(Vo.73 No.5 May 1998) <1>Kekkaku Vol.73, No.5: 329-337, 1998 LABORATORY MEDIA FOR THE CULTIVATION OF TUBERCLE BACILLUS Hajime SAITO* A variety of different media for the cultivation of mycobacteria have been described but a few of them are in use today. Those currently used can be characterized by three basic types. The first is egg-based media represented by Ogawa and LoNwenstein-Jensen. The second type is agar-based media ; the most common one are Middlebrook 7H1O and 7H11. The third type is liquid media such as Middlebrook 7H9. Several weeks of incubation may be required for the isolation of M. tuberculosis on solid media. Substantial improvement in the time to detection and the recovery rate was realized by using broth-based culture system such as the BACTEC 460TB, Septi-Chek AFB, MGIT and BACTEC 9000. In the BACTEC 460TB system, the mycobacteria is detected radiometrically. The proc- essed specimen is added to a modified 7H9 medium (BACTEC 12B) containing 14C-labeled palmitic acid and an antibiotic complex, PANTA. Mycobacterial growth can be ascertained by the liberation of 14C02 and detected by BACTEC 460TB instrument. The Septi-Chek AFB is a biphasic medium which combines broth and solid media. The liq- uid medium is a modified Middlebrook 7H9 in a carbon-dioxide-enriched culture bottle. After inoculation of the sample, the bottle is capped with a slide consisting of three solid media ; a non-selective Middlebrook 7H11 agar, an egg-based medium, and chocolate agar. A novel system is the MGIT, which is a nonradiometric broth method for the detection of mycobacteria from clinical specimens. The MGIT consists of a modified Middlebrook 7H9 broth and a sensor embedded in silicone on the bottom of a tube. The appearance of orange -colored fluorescence in the sensor when excited indicates the growth of mycobacteria. MB Redox is a modified, serum-supplemented Kirchner medium containing p-indonitro- tetrazolium violet (INT) as an indicator of microbial growth. The INT is reduced by the redox system of the mycobacteria to deep violet-colored formazan. This substance is water insoluble and is reduced to the cell surface, by which bacterial clamps can be easily detected by their violet color. At present, the egg-based media are the first choice for the culture of clinical samples. However, there are advantages to each type of medium and not all strains of mycobacteria can be recovered on a single medium. Therefore, it is recommended that one representative of each type of medium be used for primary isolation ; one example in Japan may be Ogawa egg medium in combination with Middlebrook 7H11 and MGIT. Key words : Culture media, Rapid diagnosis, MGIT, BACTEC 460 system, MB Redox *From the Hiroshima Environment and Health Association, 9-1 Hirosekita-machi, Naka-ku, Hiroshima 730-8631 Japan. (Received 26 Jan. 1998) <2>Kekkaku Vol.73, No.5: 339-347, 1998 DEVELOPMENT OF THE INTRATRACHEAL INFECTION MODEL OF EXPERIMENTAL MURINE MYCOBACTERIOSIS : Comparison with the Intravenous Infection Model Norio DOI* An intratracheal infection method of experimental murine mycobacteriosis was devel- oped for an in vivo study of antimycobacterial agents. Two models of intratracheal (IT) and intravenous (IV) routes of infection with mycobacteria of the same inoculum dose were compared in terms of the mean survival days of mice or bacterial loads in organs during the course of infection. IT model with either of M. bovis Ravenel, M. tuberculosis Kurono, M. tuberculosis H37Rv or M. intracellulare N-256 exhibited a much more distinct lung-specific infection than IV model with the same dose of respective mycobacterial strains. The intratracheal infection method presented in this report dose not require any special equipment and is a much safer method for the researcher than airborne infection. In this model, following slight anesthetizing of mice, bacillary suspension was injected quantita- tively into lungs through the mouth and trachea by using a specially modified needle set with a