock out SCID-PBL/hu mice, which were transplanted with human T cells. Granulysin is an important defensive molecule expressed by human T cells and NK cells and has a cytolytic activity against microbes including Mycobacterium tuberculosis (TB) and tumors. Expression of 15kD (15K) granulysin protein and mRNA in CD8 positive T cells in the patients infected with drug sensitive (TB) or multi-drug resistant (MDR-TB) M. tuberculosis were lower than that in the healthy volunteers, suggesting that granulysin treatment might improve the tuberculous disease in human. Therefore, we established two kinds of granulysin transgenic mice (15K granulysin transgenic mice and 9K granulysin transgenic mice). It was demonstrated that 15K granulysin transgenic mice as well as 9K granulysin transgenic mice exerted in vivo anti-TB effect, including the decrease of the number of TB and augmentation of the CTL activity. These are the first findings which demonstrate in vivo effects of 15K granulysin and 9K granulysin against TB infection. Moreover, DNA vaccine expressing 15K granulysin showed a therapeutic activity against TB in mice. CONCLUSION: These data indicate that monkey, IL-2R gene-knock out SCID-PBL/hu and granulysin transgenic mice models provide useful tools for the development of novel vaccines (HVJ-Envelope/Hsp65 DNA + IL-12 DNA vaccine and granulysin vaccine) against TB.

Novel therapeutic vaccine: granulysin and new DNA vaccine against Tuberculosis.

PURPOSE: Multi-drug resistant (MDR) Mycobacterium Tuberculosis (M.TB) is a big problem in the world. We have developed novel TB therapeutic vaccines. METHODS AND RESULTS: DNA vaccine expressing mycobacterial heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. M. TB, MDR-TB or extremenly drug resistant (XDR-TB) was injected i.v. into DBA/1 mice, and treated with the vaccine three times. This HVJ-E/Hsp65DNA+IL-12DNA vaccine provided strong therapeutic efficacy against MDR-TB and XDR-TB (prolongation of survival time and the decrease in the number of TB) in mice. Therapeutic effect of this vaccine on TB infection was also demonstrated in chronic TB infection murine model using aerosol infection intratracheally. On the other hand, granulysin protein produced from CTL has lethal activity against TB. Granulysin protein vaccine also exerted strong therapeutic effect. Furthermore, we extended our studies to monkey model, which is currently the best animal model of human TB. Hsp65DNA+IL-12 DNA vaccine exerted strong therapeutic efficacy (100% survival and augmentation of immune responses) in the TB-infected monkeys. In contrast, the survival of the saline control group was 60% at 16 week post-challenge. HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the erythrocyte sedimentation rate, and augmentated the immune responses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. CONCLUSION: These data indicate that novel vaccines might be useful against TB including XDR-TB and MDR-TB for human therapeutic clinical trials.

[Comparison of conventional tuberculin skin test and QFT-2G, a new method for diagnosis of tuberculosis infection, with the use of the contact score].

BACKGROUND: QuantiFERON TB-Gold (QFT) has recently been developed as a new method for diagnosing tuberculosis (TB) infection. To evaluate the usefulness of QFT, we analyzed the relationship between QFT and the closeness of contact with a source of infection, in comparison with that of the tuberculin skin test (TST). METHODS: Male (n=322) and female (n=340) subjects (4-75 years old) who had contact with an index case received QFT and TST. The diagnostic criterion for TB infection with TST was defined as a test with an erythema diameter of > or = 30 mm. The closeness of contact with an index case was quantified in the "contact score," based on the information obtained with a questionnaire. RESULTS: There was a significant positive correlation between the contact score and QFT-positive rate, while there was no such relationship for TST positivity. The odds ratios for positive QFT rate for the subjects in the 3rd and 4th quartile groups of contact score (taking the QFT-positive rate in the lowest score quartile as unity) were 3.40 (95% confidence interval: 1.07-10.76, p<0.05) and 7.62 (95% confidence interval: 2.60-22.37, p<0.01), respectively. These odds ratios were also significantly greater than unity after adjustment for age, sex, history of BCG vaccination and history of health care-related jobs. There was a wide difference in the QFT-positive rates between the 2nd and 3rd quartiles of contact score (3.5% vs. 11.9%). The borderline value of the contact score between these two quartiles corresponded to 200, which could be a cutoff value for defining a high-risk contact. CONCLUSION: The QFT-positive rates correlated well with closeness of contact, while TST showed a poor correlation. Thus, QFT is considered more useful than TST for diagnosing tuberculosis infection.

