(Vol.77 No.8 August 2002) <1> Kekkaku Vol.77,No.8:536-546,2002 Original Article MICROPLATE HYBRIDIZATION METHODS OF REDUCED UNSPECIFIC REASSOCIATION CONSIDERING ¢Tm-Comparison between Relative Relatedness of DNA and Key Characters for Mycobacterium szulgai Yutaka FUKASAWA Abstract It has been recognized that colorimetric microdilution plate hybridization method(DDH) shows equivocal identification results for some strains of Mycobacterium gordonae, and that chemotaxonomic identification method reveals some intermediate pattern between Mycobacterium szulgai and M.gordonae. In the present study, the results obtained by chemotaxonomic identification method on 25 strains of M.szulgai were compared with those obtained by DDH method. In chemotaxonomic methods, 8 of 25 M.szulgai strains showed negative results on 14 days' tween80 hydrolysis, 14 strains revealed negative nitrate reduction by the Tsukamura's method but all were positive by the Virtanen's method. Smooth colonies were found in 3 strains including M.szulgai type strain, JATA3201(ATCC35799). Relative relatedness (relative color index) of genomic DNA was measured by DDH instead of spectrophotometric genomic DNA-DNA relatedness. A relative relatedness of 25 M.szulgai strains tested showed higher levels than 80%, but the inter-species relatedness to the other mycobacteria also showed high levels of 50-75%, when hybridizing temperature was set at 40Ž. At 56Ž, intra-species relative relatedness in 4 strains were lower than 50%, indicating that this condition is not appropriate. When hybridization temperature raised to 56Ž after overnight at 40Ž, a relative relatedness among 25 strains were again high (>80%), and those to the other Mycobacterial species were lower than 70%. When hybridized at 56Ž after over night at 45Ž, an intra- species relative relatedness again showed higher levels than 70% in all 25 strains, and interspecies percentiles were lowered satisfactorily to <25%. In conclusion, through avoiding reassociation of nonspecific DNA fragments during the hybridization process, 45Ž overnight followed by 56Ž hybridization (¢DDH method) was found to be the better condition for identification and classification of Mycobacterium szulgai. Key words:Mycobacterium, Identification, Classification, DDH, Microplate hybridization, Relative relatedness, ¢Tm Main Office, Japan Anti-tuberculosis Association Correspondence to:Yutaka Fukasawa, Department of Microbiology and Bioinformatics, Gifu University Graduate School of Medicine, 40, Tsukasa-machi, Gifu-shi, Gifu 500-8705 Japan. (E-mail:h2801105@guedu.cc.gifu-u.ac.jp) <2> Kekkaku Vol.77,No.8:547-554,2002 Original Article RELATIVE RELATEDNESS OF 11 MYCOBACTERIA SEPCIES BY MICROPLATE HYBRIDIZATION METHODS CONSIDERING ¢Tm Yutaka FUKASAWA Abstract Intra-species variance within mycobacterium xenopi, Mycobacterium gordonae or Mycobacterium szulgai has been reported in identification employing chemotaxonomic characteristics, 16s rRNA gene sequences or relative relatedness (relative color index) of genomic DNA-DNA Hybridization. Genomic DNA-DNA reassociation at the constant temperature was found to be unreliable for classification of mycobacterial species. However, nonspecific DNA reassociation could be avoided by hybridization at 56Ž after 45Ž overnight. and this technique was named ¢DDH method in the preceding paper. The present report shows relative relatedness (relative color index) of genomic DNA in ¢DDH method among mycobacterial species. Relative relatedness was below 70% among BCG, M.kansasii, M.simiae, M.asiaticum, M.szulgai, M.gordonae, M.xenopi and M.nonchromogenicum. The results satisfied the criteria for bacterial classification, which was proposed by the International Committee for Systematic Bacteriology in 1987. In regard to Mycobacterium avium complex, relative relatedness between M.avium and M.intracellulare were approximately 75%. It appeared that M.avium and M.intracellulare could be classified into one species. It has been recognized, moreover, that there are intermediate strains between M.avium and M.intracellulare. Previously, numerical classification raised a concept of Mycobacterium avium- intracellulare-scrofulaceum complex. The present study revealed that relative relatedness of M.avium and of M.intracellulare to M.scrofulaceum were around 75%, while the percentiles of M.scrofulaceum relative to M.avium and that to M.intracellulare were both less than 50%. The relative relatedness of M.ulcerans against M.marinum was nearly 65%, whereas the relative relatedness of M.marinum against M.ulcerans was approximately 90%. The data may be partly explained by the horizontal gene