short fine polyethylene tube. This IT model may be useful not only for the in vivo assessment of anti-mycobacterial agents but also for the comparison of virulence among various mycobacterial strains. Key words : Intratracheal infection, Intravenous infection, Mouse, Mycobacterium tu- berculosis, Mycobacterium intracellulare *From the Department of Basic Research, Research Institute of Tuberculosis, Japan Anti- Tuberculosis Association, 3-1-24, Matsuyama, Kiyose, Tokyo 204-0022 Japan. (Received 27 Oct. 1997/ Accepted 12 Dec. 1997) <3>Kekkaku Vol.73, No.5: 349-353, 1998 DIAGNOSTIC USEFULNESS OF THE TUBERCULlN REACTION BY PPD-B IN MYC0BACTERIUM AVIUM COMPLEX DISEASE Yasuko HARADA*, Susumu HARADA, Akira KAJIKI, Yoshinari KITAHARA, Masahiro TAKAMOTO and Tsuneo ISHIBASHI This study was carried out to clarify the diagnostic usefulness of the skin tests of pu- rified protein derivatives from M.intracellulare (PPD-B) and M. tuberculosis (PPDs). Study subjects consisted of 41 patients with primary infection type of M. avium complex (MAC) disease, 36 patients with pulmonary tuberculosis (TB) and 29 patients with other bacterial respiratory infections including COPD (OB). The patients were sorted out to middle (50-69 y.o.) and old (70-89 y.o.) age groups of each disease. The size of skin red- ness elicited 48 hours after the PPD-B and PPDS intradermal injections were compared among them. The results were as follows. 1) Both PPD-B and PPDs skin reactions were larger in the middle age group than in the old one for each disease. 2) In PPDs skin tests, the reaction of TB group was the largest among the three dis- eases. in PPD-B skin tests, that of MAC was the largest. 3) In TB group, PPDs skin reaction.was significantly larger than that of PPD-B, while in MAC and OB groups there was no significant difference between the skin reactions of PPDs and PPD-B. 4) Defining significant positive reaction to PPD-B as PPD-B skin reaction exceeding 10mm and larger than that of PPDs skin reaction, the rate of significant positive reac- tion to PPD-B was significantly higher in MAC than TB in both age groups. These results showed that the simultaneous skin tests of PPD-B and PPDs were a use- ful aid in the diagnosis of MAC infection disease when mycobacterial infection diseases were clinically suspected by bacteriological or chest readiographic examinations. Key words : Tuberculin test, PPD-B, PPDs, Mycobacterium avium complex *From the Department of Internal Medicine, National Ohmuta Hospital, 1044-1, Tachibana, Ohmuta, Fukuoka 837-0911 Japan. (Received 8 Sep. 1997/ Accepted 17 Dec.1997) <4>Kekkaku Vol.73, No.5 :355-359, 1998 A PREVENTABLE CASE WHO DIED OF MILIARY TUBERCULOSIS AFTER RECEIVlNG CONTACT EXAMlNATION Yuka SASAKI*, Fumio YAMAGISHI, Fumio MIZUTANI, Takenori YAGI, Yuji TADA and Seiichirou SAKAO A 30-year-old man was admitted to our hospital because of headache and fever. His consciousness on admission was clouding. Sputum examination was positive for acid fast bacilli which later identified as Mycobacterium tuberculosis. Chest X-ray and computed tomogram on admission showed multiple cavitary lesions on bilateral upper lung fields and bilateral diffuse nodular shadow. He was diagnosed as miliary tuberculosis with tuberculous meningitis. His mother admitted because of pulmonary tuberculosis four months ago, and her sputum examination was smear positive for acid fast bacilli, Gaffky 4, and she complained of cough for 6 months before admission. Because of this situation, he rapidly underwent the contact examination with chest X-ray, but not exam- ined by tuberculin skin test because he was 30-year-old. As then chest X-ray was normal, he was not indicated of chemoprophylaxis, and he died of miliary tuberculosis and tuberculous meningitis 4 months after the contact examination. Key words : Miliary tuberculosis, Family infection, Contacts examination *From the Thoracic Department of National Chiba Higashi Hospital, Nitona cho 673, Chu-ou ku, Chiba City, Chiba 260-8712 Japan. (Received 