(Vol.76 No.9 September 2001) <1> Kekkaku Vol.76,No.9:603-614,2001 SEROLOGICAL DIAGNOSIS OF PULMONARY TUBERCXULOSIS AND NONTUBERCULOUS PULMONARY MYCOBACTERIOSIS 1*Ariyoshi KONDO, 2Norihiro OKETANI, 2Michio MARUYAMA, 2Yasuharu SAITO, 2Hiromi MIYAO, 2Michiko OHNO, 2Koichi WADA, 2Toshimasa TSUCHIYA, 3Tadashi NAGAI, 3Kuniko TSUCHIYA, and 3Yasushi WATANABE 1Niigata Tetsudo-Kenshin Center, East Japan Railway Company, 2Department of Respiratory Medicine and 3Department of Clinical Laboratory, National Nishi-Niigata-Chuo Hospital Objective:The present study was undertaken to evaluate the utility of the serodiagno- sis of pulmonary tuberculosis and nontuberculous pulmonary mycobacteriosis by ELISA using a Pathozyme-Myco kit (Myco kit) and a Pathozyme-TB complex kit (TB kit) (OMEGA Diagnostics Ltd.). Study population:The subjects comprised 256 healthy volunteers (HV, healthy hospital employees), 66 patients with sputum-positive active pulmonary tuberculosis (ap TB), 14 patients with healed pulmonary tuberculosis (hp TB), 24 patients with nontuberculous pulmonary mycobacteriosis (NTM) and 32 patients with pulmonary diseases other than mycobacteriosis. Results:1) The serum IgG antibody titers determined with the Myco kit were signifi- cantly higher in the ap TB group(p<0.01), the hpTB group (p<0.01), and the NTM group (p<0.01) than those in the HV and the other pulmonary disease group. At a cut-off value of the mean+2SD of the values obtained in the HV, the positive rate was 47.0% in patients with apTB, 50.0% in those with NTM, 21.4% in those with hpTB, 3.1% in those with other pulmonary diseases, and 1.6% in the HV. Analysis of ROC curves showed that the HV and the pulmonary mycobacteriosis group (apTB and NTM) were best distinguished by a cut-off value of -0.280 OD (log), with the sensitivity and the specificity being 83.3% and 78.5%, respectively. It was impossible to distinguish apTB form NTM. 2) The serum IgG antibody titers determined with the TB kit were signifi- cantly higher in the apTB group than those in the HV (p<0.01), the NTM group (p<0.05) and the other pulmonary disease group (p<0.01). No significant difference was observed between the HV and the patients with NTM or those with other pulmonary dis- eases. Although the positive rate of the test was low in the apTB group (42.4%), there was a significant difference between apTB and NTM (12.5%) (p<0.05), siggesting that apTB could be distinguished form NTM. 3) Since the serum antibody titers determined by the Myco kit showed no significant difference between apTB and NTM, and there was also no difference in the positivity between the two diseases, we performed serologic examination using the Myco kit to detect both diseases as pulmonary mycobacteriosis. After diagnosing pulmonary mycobacteriosis by the Myco kit, we then used the TB kit to separate apTB form NTM. In this case, the sensitivity and specificity of the test were 55.6% and 85.7%, respectively. Better methods should be developed to distinguish apTB from nontuberculous mycobacteriosis. Key words:Pulmonary tuberculosis, Nontuberculous pulmonary mycobacteriosis, Serodiagnosis, 38kDa antigen, Receiver operating characteristic (ROC) curve analysis, Pathozyme-Myco kit, Pathozyme-TB complex kit *1-1-1, Saiwai-Nishi, Niigata-shi, Niigata 950-0908 Japan. (Received 13 Oct. 2000/Accepted 5 Jun. 2001) <2> Kekkaku Vol.76,No.9:615-618,2001 THE USEFULNESS OF TWO-STEP TUBERCULIN SKIN TESTING IN NOSOCOMIAL INFECTION CASES 1*Shuichi YANO, 2Shinji SHISHIDO, 1Kanako KOBAYASHI, 1Yuji KAWASAKI, 1Shinji SAITO, and 1Kazuhiro KATO 1Department of Pulmonary Medicine, National Matsue Hospital, 2Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association The usefulness of the two-step tuberculin skin test in the preceding year for the contact investingation was reported, when a pulmonary tuberculosis patient was discovered in our hospital. Four persons showed stronger reaction than the results in two-step tuberculin skin test in the preceding year after nosocomial infection. One of them was diagnosed as pulmonary tuberculosis by chest radiograph, and anti- tuberculosis chemotherapy was started. Other three started chemoprophylaxis. The chemoprophylaxis subjects might incrase 4, if basic value of tuberculin reaction had not been available by two-step tuberculin skin test in the previous year. We could identify persons in whom tuberculin reaction became, stronger by comparing the test results after exposure with the result of two-step tuberculin skin test in the preceding year. It is considered that two-step tuberculin skin test is very useful to efficiently execute the protective measure. Key words:Two-step tuberculin skin test, Nosocomial infection case *5-8-31, Agenogi, Matsue-shi, Shimane 690-8556 Japan. (Received 1 Mar. 2001/Accepted 5 Jun. 2001) <3> Kekkaku Vol.76,No.9:619-624,2001 A STUDY