(Vol.73 No.2 February 1998) <1>Kekkaku Vol.73, No.2: 47-52, 1998 USEFULNESS OF MYCODOT TEST FOR THE DETECTION OF ANTI-MYCOBACTERIAL ANTIBODIES AS AN AID IN THE DIAGNOSIS OF EXPERIMENTAL MYCOBACTERIUM TUBERCULOSIS AND MYCOBACTERIUM A VIUM COMPLEX INFECTIONS IN MICE Katsumasa SATO, Toshiaki SHIMIZU, Tatsuya AKAKI, Haruaki TOMIOKA* The MycoDot(TM) is a new diagnostic kit for tuberculosis which was devised by DynaGen Inc., USA. The MycoDot test is based on the detection of anti-mycobacterial antibodies in the serum samples of patients by employing plastic combs coated with lipoarabinomannan (LAM) antigen which is a highly immunogenic lipopolysaccharide presenting in the cell wall of all species of mycobacteria. It has been reported that healthy infected and BCG- vaccinated individuals do not react to the MycoDot test, while a positive reaction occurs in patients with active tuberculosis or atypical mycobacteriosis with good sensitivity and specificity. In this study, we evaluated the efficacy of MycoDot test for the detection of anti-LAM antibodies in sera of mice infected with Mycobacterium tuberculosis or M.avium complex (MAC). By using the MycoDot test, anti-LAM antibodies were posi- tive in 2 out of 4 mice infected with M.tuberculosis 2 weeks before, while all of M.intracellulare-infected mice were negative at the same phase of infection. On the other hand, anti-mycobacterial (MB) antibodies were detected in the serum samples of mice in- fected with M.irbtracellulare as well as M.tuberculosis by home-made ELISA testing using whole cells of test mycobacteria as antigen. In the next experiment, mice were infected with M.avium. All the serum samples of mice obtained at 13 weeks after infection were negative for anti-LAM antibodies in MycoDot test, whereas they reacted positively to anti-MB antibodies in ELISA test. These results indicate that the MycoDot test is capa- ble of detecting M.tuberculosis infection but not MAC infection induced in mice. Key words : Serological diagnosis, MycoDot, M.tuberculosis, M.avium complex *From the Department of Microbiology and Immunology, Shimane Medical University, Izumo, Simane 693-8501 Japan. (Received 23 Jun. 1997/ Accepted 18 Nov. 1997) <2> Kekkaku Vol.73, No.2: 53-64, 1998 THERAPEUTIC EFFICACY OF BENZOXAZlNORIFAMYClN KRM-1648 AGAlNST EXPERIMENTAL MURINE TUBERCULOSIS : (1) A Study on the Efficacy of Short Course Treatment with the Intratracheal and Intravenous Infection Models Norio DOI* Objectives : This study aims to compare in vivo activity of benzoxazinorifamycin KRM- 1648 (KRM) with those of rifampicin (RFP) and rifabutin (RBT) against experimental murine tuberculosis. Study design : Mice were infected with Mycobacterium tuberculosis or M. bovis by the intratracheal (IT) or intravenous (IV) routes, and treated for 10 days with various doses of each drug starting from the 8th or 11th day after the TB-infection. Results : (A) A rapid test for in vivo evaluation of three rifamycins was conducted by examining the survival days of treated mice infected with 10(6) cfu of M. bovis Ravenel. Mice treated with KRM exhibited 2.1`3.7 times longer survival times, in comparison with those treated with RFP or RBT. (B) In the IT-model of M. bovis Ravenel infection, three rifamycin derivatives gave ' "drstmctrve dose response curves" in the correlation of dose sizes with the mean survival times or "log(10)CFU/lungs reductions" . (C) In M. tuberculosis Kurono infection models, the ranking of the anti-TB activity of the three rifamycins in each organ was as follows : IT- and IV-lungs : KRM tRFPFRBT, IV-spleen : KRMRBT>RFP, IV-liver : KRMRBT>RFP. (D) Based on the results of "log(1O)CFU reductrons" in different organs in M. tuberculosis Kurono mfectron models "charactenstrc in vivo activity patterns of each rifamycin" were obtained. (E) The therapeutic efficacy of KRM in lungs was greater than in spleen and liver with any dose. In contrast, RBT