transfer mechanism. Key words:Mycobacterium, Identification, Classification, Microplate hybridization, ¢Tm, Relative relatedness Main Office, Japan Anti-tuberculosis Association Correspondence to:Yutaka Fukasawa, Department of Microbiology and Bioinformatics, Gifu University Graduate School of Medicine, 40, Tsukasa-machi, Gifu-shi, Gifu 500-8705 Japan. (E-mail:h2801105@guedu.cc.gifu-u.ac.jp) <3> Kekkaku Vol.77,No.8:555-561,2002 Original Article TUBERCULOSIS CONTROL IN KOBE CITY-ANALYSES OF ADVISORY CONTENTS AND DISQUALIFIED CASES BY INTEGRATED TUBERCULOSIS ADVISORY COMMITTEE Chika SHIRAI Abstract The incidence of tuberculosis (TB) in Kobe City has been higher in comparison with that of Japan. thereby Kobe City ought to enforce anti-tuberculosis activities. Nine Wards public health departments of Kobe City used to convene their own tuberculosis advisory committees until April 1998, when a centralized committee was established to examine all TB patients. The new committee was authorized to issue clinical advice to registered physicians whenever necessary in order to improve treatment standard of TB. The author analyzed the contents of issued documents and observed any change in statistics before and after establishment of the committee. During the past 3 years, the new committee had sent 2,221 advisory documents to physicians, which occupied 35% of all 6,305 applied cases. Main suggestions included proper choice of anti-tuberculosis agents, adequate duration of treatment and reference to the results of laboratory examinations. In addition, 140 cases were rejected for medical expenses subsidy by TB Law to avoid unnecessary long-term treatment. Main reasons of rejection were long-term treatment despite negative bacilli too more than one year, continuous single INH administration following the completion of the standard treatment and unnecessary treatment for the cases with inactive findings on chest X-ray. Since the establishment of the integrated committee, the statistics of TB has demonstrated a significant decrease in the incidence and the prevalence of TB with its annual reduction of 5.6, and 11.5 per 100,000 respectively. The proportion of bacteriologically confirmed cases among newly registered patients has increased, and the diagnostic accuracy was improved due to the decrease in the unknown bacteriology case. The proportion of the case treated by INH alone fall down rapidly and it is lower than that of the whole country. These data demonstrate that the integrated tuberculosis advisory committee contributes to standardize antitubercular treatment through dissemination of proper guidance on TB diagnosis and treatment to all medical facilities in the city. Key words:Tuberculosis advisory committee, Statistics of tuberculosis, Advisory documents to physicians, Treatment standard Public Health Center of Kobe City Correspondence to:Chika Shirai, Public Health Center of Kobe City, 5-1-1, Kumoi-dori, Chuo-ku, Kobe-shi, Hyogo 651-8570 Japan. (E-mail:dfbvv106@kcc.zaq.ne.jp) <4> Kekkaku Vol.77,No.8:563-567,2002 Case Report A CASE OF PERFORATIVE PERITONITIS COMPLICATED WITH LUNG AND INTESTINAL SEVERE TUBERCULOSIS 1Fuminobu KURODA, 2Takenori YAGI, 2Fumio YAMAGISHI, 2Yuka SASAKI, 2Tomoko HAMAOKA, and 1Hiromi HIGURASHI Abstract A 27-year-old man was admitted to our hospital in September 18, 2000, complaining of fever, cough, appetite loss and body weight loss. He was diagnosed as advanced lung tuberculosis, because of chest X-ray findings and positive acid-fast bacilli in his sputum. He was administrated rifampicin (RFP), isoniazid (INH), and ethambutol (EB), Two days after starting treatment he complained of abdominal pain and the signs of perforating peritonitis. Emergency laparotomy was performed and we observed multiple ulcers and a perforation of ileum. We resected a part of distal ileum and ascending colon and made ileostomy. Histopathologic examination of resected ileum and colon showed multiple ulcers and epithelioid cell granulomas with caseous necrosis. Many acid bacilli were identified from the lesion by specially stained tissue sections. He was administrated streptomycin and INH by injection post-operatively while oral administration was impossible. Six days after the first operation, we found the signs of perforation in another part of the ileum. So we were obliged to perform second laparotomy and resect the part involved. Five days after the second operation, he was able to take RFP, INH, and levofloxacin per oral route. On February 8, 2001 we performed ileocolonal reconstruction with side to side anastomosis and closed ileostomy at the third laparotomy. He had continued