ON RELATION BETWEEN ACTIVE PULMONARY TUBERCULOSIS AND UNDERLYING DISEASES 1*Mouka TAMURA, 1Reirou SHIRAYAMA, 1Reiko KASAHARA, 1Ryuji MIYAZAKI, 2Masanori YOSHIKAWA, 2Katsuhiko TSUKAGUCHI, 2Takahiro YONEDA, and 2Nobuhiro NARITA 1*Department of Internal Medicine, Nishinara National Hospital, 2Second Department of Internal Medicine, Nara Medical University A study was made on the relation between active pulmonary tuberculosis and underly- ing diseases in 119 tuberculosis patients. Out of total 119 patients, 87 patients (73.1%) had underlying diseases. The most common underlying disease was diabetes mellitus in 34 patients (39.1%), followed by HCV (+) chronic hepatitis, sequela of cerebral infarction, hypertension and gastric ulcer. In patients who had underlying diseases, the mean age was higher, proportion of sputum smear positive cases was higher, albumin was lower, and period until sputum culture negative conversion was longer. In patients who had diabetes mellitus, proportion of cases with cavity on chest X-P was higher, and in patients who had sequela of cerebral infarction or hypertension, mean age was higher. In patients who had diabetes mellitus and whose HbA(1C) was †9%, proportion of smear positive cases was higher, albumin was lower and period until culture negative conversion was longer than in patients who had diabetes mellitus and whose HbA1c was < 9%, suggesting that control of blood sugar in diabetes mellitus related to severity of pulmonary tuberculosis. In patients who had diabetes mellitus and whose albumin was < 3g/dl, period until culture negative conversion was longer than in patients who had diabetes mellitus and whose albumin was †3g/dl. In patients who had underlying diseases, these diseases caused decline of tuberculous immunity and nutritional disturbance represented by lower albumin also promoted decline of tuberculous immunity. It is suggested that the underlying diseases affected the onset and progression of pulmonary tuberculosis. Key words:Active pulmonary tuberculosis, Underlying disease, Diabetes mellitus, Hemoglobin A(1C), Albumin *2-789, Shichijo, Nara-shi, Nara 630-8053 Japan. (Received 8 Nov. 2000/Accepted 6 Jun. 2001) <4> Kekkaku Vol.76,No.9:625-634,2001 TUBERCULOSIS OUTBREAK IN A JUNIOR HIGH SCHOOL IN KOCHI CITY -Studies on Factors Relating to Extent of Tuberculosis Infection and the Efficacy of Isoniazid Chemoprophylaxis- *Makoto TOYOTA and Shigeharu MORIOKA *Kochi City Health Center A 15-year-old girl, third-grade student of a junior high school (the index case) was found to have smear-positive, cavitary pulmonary tuberculosis. Contact investigation was conducted, including tuberculin skin test and chest X-ray examinations over 700 persons. Tuberculin skin test revealed an excess of strongly-positive reactors in the third- grade students. During 2 years after the detection of the index case, a total of 31 tuberculosis patients were newly diagnosed, and out of them 8 were culture positive and restriction fragment length polymorphism (RFLP) analysis of the 8 strains and that of the index case demonstrated an identical pattern. A delay in diagnosis of the index case and poor ventilation of the classrooms were attributable to this rather large outbreak. In addition the source case seems to be highly infectious, because transmission following only sporadic contact was documented. Among the third-grade students and school staff, 129 persons were strongly-positive reactors to tuberculin skin test. Out of them one hundred five persons completed isoniazid chemoprophylaxis of recommended six months, and the others didn't receive chemoprophylaxis because most of them were aged above 30 years. All of them were followed up for 2 years after the detection of the index case, and out of 105 persons who received chemoprophylaxis, 2 cases (1.9%) were newly diagnosed as tuberculosis, while out of 24 persons without chemoprophylaxis, 6 cases (25%) developed tuberculosis. Key words:Tuberculosis outbreak, Junior high school, Contact investigation, Highly infectious case, RFLP analysis, Chemoprophylaxis *2-4-1, Marunouchi, Kochi-shi, Kochi 780-0850 Japan. (Received 23 Feb. 2001/Accepted 22 Jun. 2001) <5> Kekkaku Vol.76,No.9:635-639,2001 THE EFFECTIVENESS OF STEROID AS ADJUNCTIVE THERAPY IN THE MANAGEMANT OF TWO PATIENTS WITH FEBRILE REACTION DUE TO ANTITUBERCULOUS DRUGS *Shuichi YANO, Kanako KOBAYASHI, Yuji KAWASAKI, Kazuhiro KATO, and Shinji SAITO *Department of Pulmonary Medicine, National Matsue Hospital Antituberculuous treatment could be continued by using steroid as adjunctive therapy in two patients showing feabrile reaction to antituberculous drugs in spite of desensitization therapy. Case 1(39-year-old man) was admitted to our hospital with positive sputum-smear and bilateral cavitary tuberculosis (bII2) on chest X-ray. He showed fever of 38-39Ž after 8 