6 Nov. 1997/ Accepted 13 Jan. 1998) <5>Kekkaku Vol.73, No.5: 361-363, 1998 K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE Chairpersons : Fujiya KISHI* Mitsunori SAKATANl** Symposium Topics and Presenters : 1 . Mechanisms of host resistance to Mycobacterium avium comples and Mycobacte- rium tuberculosis infection : Haruaki TOMIOKA, et al. (Shimane Medical Univer- sity) (Vol.73, N0.2 : 71-76) 2 . Establishment of new diagnostic technique and its clinical application for nontuberculous mycobacterial infections. : Hironobu KOGA (Sankohkai Miyazaki Hospital) 3 . The short term effects of multidrug chemotherapy for pulmonary Mycobacterium avium-intracellulare complex (MAC) infection : Atsuyuki KURASHIMA (National Tokyo Hospital) Additional Comment (1). The view of development of new drugs against nontuberculous mycobacterial infections : Shin KAWAHARA, et al. (National Minami-Okayama Hospital) (Vol.73, N0.2 : 77-82) Additional Comment (2). Long-term prognosis of Mycobacterium avium complex dis- ease : Eriko SHIGETO (National Hiroshima Hospital) (Vol.73, N0.2 : 83-85) 4 . Infection with Human Immunodeficiency Virus (HIV) and nontuberculous myco- bacteriosis : Seiji MIZUTANl (JATA, Fukujuji Hospital) (Vol.73, N0.2 : 87-92) Additional Comment. Geographical distribution of Mycobacterium avium complex in environment and serovars of Mycobacterium avium complex isolates from patients with and without AIDS : Hajime SAITO (Hiroshima Environment and Health Asso- ciation), et al. In the past decade, the non-tuberculous mycobacterial (NTM) infection was a rare disease but now is the common and increasing pulmonary infection in Japan. It is sus- pected that about 16% of patients with positive AFB in their sputa are the NTM pa- tients. It is well known that disseminated M. avium complex infection is an important complication of HIV disease. It will be worth to summarize some basic and clinical topics of this infectious disease in this symposium. The profiles of the antimicrobial action of microbicidal effector molecules against Mycobacterium avium complex (MAC) were presented by Dr. Tomioka, who reported the collaborating effect of reactive nitrogen intermediates with free fatty acids is crucial for macrophage-mediated intracellular killing of AFB. In addition, the auther discussed some important roles of type-K alveolar epithelial cells as a target cell for primary invasion of AFB in the host lungs. Dr. Koga reviewed the clinical usefullness of Amplicor, one of commercially available PCR detection kits for MAC. The MAC was detected in clinical samples such as sputum, bronchial washing fluids and gastric juice with 5 to 10% in frequency. The 60% to 80% of MAC positive cases were finally diagnosed as MAC disease and treated. He recom- mended that the positive result of genetic diagnostic method such as Amplicor should be carefully considered for clinical use. Dr. Kurashima summarized the short term effects of chemotherapy for pulmonary MAC infection. The auther analysed the 170 MAC cases treated with multi-drug chemotherapies which had not changed for 6 months or more. At 2 months after starting chemotherapy, 57.l% of patients obtained negative conversion but at 6 months only 28.9% remained bacilli negative. He recommended the regimen with combination of more than 3 drugs including aminoglycosid and clarithromycin. Two authers supplemented additional comments. Dr. Shigeto reported the long-term prognosis of MAC disease, indicating 64.7% obtained negative conversion and 35.5% re- mained bacteriologically negative in 71 patients who treated with at least 3 drugs and for more than 12 months. In addition, in the 12 patients who underwent surgical therapy, only one patient deteriorated. Dr. Kawahara precisely reported the basic preclinical evaluation of promising new drugs including new quinolones and new macrolides and Rifamycins. He also reviewed the clinical impact of these drugs. The present status of HIV infection and NTM in Japan was presented by Dr. Mizutani. All