Moxifloxacin resistance and genotyping of Mycobacterium avium and Mycobacterium intracellulare isolates in Japan.

BACKGROUND: Although fluoroquinolones are considered as alternative therapies of pulmonary Mycobacterium avium complex (MAC) disease, the association between fluoroquinolone resistance and MAC genotypes in clinical isolates from individuals not previously treated for MAC infection is not fully clear. METHODS: Totals of 154 M. avium isolates and 35 Mycobacterium intracellulare isolates were obtained from treatment-naïve patients with pulmonary MAC disease at the diagnosis of MAC infection at 8 hospitals in Japan. Their susceptibilities of moxifloxacin were determined by broth microdilution methods. Moxifloxacin-resistant isolates were examined for mutations of gyrA and gyrB. Variable numbers of tandem repeats (VNTR) assay was performed using 15 M. avium VNTR loci and 16 M. intracellulare VNTR loci. RESULTS: Moxifloxacin susceptibility was categorized as resistant and intermediate for 6.5% and 16.9%, respectively, of M. avium isolates and 8.6% and 17.1% of M. intracellulare isolates. Although the isolates of both species had amino acid substitutions of Thr 96 and Thr 522 at the sites corresponding to Ser 95 in the M. tuberculosis GyrA and Gly 520 in the M. tuberculosis GyrB, respectively, these substitutions were observed irrespective of susceptibility and did not confer resistance. The VNTR assays showed revealed three clusters among M. avium isolates and two clusters among M. intracellulare isolates. No significant differences in moxifloxacin resistance were observed among these clusters. CONCLUSIONS: Although resistance or intermediate resistance to moxifloxacin was observed in approximately one-fourth of M. avium and M. intracellulare isolates, this resistance was not associated with mutations in gyrA and gyrB or with VNTR genotypes.

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.

[A case of cryptococcal empyema successfully treated by debridement by medical thoracoscopy with local anesthesia].

Cryptococcal empyema is a rare disease which usually occurs in immunocompromised patients. We describe a 57-year-old man with diabetes mellitus with a mass-like shadow in the right middle lung field. Transbronchial lung biopsy of the right lung revealed numerous yeast-like fungi in fibrotic and necrotic lesions. These findings, together with positive serum cryptococcal antigen yielded a diagnosis of pulmonary cryptococcosis secondary to diabetes mellitus. Despite treatment with several anti-fungal drugs, and dyspnea and pleural effusion developed. He was referred to our hospital for further examination and therapy. The presence of positive cryptococcal antigen and numerous yeast-like fungi were confirmed cytologically in the pleural effusion. Therefore, we suspected that pulmonary cryptococcosis had perforated into the thoracic space and empyema had developed. Because antifungal drugs were ineffective, debridement of the fibrinopurulent material by medical thoracoscopy and chest drainage were performed. The clinical symptoms of this patient improved with antifungal treatment for 1 year, and we successfully treated the cryptococcal empyema without recurrence. Debridement by medical thoracoscopy and chest drainage were useful for this case of cryptococcal empyema.

[Clinical application of testing methods on acid-fast bacteria].