days of HREZ treatment. Desensitization therapy of RFP with the combined use of prednisolone was enforced, since the exothermic reaction continued in spite of stopping the 4 medicines, and the treatment became possible. Case 2(40-year-old women) was admit- ted with positive sputum-smear and bilateral cavitary tuberculosis (bII2) on chest X- ray. The administration of drugs was stopped as temperature rose to 38-39Ž after 12 days of HREZ treatment. Though the DLST of INH was positive, the treatment with INH became possible by the combined use of PSL. Key words:Antituberculous drugs, Steroid, Pyrexia *5-8-31, Agenogi, Matsue-shi, Shimane 690-8556 Japan. (Received 14 Feb. 2001/Accepted 6 Jun. 2001) <6> Kekkaku Vol.76,No.9:641-645,2001 The 76th Annual Meeting Special Lecture UNDERSTANDING IMMUNITY OT TUBERCULOSIS: GUIDELINES FOR RATIONAL VACCINE DEVELOPMENT *Stefan H.E. Kaufmann *Department of Immunology, Max-Planck-Institute for Infection Biology Mycobacterium tuberculosis has chosen macrophages as its preferred habitat. The tubercle bacillus is capable of arresting phagosomal maturation at an early stage, and of resisting anti-bacterial effector mechanisms, enabling M.tuberculosis to persist in resting macrophages. Upon activation by T lymphocytes, macrophages express increased anti-bacterial activities. The activated macrophages are major effector cells of the host defense against tuberculosis, and achieve mycobacterial containment in destinct granulomatous lesions. Although containment fails to fully eradicate the pathogen in most cases, it is generally sufficient to prevent the outbreak of clinical disease. Accordingly, T lymphocytes are central mediators of the spe- cific immune response against tuberculosis. The T cell compartment comprises several populations that together contribute to the control of M.tuberculosis. Of major importance are major histocompatibility complex (MHC) class II-re- stricted CD4 T cells. In addition, MHC class I-restricted CD8 T cells, ƒÁƒÂ T cells and CD1- restricted T cells, participate in protection. M.tuberculosis modifies the early phagosome to its advantage. It prevents phagosomal maturation at an early stage and fusion with lysosomes. Mycobacterial proteins are shuttled by MHC class I molecules from the phagosome to the cell surface, stimulating CD4 T cells. These T cells produce cytokines of Th1 type, in particular interferon-ƒÁ (IFN-ƒÁ). IFN-ƒÁ is a major activator of antimicrobial capacities of macrophages, and therefore critical for protection against tuberculosis. Other cytokines are also required for protection. In vivo protection focuses on granulomas, where M.tuberculosis is contained. Recent experimenmts have revealed a role of tumor necrosis factor-ƒ¿ (TNF-ƒ¿) and lymphotoxin-ƒ¿3 in granuloma formation and mycobacterial containment within a productive granuloma. Although it is not fully understood how mycobacteria enter the MHC class I pathway, CD8 T cells clearly do participate in protection. This notion was originally based on experiments with ƒÀ2-microglobulin (ƒÀ2m) knockout (KO) mice. These mutant mice lack MHC class I expression and hence the functional corollary in the form of CD8 T cells. Recent findings reveal that ƒÀ2m has several functions and therefore deficiency causes a range of effects. KO mice lacking MHC class I expression (and therefore only CD8 T cells) are less susceptible than ƒÀ2m deficient mutants. This demonstrates not only that CD8 T cells indeed do participate in protection against tuberculosis, but also that susceptibility due to ƒÀ2m deficiency is attributable to a number of reasons. CD8 T cells can produce IFN-ƒÁ, but more importantly, express cytolytic activities. They can lyse infected macrophages and directly attack mycobacteria. It is therefore likely that cytolytic T cell functions contribute to protection against tuberculosis. Cognates of group 1 CD1 protein family present glycolipids that are abundant in the mycobacterial cell wall. Group 1 CD1 molecules are present in guinea pigs and humans but not in mice, hampering accurate analysis of their role in protecion. ƒÁƒÂT cells expressing the VƒÁ2ƒÂ2 chain combination react with phospholigands abundant in mycobacteria. They probably recognize phospholigands in the absence of any host presentation molecules. Both CD1 restricted and ƒÁƒÂT cells produce TFN-ƒÁ and express cytolytic activity and can contribute to protection against tuberculosis in this man- ner. Because of the central role of the T cell compartment in protective immunity against tuber- culosis, it represents a major target for activation by a vaccine. Natural infection with M.tuberculosis is well controlled in the 2 billion individuals infected with this pathogen. Annually, 8 million of the infected individuals develop tuberculosis, often after long incubation periods, during which dormant