chemotherapy and went back to Korea in April 7, 2001. Although intestinal tuberculosis has sharply declined in Japan thanks to development of effective antituberculous drugs, we should keep in mind that it could be a possible cause of the acute abdomen. Key words:Intestinal tuberculosis, Laparotomy, Perforation, Peritonitis, Ileostomy, Foreigner 1Department of Chest Medicine, School of Medicine, Chiba University, 2Division of Thoracic Disease, National Chiba-Higashi Hospital Correspondence to:Fuminobu Kuroda, Department of Chest Medicine, School of Medicine, Chiba University, 1-8-1, Ino-hana, Chuo-ku, Chiba-shi, Chiba 260-8677 Japan. (E-mail:fkuroda@insei.m.chiba-u.ac.jp) <5> Kekkaku Vol.77,No.8:569-571,2002 Short Report CHEMOTHERAPY WITH ISONIAZID AND RIFAMPICIN FOR PULMONARY TUBERCULOSIS Kenji KAWAKAMI Abstract Two-drug regimen of chemotherapy with isoniazid and rifampicin for pulmonary tuberculosis is the one of the standard methods of treatment for active pulmonary tuberculosis in Japan since 1996, while ATS/CDC and WHO/IUATLD recommended the three-drug or four-drug regimen containing chemotherapy pyrazinamide unless the prevalence of drug resistance, among new tuberculosis cases is low. We sent a questioners of 141 tuberculosis centers in Japan to investigate the rate of each standard chemotherapy regimen employed and reasons for selecting the two-drug regimen in each center. Of 3840 newly diagnosed cases in the 57 centers, 47.4% were treated with the four-drug regimen and 37.1% with the three- drug regimen, and two-drug regimen was employed in only 2.6% Sputum smear-negative status was the major reason for selecting the two-drug regimen of chemotherapy. Though the two-drug regimen was used in few cases, taking into account the prevalence of primary resistance to isoniazid in Japan in 1997 was 4.4%, it was concluded to discontinue recommending the two-drug regimen as one of standard methods of treatment for pulmonary tuberculosis. Key words:Pulmonary tuberculosis, Chemotherapy, Standard treatment, Isoniazid, Rifampicin Department of Respiratory Disease, Kawatana National Hospital Correspondence to:Kenji Kawakami, Department of Respiratory Disease, Kawatana National Hospital, 2005-1, Shimogumi-go, Kawatana-cho, Higashisonogi-gun, Nagasaki 859-3615 Japan. (E-mail:kkawamam@kwthp.hosp.go.jp) <6> Kekkaku Vol.77,No.8:573-584,2002 The 77th Annual Meeting Special Lecture PROSPECTS FOR DEVELOPMENT OF NEW ANTITUBERCULOUS DRUGS Haruaki TOMIOKA Abstract Tuberculosis (TB) is a growing international health concern, since it is the leading infectious cause of death in the world today. Moreover, the resurgence of TB in industrialized countries and the worldwide increase in the prevalence of Mycobacterium avium complex(MAC) infections in immunocompromised hosts have prompted the quest for new antimycobacterial drugs. In particular, the appearance of multidrug-resistant(MDR) strains of M. tuberculosis, which exhibit in vitro resistance to at least two major antituberculous drug (usually INH and RFP) and cause intractable TB, has greatly contributed to the increased incidence of TB. Because of the global health problems of TB, the increasing rate of MDR-TB and the high rate of a co-infection with HIV, the development of potent new antituberculous drugs without cross-resistance with known antimycobacterial agents is urgently needed. In this article, I reviewed the following areas. First, I briefly reviewed some new findings (mainly reported after 2000) on the pharmacological status of rifamycin derivatives (rifabutin, rifapentine, and rifalazil), fluoroquinolones (ciprofloxacin, ofloxacin, sparfloxacin, levofloxacin, gatifloxacin, sitafloxacin, moxifloxacin, and others), and new macrolides (clarithromycin, azithromycin, roxithromycin). Second, I decribed other types of agents which are being developed as antimycobacterial drugs. Some of the agents discussed are already under preliminary clinical investigation, and others appear to be promising candidates for future development. In this review, the status of the development of new antimycobacterial, especially antituberculous agents including oxazolidinone(PNU-100480), 5'-nitroimidazole(CGI 17341), 2-pyridone (ABT-255), new riminophenazines, nitroimidazopyran(PA-824), new ketolides(ABT-773, telithromycin) and defensins(human neutrophil peptide-1), was examined. Third, the development of new antitubercular drugs was discussed according to the potential pharmacological target. New critical information of the whole genome of M.tuber- culosis recently elucidated and increasing knowledge on various mycobacterial virulence genes will promote the progression