(Vol.74, No.5 May 1999) <1> Kekkaku Vol.74, No.5: 425-432,1999 THE PREVALANCE SURVEY ON CHRONIC TUBERCULOSIS PATIENTS WITH BACILLI DISCHARGE IN OSAKA PREFECTURE PULMONARY MYCOBACTERIUM AVIUM COMPLEX DISEASE Kayo MUI*, and Kazuhiko KAMEDA This survey was made at the end of 1996 in Osaka prefecture including three ordinance designated cities of Osaka, Sakai and Higashiosaka. As of December 31 1996, 109 or 3.3% of active tuberculosis cases were found to be chronic tubercle bacilli excreters in Osaka city, and 128 or 3.6% in Osaka prefecture other than Osaka city, respectively. In the area called Airin at Nishinari-ward, Osaka, which has been an area with high prevalance of the disease, 33 or 5.8% were chronic tubercle bacilli excreters, and the rate was slightly higher than the other areas. Compared to the survey conducted 10 years ago, while the number of the chronic tuber- cle bacilli excreters decreased by half to 109 from 200 in Osaka city, to 128 from 211 in Osaka prfecture excluding Osaka city, the rate of chronic excreters to total active tuber- culosis showed almost no change. Various factors such as difficult living conditions often attributed to defaulting of ac- tive tuberculosis patients from their treatment, thus resulting to development of chronic excretion. It is highly recommended to apply DOTS strategy for the completion of their treatment. Public health centers that have personal clinical records of each patient should be familiar with the results of their bacillus examinations. They should also be required to cooperate with medical institutions to cope with those who need retreatment. Key words:Chronic tubercle bacilli excreters, Defaulting, Homeless peoples, Directly Observed Therapy, Short-course(DOTS), High prevalance area *From Naniwa Public Health Center, Shikitsu-higashi 1-1-30, Naniwa-ku Osaka 556-0012 Japan. (Received 10 Oct. 1998/Accepted 30 Nov. 1998) <2> Kekkaku Vol.74, No5: 433-439, 1999 EVALUATIONS OF MTD AND AMPLICOR(TM) MYCOBACTERIUM FOR DIRECT DETECTION OF MYCOBACTERIA FROM CLINICAL SPECIMENS Akimasa SATO*, Toshiaki SONOBE, Miki OKAZAKI, and Bun-ichi UMEDA MTD (GEN-PROBE AMPLIFIED MYCOBACTERIUM TUBERCULOSIS DIRECT TEST(TM)) for Mycobecterium tuberculosis, and Amplicor(TM) Mycobacterium for Mycobacteria (AMP-M. tb for M.tuberculosis, AMP-M. av for M.avium and AMP-M. in for M. intracellulare) were used for the detection of relevant Mycobacterium. Their sensitivity and specificity were evaluated. Total 244 clinical specimens including 164 sputa were ex- amined by the above two tests. The results were compared with those obatined by the conventional methods. Of 244 samples, number of the M.tuberculosis positive samples by microscopy, clutural test, MTD and AMP-M. tb were 32, 33, 38 and 35, respectively. Among 33 culture posi- tive samples, 25 were MTD positive and 26 were AMP-M. tb positive. Therefore, sensi- tivity of MTD and AMP-M. tb were 75.8% and 78.8%, and their specificity were 98.3% and 95.7%, respectively. When only sputa were used for the tests as the clinical speci- mens, both sensitivity of MTD and AMP-M. tb were increased to 94.4%. For MAC, positive samples of M.avium cmplex by culture, M.avium by AMP-M. av and M.intracellulare by AMP-M. in were 13, 16, and 8, respectively. Sensitivity and specificity of AMP-M. av/M. in were 100% and 95.2%, respectively. Clinical findings of the patients whose MTD tests were positive but negative by culture were reexamined. Three of 9 specimens were also positive in AMP-M. tb. From the records of the isolations of tubercle bacilli or other important pathogens from the other kind of clinical specimens, smear tests and patients' response to tuberculosis chemother- apy, four of 9 specimens were confirmed as true positive, three were suspected as posi- tive, and two other specimens were false positive which might be caused by contamination. From these observations, it could be concluded that MTD and AMP-M. tb are more sensitive than conventional culture method, and MTD is more sensitive than AMP-M. tb but needs more careful treatment to avoid the contamination. Key words:Mycobacterium tuberculosis, Mycobacterium avium complex, MTD(TM), Amplicor(TM) Mycobacterium, nucleic acid amplification. *From Kobe Institute of Health, 4-6 Minatojima- Nakamachi, Chuo-ku, Kobe 650-0046 Japan. (Received 20 Apr. 1998/Accepted 10 Dec. 1998) <3> Kekkaku Vol.74, No.5: 441-445, 1999 pncA GENE MUTATIONS IN CLINICAL ISOLATES OF