exhibited more remarkable in vivo activity in the spleen and liver than in lungs. Conclusion : The prominent in vivo activity of KRM may allow small dose for effective therapy ; 1/3 dose or less in comparison with those of RFP or RBT, or intermittent therapy of tuberculosis. Key words: Intratracheal infection. Mycobacterium tuberculosis, Mouse, KRM-1648, Rifampicin, rifabutin *From the Department of Basic Research, Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24, Matsuyama, Kiyose, Tokyo 204-8533 Japan. (Received 26 Sep. 1997/ Accepted 26 Nov. 1997) <3> Kekkaku Vol.73, No.2: 65-70, 1998 The 72nd Annual Meeting President Lecture BIOCHEMICAL AND IMMUNOLOGICAL PROPERTIES OF THE FRACTIONS OF TUBERCLE BACILLI Ichiro AZUMA* In 1954, Dr. Yuichi Yamamura and his coassociates, National Sanatorium Toneyama Hospital, has reported the formation of experimental tuberculous cavity in rabbits. This work was a milestone of the researches in the field of cellular immunology. In 1960, I have started my scientific carrier in Kyushu University Medical School as the post-gradu- ate student under the guidance of Prof. Yamamura. Since then, I have worked on the field of biochemical and immunological properties of tubercle bacilli. The arabinose mycolate, polysaccharides, cell-wall skeleton (CWS) and trehalose dimycolate (TDM) were purified from tubercle bacilli and their Chemical structures and biological activities were investi- gated in detail in our laboratory. In 1971, we have reported that adjuvant active principle of tubercle bacilli was a CWS fraction, and the details of chemical structure and adjuvantity of mycobacterial CWS were investigated in our research group. The application of BCG-CWS to cancer immunotherapy was also studied. In 1974, Prof. E. Lederer and his coworkers, University of Paris, have reported that N-acetyl muramyl-L-alanyl-D-isoglutamine (MDP) was the minimum adjuvant-active structure of bacterial cell wall. We 'have synthesized several hundreds of MDP derivatives and selected, MDP-Lys(L18), romurtide, as the candidate of cytokine inducer for the clinical application. Romurtide is applied in cancer patients for the recovery of the number and functions of monocytes, neutrophils and platelets. These results suggest that the tubercle bacilli, especially, CWS and related synthetic MDP derivatives, are effective for the potentiation of host resistance against infectious dis- eases and cancer. Key words : Tubercle bacilli, Cell-wall skeleton (CWS), Adjuvant activity, Muramyl dipeptide (MDP), Cytokine inducer *From the Institute of Immunological Hokkaido University, N-15, W-7, Kita-ku, Sapporo 060-0815 Japan. (Received 10 Dec. 1997) <4> Kekkaku Vol.73, No.2: 71-76, 1998 The 72nd Annual Meeting Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 1 . MECHANISMS OF HOST RESISTANCE TO MYCOBACTERIUM AVIUM COMPLEX AND MYCOBACTERIUM TUBERCULOSIS INFECTIONS Haruaki TOMIOKA*, Katsumasa SATO, Toshiaki SHIMIZU and Tatsuya AKAKI In order to know profiles of the antimicrobial action of some microbicidal effector molecules against Mycobacterium avium complex (MAC) and M. tuberculosis (MTB), profiles of collaborating effects among reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) were studied. RNI and FFA ex- erted synergistic effects in killing MAC and MTB, while the combination of ROI (H(2)0(2) -me- diated halogenation system) with FFA conversely caused antagonism. The combination of RNI with ROI displayed additive effects in killing MTB, whereas the same combination showed antagonistic effects against MAC. Murine peritoneal macrophages (Ms) pro- duced and/or released these three antimicrobial effectors in the order ROI, FFA, and RNI in response to cellular stimulation induced by their contact with MAC or MTB organ- isms. These findings indicate that the collaborating effect of RNI with FFA is crucial for M-mediated intracellular killing of MAC and MTB. Secondly, we examined