of 32 reported cases are male, in which 23 were with MAC and 6 were with M. kansasii disease. The 3 cases in 4 without bacteremia were patients with M. kansasii. The Mean count of CD 4+ Iymphocytes in the peripheral bloods of these 32 patients was only 11/mm3 . As additional comments, the geographical distribution in environment and serovars of MAC were reported by Dr. Saito. It was not found that the difference in the geo- graphic distribution between soil MAC and disease-associated MAC. The 55% of soil MAC reacted with the AccuProbe MAC but not with the AccuProbe M. avium and M. intracellulare. The auther also demonstrated that 38 MAC strains isolated from 38 Japa- nese AIDS patients were all M. avium and serovars of Japanese MAC from AIDS patients are similar to those of the American MAC strains from such patients. *From the National Sapporo Minami Hospital, Shirakawa 1814, Minami-ku, Sapporo 061-2276 Japan. **From the National Kinki-Chuo Hospital, Nagasone-cho 1180, Sakai 591-8555 Japan. (Received 30 Mar. 1998) <6>Kekkaku Vol.73, No.5:365-370, 1998 The 72nd Annual Meeting Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 2. ESTABLISHMENT OF NEW DIAGNOSTIC TECHNIQUE AND ITS CLlNICAL APPLICATION FOR NONTUBERCULOUS MYCOBACTERIAL INFECTIONS Hironobu KOGA* The clinical futures of 52 patients with pulmonary Mycobacterium avium complex in- fection (MAC group), 7 M.kansasii infection (MK group), and 5 M.chelonae infection (MC group) were studied. Aged female was dominant in MAC and MC group, while middleaged man was dominant in MK group. Although body mass index (BMI) was the lowest in MAC group, there was no difference between the groups in clinical sign, symp- tom, and laboratory data. Type L radiological finding was dominant in MAC group, while type K in MK and MC groups. Bacteriological eradication rate of causative organ- isms was 86% in MK group, 54% in MAC group, and 40% in MC group. However, the relation between clinical efficacy of chemotherapy, BMI, serum protein level, and cavity formation in chest radiogram was not observed. MAC was detected in clinical samples such as sputum, bronchial washing fluid, and gastric juice with 5 to 10% in frequency by Amplicor, one of commercially available PCR detection kits for mycobacteria. Furthermore, 60% to 80% of MAC positive cases were diagnosed and treated as nontuberculous mycobacteriosis. Although MAC is occasionally exist in oral cavity or clinical environment, the positive result of genetic diagnostic method such as Amplicor should be carefully considered. Key words : Nontuberculous mycobacteriosis, Mycobacterium avium complex, M.kansasii, M.chelonae, Amplicor *From the Sankohkai Miyazaki Hospital, 1575-1 Kuyama, Isahaya 854-0066 Japan. (Received 4 Mar.1998) <7>Kekkaku Vol.73, No.5: 371-377, 1998 The 72nd Annual Meeting Symposium U. NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 3 . THE SHORT TERM EFFECTS OF MULTIDRUG CHEMOTHERAPY FOR PULMONARY MYCOBACTERIUM AVIUM-INTRACELLULARE COMPLEX (MAC) INFECTION Atsuyuki KURASHIMA* In performing MAC chemotherapy, we are faced with the following two problems : first, unlike for Mycobacterium tuberculosis, we have neither any suitable bacteriocidal drugs nor bacteriocidal regimens for MAC treatment ; secondly, in case of MAC, unlike in the case of Mycobacterium tuberculosis, in vitro sensitivity does not correlate with in vivo sensitivity. For these reasons, we find difficulty in planning a rational protocol for MAC treat- ment, and thus depend on previous experiences of other physicians in MAC chemotherapy. Here, we have tried to evaluate such previous experiences objectively, and studied retro- spectively the effects of previous combination chemotherapy cases of pulmonary MAC in- fections at National Tokyo Hospital. We selected 170 cases which had received the same chemotherapy continuously over a period of six months. Concentration of bacilli cultured, 8 weeks in Ogawa solid egg containing medium was translated semi-quantitatively to colony