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.

[A DRG/PPS simulation in the medical care of tuberculosis].

PURPOSE: To study the expected usefulness of the introduction of the DRG-PPS (Diagnosis-Related Group/Prospective Payment System, in which an insurer pays a fixed medical fee per hospitalization) into the current medical care of tuberculosis (TB) in Japan. METHOD: The medical fees were reviewed for all TB inpatients at 19 hospitals under the National Hospital Organization who were discharged in either June 2007 or February 2008. The sum of the fixed fee by the DRG was assumed based on the bivariate regression analysis of each patient's hospital days and his or her total actual fees during the hospital stay under the current (fee for care) system, since it was difficult to directly calculate the daily fees for every patient that would be the basis of DRG-PPS. RESULTS: Linear regression analysis estimated that the medical fees (including fees for the medical examinations and the treatments) for a hospital stay of 60 days, which is the standard for TB treatment, was 1,192,470 yen (19,870 yen per person per day) in June 2007, and 1,167,600 yen (19,460 yen per person per day) in February 2008. DISCUSSION: If we assume an average medical fee of about Y1.1-1.2 million yen for the standard hospital care of TB, the economic balance of the hospitals is negative, with a deficit of 0.6-0.7 million yen, given the estimated expenses of 1.8 million yen (i.e., 30,000 yen per person per day x 60 days). CONCLUSION: If the DRG-PPS is to be implemented based on the current medical fee rating system, the hospital administrators could not accept its introduction to the TB medical care service as it is, because it may undermine the economic management of hospitals.

Computed tomography findings in acute exacerbation of idiopathic pulmonary fibrosis.

RATIONALE: The serial computed tomography findings and prognosis of the acute exacerbation of idiopathic pulmonary fibrosis (IPF) are not yet well defined in a larger number of cases. OBJECTIVES: To evaluate the parenchymal abnormalities and prognosis using high-resolution computed tomography (HRCT) in acute exacerbation of IPF. METHODS: The study consisted of clinical, laboratory, and HRCT data before and at the time of acute exacerbation in 64 episodes of 58 patients with IPF. A semiquantitative analysis of overall extent of parenchymal abnormalities, extent of alveolar opacity (ground-glass attenuation and consolidation), and extent of fibrotic opacity (reticulation and honeycombing) on CT was performed by two chest radiologists. The newly appeared parenchymal abnormalities were also classified into three patterns: peripheral, multifocal, and diffuse. MEASUREMENTS AND MAIN RESULTS: In all patients, HRCT scans taken at the exacerbation showed typical signs of IPF and newly developing alveolar opacity. They included 34 patients of peripheral pattern, 8 of multifocal pattern, and 16 of diffuse pattern. Twenty-five patients died and 33 survived after the initial exacerbation. Worse survival was associated with patients with diffuse type compared with patients with multifocal and peripheral type. The CT patterns and overall CT extent were associated with an increased hazard of death after adjusting for age, sex, smoking, baseline diffusion capacity for carbon monoxide, baseline FVC, and disease extent on CT. On multivariate analysis, the strongest correlations were observed between CT patterns (combined diffuse and multifocal versus peripheral) and survival (odds ratio, 4.629; 95% confidence interval, 1.900-11.278; P = 0.001). CONCLUSIONS: HRCT extent and patterns are predictive of survival in acute exacerbation of IPF.

Serodiagnosis of Mycobacterium avium-complex pulmonary disease using an enzyme immunoassay kit.