mycobacteria are controlled by the im- mune system and do not cause any clinical disease. Disease develops due to exogenous insult, intrinsic genetic susceptibility or both. Since the vast majority of the population do control M. tuberculosis in a satisfactory manner, rational vaccine design can learn form the immune re- sponse in those individuals. Importantly, the immune response in the susceptible individuals needs to be also studied to gain understanding about which mechanisms are lacking in these individuals, and are therefore critical in protection against tuberculosis. The existing vaccine, Bacille Calmette-Guerin, is of insufficient efficacy due to its failure to provide adequate pro- tection against pulmonary tuberculosis in adults. It is important to define the mechanisms that BCG and natural infection fail to activate, in order to design novel vaccines better than BCG. The elucidation of the genome of M.tuberculosis provided the blue print for the identifica- tion of candidate protective antigens and virulence factors. Identification of protective antigens is particularly important for the subunit vaccine approach, while identification of virulence factors is particularly important for the whole bacterial vaccine approach. The subunit vaccine approach includes:(1)formulations comprising protein antigens with adjuvants capable of improving immunogenicity of vaccine antigens. While such vaccines are capable of stimulating CD4 T cell responses, they often fail to satisfactorily activate CD8 T cells and unconventional T cells. (2) Naked DNA vaccines have shown promise in mouse models, as they activate both CD4 and CD8 T cells. (3) Recombinant vaccines are viable carriers ex- pressing mycobacterial antigens and are able to stimulate both CD4 and CD8 T cells. All types of subunit vaccines stimulate a restricted number of T cell clones as they are comprised of only one or a few antigens. Unconventional T cells with specificity for non-proteinaceous antigens would be stimulated insufficiently, if at all. Promising subunit vaccine formulations, com- prising protein-adjuvant utilize Antigen 85 (Ag85), Mtb 8.4 or a fusion protein consisting of Ag85 and ESAT-6. Promising naked DNA vaccine candidates include Hsp60, Ag85 and Mtb 8.4. Notably, successful therapeutic vaccination with naked DNA composed of Hsp60 has been reported. Recombinant Vaccinia and Salmonella carriers expressing Ag85 also show pro- mise. The live attenuated bacterial vaccine approach includes:(1)Deletion mutants of M.tuberculosis. These should lack not only "classical" virulence factors, but also those that may impair the en- suing immune response. Promising M.tuberculosis mutants have been generates. They lack ƒ¿-crystallin, erp, isocitrate lyase, mycolic-acid cyclopropane synthase or phthiocerol dimyco- cerosate. (2)Auxotrophic mutants of M.tuberculosis or BCG. Auxotrophic mutants of BCG benefit from improved safety. Auxotrophic BCG mutants for methionine, leucine and isoly- sine, as well as M.tuberculosis auxotrophic mutants for methionine, proline and tryptophane have been described. (3)Recombinant BCG strains with improved immunogenicity. The weak immunostimulatory capacity of BCG could be improved by the introduction of a cytolysin, which facilitates MHC class I antigen processing. Alternatively, recombinant BCG strains expressing cytokines have been constructed. (4)Recombinant BCG over-expressing important antigens should also be considered. A recombinant BCG strain over-expressing Ag 85 has achieved better protection than wild-type BCG. By combining different approaches, further improvements could be achieved, for example, prime-boost regimens with viable bacterial vaccines and a subsequent boost with a subunit vaccine, or vice versa. As BCG vaccination will be continued also after the introduction of a novel vaccine candidate, prime boost regimes using both BCG and the novel candidate should be carefully considered. Learning form the immune response induced by BCG vaccination and by natural infection with M.tuberculosis, and comparing responses between susceptible and resistant individuals will further increase our knowledge about protective mechanisms against tuberculosis. Using global transcriptome analysis will facilitate the characterization of the protective "signature" of sur- rogates of protection. These studies will be performed both in the human system and in various animal models, mostly mice, but also guinea pigs and non-human primates. In sum- mary, although we are far from considering a selected vaccine candidate, recent achievements in genomics, proteomics and immunology have paved the way for the rational development of a vaccine more satisfactory than BCG. Key words:Tuberculosis, Vaccination, T lymphocyte, Cytokine, BCG *Schumannstr. 21-22, 10117 Berlin, Germany (Received 28 Jun.2001)