forming units (CFU) according to the Japanese guideline for acid fast bacilli test. Having set a mean CFU of 3 times sputum culture before treatment as 100%, we cal- culated a six month sequential bacillary response to a regimen and plotted the bacillary response curves. The response curve of the total 170 MAC treatment cases exhibits a minimum point of 42.9% at 2 months, and subsequently rises to 71.1% after six months compared to the state before treatment. The response curves of various regimens of multidrug chemotherapy indicate that combinations of more than 3 drugs including aminoglycosid and clarithromycin are most effective. However, although some effectiveness is indicated, neither the present drugs nor regi- mens are capable to achieve a bacteriocidal effect in MAC treatment. Key words : Non tuberculous mycobacteriosis, Mycobacterium avium - intracellulare complex, Chemotherapy, Multidrugchemotherapy, Clarithromycin, Aminoglycosid *From the Department of Pulmonary Medicine, National Tokyo Hospital, 3-1-1, Takeoka, Kiyose City, Tokyo 204-8585 Japan. (Received 9 Mar. 1998) <8>Kekkaku Vol.73, No.5: 379-383, 1998 The 72nd Annual Meeting Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 4'. GEOGRAPHICAL DISTRIBUTION OF MYCOE:ACTERIUM AVIUM COMPLEX IN ENVIRONMENT AND SEROVARS OF MYCOBACTERIUM A VIUM COMPLEX ISOLATES FROM PATIENTS WITH AND WITHOUT AIDS Hajime SAITO*, David DAWSON, Masanori KAI and Kazuo KOBAYASHI Mycobacterium avium complex (MAC) organisms have been isolated from water and soil. It is now generally accepted that environmental sources, especially natural waters, are the reservoirs for most human infections caused by MAC. Previously, we reported that M. avium and M. intracellulare were distributed predominantly in the eastern and western part of Japan, respectively. To clarify the factor(s) of the difference, the fol- lowing experiment was undertaken. MAC was isolated from soil samples collected in Tokai, Kinki and Chugoku districts, by the method of lchiyama et al. MAC isolates were identified by AccuProbe Confirma- tion and Identification tests, together with some conventional tests. Seven (11.7%) of 60 isolates were identified as M. avium, twenty (33%) as M. intracellulare and 33 (55%) as MAC-like organisms. Therefore, it was not found that the difference in the geographical distribution between soil MAC and disease-associated MAC. MAC-like strains possess MAC-specific antigen, and biological and biochemical features of MAC. They reacted with the AccuProbe MAC but not with the AccuProbe M. avium and M. intracellulare. Se- quencing analysis of 16s RNA gene implies that MAC-like strains show an intermediate sequence pattern of M. avium and M. intracellulare. HPLC patterns of these strains were compatible with those of MAC. It is known that the major serovars of MAC isolates from patients with or without AIDS are different. Serological aspects in this respect are not yet elucidated well in Japan. The major serovars of MAC from patients with AIDS are 4 and 8 in the USA and Australia, 6 and 4 in Sweden, and 8/21 and 8 in Germany. On the other hand, the major serovars of MAC from patients without AIDS are 8 and 16 (15) in the USA and 6 and 1 in Sweden. According to our recent study the major serovars of MAC from non-AIDS patients are 1 and 8 in the eastern part and 16 and 14 in the western part of Japan. In the present study, 38 MAC isolates from sputum, stool and blood of AIDS patients were identified. All of the isolates were identified as M. avium. Serovars of 15 strains were 4 (4 strains), 8 (3 strains), 9 (3 strains), 3 (2 strains), Darkin (2 strains) and 1 (1 strain). Therefore, it seems that the major serovars of the Japanese MAC strains from AIDS patients are similar to those of the American MAC strains from such patients. Key words : Mycobacterium avium complex, Mycobacterium avium, Mycobacterium intracellulare, AIDS, Serovar , *From the Hiroshima Environment and Health association, Hiroshima 730-8631 Japan. (Received 26 Mar.1998)