in the identification of genes that code for new drug targets. Using such findings on mycobacterial genomes, drug development using quantitative structure-activity relationship may be possible in the near future. In this review, I described the screening of drugs that have an inhibitory activity against dTDP-rhamnose synthesis of M.tuberculosis, as a new drug target of the organism. In addition, I discussed the usefulness of antisense oligo DNAs specific to mycobacterial genes encoding oligo DNAs specific to mycobacterial genes encoding certain metabolic enzymes or virulence factors that play roles in the bacterial escape from antimicrobial mechanisms of host macrophages. Fourth, I reviewed the drug vehicles which enable efficacious drug delivery to their target in vivo. The usefulness of poly (DL-lactide-co-glycolide) microsphere technology, which enables the encapsulated drugs to deliver the requested doses of them for prolonged time periods by a single shot without causing any toxicity and, moreover, enables the highly targeted delivery of the drugs to host macrophages, was discussed. Fifth, I described adjunctive immunotherapy for the management of patients with mycobacterial infections by giving certain immunomodulators in combination with antimycobacterial drugs. Adjuvant clinical trials using IL-2 or GM-CSF have been found to be efficacious to some extent in improving patients with tuberculosis or disseminated MAC infections. However, it seems that these immunopotentiating cytokines as well as IFN-ƒÁ and IL-12 are not so promising for the therapeutic agents of mycobacterial infections because of the possible induction of immunosuppressive cytokines during adjuvant therapy and, in some cases, severe side-effect. Thus, the development of new classes of immunomodulators other than cytokines, particularly those with no severe side-effect, is needed. This review dealt with ATP and its analogues which potentiate macrophage antimycobacterial activity via a purinergic P2X(7) receptor. Finally, I described the roles of type II alveolar epithelial cells in the establishment of mycobacterial infections in the host lungs and the profiles of drug susceptibilities of M.tuberculosis and MAC organisms replicating within the type II pneumocytes. These findings are useful to precisely assess or predict the in vivo therapeutic activity of a given antimycobacterial drug from its in vitro activity. In this article, I have thoroughly reviewed the status of the development of new antimycobacterial drugs. There are a number of difficulties in the drug-design for the development of new drug formulations with increased potential for antimycobacterial effects, excellent pharmacokinetics, and tolerability. It should be emphasized that the most urgent goal of chemotherapy of tuberculosis and MAC infections, especially that associated with HIV infection, is to develop highly active, low-cost drugs which can be used not only in industrialized countries but also in developing countries, since the incidences of AIDS- associated intractable tuberculosis is rapidly increasing in the latter. Key words:Antituberculous drugs, Drug screening, Drug targets, Mycobacterium tuberculosis, Mycobacterium avium complex Department of Microbiology and Immunology, Shimane Medical University Correspondence to:Haruaki Tomioka, Department of Microbiology and Immunology, Shimane Medical University, 89-1, Enya-cho, Izumo-shi, Shimane 693-8501 Japan. (E-mail:tomioka@shimane-med.ac.jp) <7> Kekkaku Vol.77,No.8:585-588,2002 Memorial Lecture by the Imamura Award Winner, 2002 STUDY ON THE ROLES OF CYTOKINES INVOLVED IN MYCOBACTERIAL INFECTION Isamu SUGAWARA Abstract The roles of various cytokines in early-phase mycobacterial infection were investigated utilizing murine tuberculosis models. Among them, IFN-ƒÁ and TNF-ƒ¿ are very important in protective immunity against mycobacterial infection. This finding is closely associated with human tuberculosis. It is reported that persons with IFN-ƒÁ receptor 1 deficiency and patients with rheumatoid arthritis and Crohn's disease are susceptible to Mycobacterium tuberculosis. It is expected that a novel immunotherapy and a diagnostic method of tuberculosis are developed by clarifying roles of various cytokines immunologically in early-phase mycobacterial infection. Key words:Cytokine, Aerosol infection, M.tuberculosis, IFN-ƒÁ, TNF-ƒ¿ Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association Correspondence to:Isamu Sugawara, Department of Molecular Pathology, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24, Matsuyama, Kiyose-shi, Tokyo 204-8533 Japan. (E-mail:sugawara@jata.or.jp)