RATIONALE: The diagnosis of Mycobacterium avium-complex pulmonary disease (MAC-PD) and/or its discrimination from pulmonary tuberculosis (TB) is sometimes complicated and time consuming. OBJECTIVES: We investigated in a six-institution multicenter study whether a serologic test based on an enzyme immunoassay (EIA) kit was useful for diagnosing MAC-PD and for distinguishing it from other lung diseases. METHODS: An EIA kit detecting serum IgA antibody to glycopeptidolipid core antigen specific for MAC was developed. Antibody levels were measured in sera from 70 patients with MAC-PD, 18 with MAC contamination, 37 with pulmonary TB, 45 with other lung diseases, and 76 healthy subjects. MEASUREMENTS AND MAIN RESULTS: Significantly higher serum IgA antibody levels were detected in patients with MAC-PD than in the other groups (P < 0.0001). Setting the cutoff point at 0.7 U/ml resulted in a sensitivity and specificity of the kit for diagnosing MAC-PD of 84.3 and 100%, respectively. Significantly higher antibody levels were also found in patients with nodular-bronchiectatic disease compared with fibrocavitary disease in MAC-PD (P < 0.05). There was a positive correlation between the extent of disease on chest computed tomography scans and the levels of antibody (r = 0.43, P < 0.05) in patients with MAC-PD. CONCLUSIONS: The EIA kit is useful for the rapid diagnosis of MAC-PD and for differentiating MAC-PD from pulmonary TB and, if validated by studies in other populations, could find wide application in clinical practice.

Characteristics of a large cohort of patients with autoimmune pulmonary alveolar proteinosis in Japan.

RATIONALE: Acquired pulmonary alveolar proteinosis (PAP) is a syndrome characterized by pulmonary surfactant accumulation occurring in association with granulocyte/macrophage colony-stimulating factor autoantibodies (autoimmune PAP) or as a consequence of another disease (secondary PAP). Because PAP is rare, prior reports were based on limited patient numbers or a synthesis of historical data. OBJECTIVES: To describe the epidemiologic, clinical, physiologic, and laboratory features of autoimmune PAP in a large, contemporaneous cohort of patients with PAP. METHODS: Over 6 years, 248 patients with PAP were enrolled in a Japanese national registry, including 223 with autoimmune PAP. MEASUREMENTS AND MAIN RESULTS: Autoimmune PAP represented 89.9% of cases and had a minimum incidence and prevalence of 0.49 and 6.2 per million, respectively. The male to female ratio was 2.1:1, and the median age at diagnosis was 51 years. A history of smoking occurred in 56%, and dust exposure occurred in 23%; instances of familial onset did not occur. Dyspnea was the most common presenting symptom, occurring in 54.3%. Importantly, 31.8% of patients were asymptomatic and were identified by health screening. Intercurrent illnesses, including infections, were infrequent. A disease severity score reflecting the presence of symptoms and degree of hypoxemia correlated well with carbon monoxide diffusing capacity and serum biomarkers, less well with pulmonary function, and not with granulocyte/macrophage colony-stimulating factor autoantibody levels or duration of disease. CONCLUSIONS: Autoimmune PAP had an incidence and prevalence higher than previously reported and was not strongly linked to smoking, occupational exposure, or other illnesses. The disease severity score and biomarkers provide novel and potentially useful outcome measures in PAP.

[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.

[The QFT-test, a new tool for control of nosocomial infection of tuberculosis].

In Japan, nosocomial transmission of tuberculosis from patients to hospital workers is not rare yet. The morbidity rate of tuberculosis among workers in national hospitals is higher (45.7 in 2003-2005) than the Japanese average rate (24.8 in 2003). The rate is especially high among nurses, indicating 73.2 in 3 years from 2003 to 2005. Although the indivisuals with latent tuberculosis infection (LTBI) are usually detected by tuberculin skin test in contact investigation, determination is not strict in BCG-vaccinated indivisuals. A novel diagnostic method (QFT-2G; QFT-test) can detect TB infection regardless past history of BCG vaccination. The tuberculin skin test and QFT-test were concurrently studied with 259 workers in National Hospital Organization Kinki-chuo Chest Medical Center. It is conjectured from the study-results that the QFT-test is a more accurate tool for detecting LTBI. Similar studies as 3 contact investigations in Kobe-city also came to the same inference. Although QFT-test is still new, and some questions remain to be answered, that is a useful test in medical examination for hospital workers, providing a new tool for control of nosocomial infection of TB.