Clinical bacteriology pertaining to acid-fast bacteria has made marked advances over the past decade, initiated by the development of a DNA probe kit for identification of acid-fast bacteria. Wide-spread use of nucleic acid amplification for rapid detection of tubercle bacillus contributed more greatly than any other factor to such advances in this field. At present, 90% of all kits used for nucleic acid amplification in the world are consumed in Japan. Unfortunately, not a few clinicians in Japan have a false idea that the smear method and nucleic acid amplification are necessary but culture is not. In any event nucleic acid amplification has exerted significant impacts on the routine works at bacteriology laboratories. Among others, collecting bacteria by pretreatment with NALC-NaOH has simplified the introduction of the collective mode smear method and liquid media. Furthermore, as clinicians have become increasingly more experienced with various methods of molecular biology, it now seems possible to apply these techniques for detection of genes encoding drug resistance and for utilization of molecular epidemiology in routine laboratory works. Meanwhile, attempts to diagnose acid-fast bacteriosis by checking blood for antibody have also been made, primarily in Japan. At present, two kits for detecting antibodies to glycolipids (LAM, TDM, etc.) are covered by national health insurance in Japan. We have an impression that in Japan clinicians do not have adequate knowledge and skill to make full use of these new testing methods clinically. We, as the chairmen of this symposium, hope that this symposium will help clinicians increase their skill related to new testing methods, eventually leading to stimulation of advances in clinical practices related to acid-fast bacteria in Japan. 1. Smear microscopy by concentration method and broth culture system: Kazunari TSUYUGUCHI (Clinical Research Center, National Hospital Organization Kinki-chuo Chest Medical Center) Smear microscopy and culture still remain the cornerstone to diagnose tuberculosis. However, the classical methods in Japan using direct microscopy and Ogawa solid media were not sufficient for clinical use. In recent years substantial advance has been made in these fields. Concentration of clinical samples by centrifugation improves the sensitivity of smear microscopy with excellent reproducibility. The Mycobacteria Growth Indicator Tube (MGIT) system using liquid media yields high sensitivity and rapidity. Using these methods, more and more tuberculosis cases would be correctly diagnosed and treated adequately based on drug susceptibility testing. 2. New technologies for anti-tuberculosis drug susceptibility testing: Satoshi MITARAI (Bacteriology Division, Reference Centre for Mycobacterium, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association) Several new technologies have been developed to obtain anti-tuberculosis drug susceptibility testing (AST) results rapidly, utilising liquid culture and molecular technologies. Mycobacterium Growth Indicator Tube (MGIT), as a popular liquid culturing and AST system, was evaluated for its accuracy and usefulness. As for isoniazid, MGIT showed 12.6% of discordant result comparing with standard method. These MGIT resistant and Ogawa susceptible strains had relatively high MICs ranging 0.13 to 2.0 microg/ml. The molecular detection of resistant gene mutation is also a useful method to estimate drug resistance rapidly. The rpoB mutation detection is reliable with high sensitivity and specificity. 3. Nucleic acid amplification and novel diagnostic methods: Shunji TAKAKURA (Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine) Sensitivities of nucleic acid amplification tests (NAATs) for the diagnosis of tuberculosis meet clinical requirement that patients with high-risk of transmission should be identified within a day. Comparison of the performance of various NAATs is difficult because of the difference in sample processing and in samples tested among methods and reports. Considering the limitations of NAATs (low sensitivity compared with culture, inability to differentiate dead bacilli from the living), further advances would be expected when novel technologies could confer additional information, such as drug susceptibility, quantity, viability, and genotype. 4. Serodiagnosis of Mycobacterium avium complex lung disease: Seigo KITADA (Department of Internal Medicine, National Hospital Organization Toneyama National Hospital) Mycobacterium avium complex (MAC) organisms are ubiquitous in environment and a contamination in respiratory tracts is sometimes observed, and that complex the diagnosis. We developed a serodiagnostic method for MAC disease using an enzyme immunoassay with the MAC-specific glycopeptidolipid (GPL) core as antigen. A significant increase in GPL core antibodies was detected in sera of patients with MAC pulmonary diseases compared to patients who were colonized with MAC, patients with M. kansasii disease and tuberculosis and healthy subjects. The serodiagnosis is useful for diagnosis of MAC lung disease. 5. Molecular epidemiologic tools for tuberculosis: IS6110 RFLP, Spoligotyping, and VNTR: Tomoshige MATSUMOTO, Hiromi ANO, Tetsuya TAKASHIMA, Izuo TSUYUGUCHI (Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) We have performed molecular typing on about 1,300 culture positive clinical isolates that made up the majority of tuberculosis strains in part of southeast Osaka since 2001 until now. By spoligotyping, about 75% of entire strains belonged to the Beijing strain. Particular spoligotyping descriptions, which were not described in SpolDBIII, were found in the strains with lower than 6 copies of IS6110 RFLP. We described them as Osaka type. We could also show that direct typing from Tb PCR positive sputum of patients with tuberculosis was possible by VNTR and that VNTR with 16 loci was useful in tuberculosis typing in Osaka.

Successful treatment with faropenem and clarithromycin of pulmonary Mycobacterium abscessus infection.

Mycobacterium abscessus accounts for 80% of rapidly growing mycobacterial pulmonary infections and can be lethal. Treatment is difficult because of the paucity of effective drugs. We describe a patient with pulmonary M. abscessus infection who was treated with a regimen that included faropenem, a novel oral penem, and clarithromycin. He showed favorable responses to the treatment for more than 12 months. In vitro, faropenem had considerable inhibitory activities against 56 strains of rapidly growing mycobacteria, including M. peregrinum, M. chelonae, M. fortuitum, and M. abscessus (stated in order of increasing minimal inhibitory concentrations). Thus, faropenem has the potential to be used as an adjunctive drug with clarithromycin for the treatment of infection with rapidly growing mycobacteria, including M. abscessus.