the modes of bacterial growth of MAC and MTB in murine peritoneal Mcs and A-549 type U al- veolar epithelial cell line. The growth rate of these organisms was much larger in A-549 cells than in Mcs. In addition, the growth rate of high-vilulence MAC (N-260 strain) was significantly larger than that of low-virulence MAC (N-444 strain), when they were residing in Ms or A-549 cells. Although a high-virulence MTB (strain Kurono) also showed much more rapid growth in Ms than did low-virulence MTB (strain H(37)Ra), such a phenomenon was not observed for their intracellular growth in A-549 cells. MTB exhibited strong cytotoxic effects against Ms but not against A-549 cells when resided in these cells. On the other hand, MAC organisms did not cause cytotoxicity even in Ms. Although MAC and MTB infections caused significant increase in RNI production by Ms but not by A-549 cells, there was no significant relationship between the degree of M RNI production by a given mycobacterial organism and its virulence. These find- ings indicate some important roles of type K alveolar epithelial cells as a target cell for primary invasion and transient growth of mycobacterial organsisms in the host lungs. Key words : Mycobacterium avium complex, Mycobacterium tuberculosis, Antimicrobial effectors, Macrophages, Type K alveolar epithelial cells *From the Department of Microbiology and Immunology, Shimane Medical University, Izumo, Shimane 693-8501 Japan. (Received 9 Jan. 1998) <5> Kekkaku Vol.73, No.2: 77-82, 1998 The 72nd Annual Meeting Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 3 -(1) THE VIEW OF DEVELOPMENT OF NEW DRUGS AGAlNST NONTUBERCULOUS MYCOBACTERIAL INFECTIONS Shin KAWAHARA* and Hitoshi NAGARE It is obvious that the number of patients with pulmonary nontuberculous mycobacterial infections is increasing gradually in Japan. Of these infections, M. avium complex (MAC) is the most common cause, and is known to be resistant to many antimicrobial drugs. At present, no standard regimen which is able to control MAC infections com- pletely is established. For these reasons, the development of new drugs with strong antimycobacterial activity which are not cross - resistant to conventional antimy- cobacterial drugs is urgently desired. Thus, we studied in vitro activities of various drugs which are expected to be a new promising drug against nontuberculous mycobacterial infections, and reviewed clinical im- pact of these drugs. 1) New quinolones New quinolones including ofloxacin, ciprofloxacin, Ievofloxacin and sparfloxacin (SPFX), are considered to be active against M. tuberculosis, M. kansasii, M. fortuitum, but are inactive against MAC, M. chelonae. M. abscessus. M. scrofulaceum. Both AM-1155 and Du-6859a, newer quinolones, seemed to be comparable to or more active than SPFX which is considered to be most active now. 2) New macrolides Clarithromycin (CAM) has in vitro activities against various nontuberculous mycobacteria including MAC, and also has proven to have clinical potential not only for disseminated MAC infections in AIDS but also for pulmonary MAC infections. Therefore, CAM seems to be a candidate for one of the key drugs in the treatment of MAC infec- tions. 3) Rifamycins Rifabutin (RBT) and rifapentine exhibited more potent in vitro and in vivo antimy- cobacterial activities than rifampicin. RBT has already demonstrated clinical effect against intractable tuberculosis and MAC infections. Thus, RBT is recommended for the prophylaxis of M. tuberculosis and MAC infections in AIDS patients in US. KRM-1648 displayed much more potent in vitro and in vivo activities than rifampicin against both M. tuberculosis and MAC. It is needed an effort to confirm its therapeutic efficacies. Now clinical phase study is going on in US. 4) Phenazines Clofazimine (CFZ), an effective antileprosy drug, is known to be active in vitro against various mycobacteria including MAC, and often used