[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.

[An adolescent case of pulmonary MAC infection, found 3 years later from bone marrow transplantation for myelodysplastic syndrome].

Mycobacterium avium complex (MAC) pulmonary infection is usually seen in elderly persons. We encountered a rare case of MAC pulmonary disease seen in a 19-year-old adolescent. The patient had received bone marrow transplantation at the age of 16 for myelodysplastic syndrome. Subsequently, he developed constrictive bronchiolitis and has been treated with corticosteroid and taclorimus. At age 19, small or fine nodules and a cavitary nodule in right lung were detected on the chest radiograph and computed tomography. Afterwards, Mycobacterium avium was detected by bronchoscopic examination and sputum examination and he was diagnosed as MAC pulmonary infection. MAC pulmonary infection in a young person at the age of 19 is an extremely rare case, in which constrictive bronchiolitis, immunosuppression by corticosteroid and tacrolimus, and diabetes mellitus were considered as critical predisposing factors.

[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.

[Clinical research on multi-drug resistant and extensive drug-resistant tuberculosis in Japan].

PURPOSE: To know the number of patients with MDR-TB and XDR-TB newly diagnosed annually and the number of those with continuously culture-positive in spite of continued treatment. METHODS: To fill the questionnaire sent to all the TB hospitals in Japan and to investigate the number of beds for TB, MDR-TB cases newly admitted to each hospital in 2006 and chronic bacillary cases in spite of continued treatment. RESULT: We sent the questionnaires to all the TB hospitals in Japan and 81% of 270 hospitals replied. As the result, 93 MDR-TB were newly hospitalized (12 cases were with XDR-TB). 76 cases of them were newly diagnosed MDR-TB. Almost after 1 year treatment, patients with XDR-TB showed lower negative conversion rate than other MDR-TB (42.9% vs 63.8%) and higher fatality rate (42.9% vs 7.2%). Excluding above 93 new MDR-TB cases, 103 cases with chronic MDR-TB including 44 cases with XDR-TB had been treated during the observation period, 84 case had been hospitalized and other 19 cases at OPD. DISCUSSION: Since 2000, extensive multi-resistant (XDR) TB has been a global topic. In Japan, nation-wide survey on 2002 showed the ratio of MDR and XDR were 1.9% and 0.6% respectively out of 3122 TB stains investigated. XDR/MDR rate was higher than those in other countries. Our clinical based investigation showed total XDR/MDR rate was 28.6% (56/196) and it was similar to that of previous bacteriological survey in 2002. CONCLUSION: We investigated the number of patients with MDR-TB and XDR-TB newly diagnosed in 2006 and the number of those who were continuously culture-positive. The survey showed that there were 196 patients with MDR-TB, and 56 patients of them (28.6%) were with XDR-TB. Many of them were in the districts of Kinki area and Kanto-Shinetsu area and 70% of them had been treated in the hospitals belonging to the National Hospital Organization.

Inhalational talc pneumoconiosis: radiographic and CT findings in 14 patients.

OBJECTIVE: The purpose of this study was to evaluate the radiographic and CT findings of inhalational talc pneumoconiosis. CONCLUSION: Large opacities of talc pneumoconiosis progress more often than do small opacities. The CT findings of talc pneumoconiosis overlap those of silicosis and asbestosis.

[Molecular epidemiology of Mycobacterium tuberculosis--comparison between multidrug-resistant strains and susceptible strains].

PURPOSE: Comparing multidrug-resistant M. tuberculosis strains with susceptible strains by molecular epidemiological methods. 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). RESULTS: 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. CONCLUSION: 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.

[Molecular epidemiological analysis of Mycobacterium kansasii isolates].

PURPOSE: To make molecular epidemiological analysis of Mycobacterium kansasii (M. kansasii) isolates. METHODS: 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. RESULTS: 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. DISCUSSION: 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.