as a component of combina- tion chemotherapy for disseminated MAC infections in AIDS patients in US. Recently, CFZ new analogs have been developed, and it is necessary to evaluate its activities against nontuberculous mycobacteria. Key words : New drugs, Nontuberculous mycobacterial infections, New quinolones, New macrolides, Rifamycins, Phenazines *From the Department of Internal Medicine, National Minami-Okayama Hospital, 4066 Hayashima, Okayama 701-0304 Japan. (Received 15 Dec. 1997) <6> Kekkaku Vol.73, No.2: 83-85, 1998 The 72nd Annual Meetmg Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 3 -(2) LONG-TERM PROGNOSIS OF MYCOBACTERIUM AVIUM COMPLEX DISEASE Eriko SHIGETO* This paper shows long-term course of 71 patients with Mycobacterium avium complex (MAC) disease treated in National Hiroshima Hospital between 1977 and 1991 and were observed for more than 5 years or died between 6 months and years from the onset. In the patients who were not treated with chemotherapeutic agent or treated with less than 3 drugs, about 60 per cent deteriorated and few obtained persistent negativity of bacte- ria. In patients treated with at least 3 drugs out of isoniazid, rifampicin, ethambutol, ethionamide, cycloserine, clarithromycin and new-quinolones for more than 12 months, 59.2 per cent deteriorated, while 29.6 per cent obtained negative conversion of bacteria by initial chemotherapy. However only 18.5 per cent remained negative during long term observation. In patients treated with at least 3 drugs including one aminoglycoside such as streptomycin for more than 12 months, 64.7 per cent obtained negative conversion and 35.5 per cent remained negative bacteriologically, while 23.5 per cent deteriorated. In twelve patients who were operated, only one patient deteriorated. The long-term prognosis of MAC disease under the chemotherapy mainly by anti- tuberculous agents up to this time was still poor compared to that by short-term obser- vation. Anticipated new regimens for MAC, should be multidrug, to prevent secondary drug resistance. Key words : Mycobacterium avium complex disease, Long-term prognosis, Chemotherapy *From the National Hiroshima Hospital, 513 Jike, SSaijo-cho, Higashihiroshima, Hiroshima 739-0041 Japan. (Received 15 Dec.1997) <7> Kekkaku Vol.73, No.2: 87-92, 1998 The 72nd Annual Meeting Symposium K . NONTUBERCULOUS MYCOBACTERIOSIS ; THE PRESENT STATUS AND IN THE FUTURE 4 . INFECTION WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) AND NONTUBERCULOUS MYCOBACTERIOSIS Seizi MIZUTANl* Disseminated Mycobacterium avium-inttracellulare complex (DMAC) infection is a com- mon complication of AIDS. The cumulative incidence is 40% in patient surviving 2 years after diagnosis of AIDS. AIDS patients with DMAC reduced life expectancy compared with those without. Antimycobacterial therapy with Clarithromycin (CAM) can signifi- cantly reduce bacteremia and improve symptoms, quality of life, and survival of patients with DMAC. Prophylactic therapy with Rifabutin, CAM and Azithromycin is effective and Synergic effect can be expected as Rifabutin and Azithromycin are administerd to- gether. But it is serious problem to get resistance to CAM when prophylactic therapy with CAM failed because we lose one of the most effective medicines against DMAC. It is recommended to start prophylactic therapy when CD4 Lymphocyte count falls below 50- 75/ʃ in patients who had oppotunistic infection. In Japan, 32 cases of AIDS with NTM are reported. All of them are male, and mean count of CD4+lymphocyte was 11/ʃ. Twenty three out of 32 were MAC and 6 were M. kansasii. Cases of NTM bacteremia were 9 (69.2% ) and cases of those without bacteremia were 4 (30.8% ) . Three out of 4 were cases of M. kansasii. Key words : AIDS, Nontuberculous myco-bacteriosis, Clarithromycin, Prophylaxis *From the JATA, Fukujuji Hospital, Matsuyama 3-1-24, Kiyose city, Tokyo 204-8533 Japan. (Received 12 Jan. 1998)