TUBERCULE BACILLUS BY POLYMERASE CHAIN REACTION-DIRECT SEQUENCING METHOD: IN RELATIONSHIP TO PYRAZINAMIDE RESISTANCE Shigehisa HOASHI*, Hisakazu TAI, Mayumi TAMARI We screened clinical isolates of tubercle bacillus for mutations in the pncA gene, which encodes pyrazinamidase (PZase), by polymerase chain reaction (PCR)-direct sequencing method. Sixty-eight strains of tubercle bacillus were isolated form 32 patients with pul- monary tuberculosis. The poatients were treated with antituberculous agents including pyrazinamide (PZA) for 2 months. Thirty-two of the 68 strains were isolated form spu- tum samples collected form the patients before treatment;29 strains and 7 strains were collected after 1 month and 2 months of treatment, respectively. The pncA genes in these strains, were assessed for mutations by direct sequencing of PCR products using an auto- mated sequencer. Similarly, we examined two clinical isolates (ka567 and minami22) of tubercle bacillus, determined to be deficient in PZase activity by the Wayne method. A PZA-sensitive strain (H37Rv, ATCC27294), and a PZA-resistant strain (H37Rv-PZA-R, ATCC35828) were used as negative and positive controls for mutations in the pncA gene, respectively. None of the 68 strains demonstrated any mutations in the pncA gene;how- ever, the 2 PZase-deficient strains had missense mutations in the pncA gene resulting in an amino acid substitution from His82 to Arg in clone ka567, and from Ala171 to Val in clone minami22. Key words:Drug-resistant Mycobacterium tuberculosis, Pyrazinamide, pncA gene, PCR-direct sequence, Point mutation *From the Department of International Medicine IV, Daisan Hospital, The Jikei University School of Medicine, 4- 11-1, Izumihon-chou, Komae-shi, Tokyo 201-8601 Japan. (Received 25 Sep. 1998/Accepted 11 Dec. 1998) <4> Kekkaku Vol.74, No.5: 447-452, 1999 A STUDY OF BETA-LACTAMASE ACTIVITY OF MYCOBACTERIA AND CLINICAL TRIAL OF PENICILLIN/BETA-LACTAMASE INHIBITOR COMBINATIONS IN THE TRATMENT OF DRUG-RESISTANT MYCOBACTERIUM TUBERCULOSIS Yoshihisa NAKAGAWA*, Kazuyoshi SHIMAZU, Momoko EBIHARA, and Kumiko AMAN Beta-lactamase activity was determined using a nitrocefin disc method on 34 Mycobac- terium tuberculosis (M.tuberculosis) strains and 13 nontuberculous mycobacteria strains. In the 34 M.tuberculosis strains, 23 strains showed beta-lactamase activity. In 10 Myco- bacterium avium complex strains, no beta-lactamase activity was detected. In the Myco- bacterium chelonae strains, all three strains examined showed strong beta-lactamase activity. No correlation was found between beta-lactamase activity and resistance to anti- tuberculous chemotherapeutic agents. Four patients who were persistently positive for multi-drug-resistant M.tuberculosis (MDR-TB) on sputum and positive in beta-lactamase activity, were treated with penicil- lin/beta-lactamase inhibitor combinations. In two cases, the trials were discontinued be- cause of diarrhea;the trials were continued in the remaining two for four months, but the MDR-TB was positive during the course of the therapy. Effectiveness of the therapy with penicillin/beta-lactamase inhibitor combinations against M.tuberculosis was obscure, although many of M.tuberculosis examined showed beta-lactamase activity. Key words:Beta-lactamase, Multi-drug resistant mycobacterium tuberculosis, Penicillin *From the Department of Internal Medicine, Kumamoto National South Hospital, 2338 Matsubase, Shimomashiki, Kumamoto 869-0524 Japan. (Received 10 Oct. 1998/Accepted 21 Dec. 1998) <5> Kekkaku Vol.74, No.5: 453-456, 1999 A CASE OF MIDDLE EAR TUBERCULOSIS;PCR OF THE OTORRHEA WAS USEFUL FOR THE DIAGNOSIS Tetsuro INOUE*, Nobuaki IKEDA, Takuya KURASAWA, Atsuo SATO, Kohichi NAKATANI, Takeshi IKEDA, Harukazu YOSHIMATSU, and Ryoichi AMITANI A 26-year-0ld female was admitted to our hospital with complaints of fever, cough, otorrhea and otalgia and progressive hearing loss of her left ear. Smears of her sputum were positive for acid-fast bacilli. Smears of her otorrhea were negative for acid-fast bacilli but PCR of her otorrhea was positive. Chest X-ray showed infiltrative shadows with the cavity. She was diagnosed as middle ear tuberculosis associated with pulmonary tuberculosis. After anti-tuberculous chemotherapy, fever, cough, otorrhea and pain of her left ear were improved, but her hearing level was not improved. In the case of middle ear tuberculosis, it is necessary to make an early diagnosis and treatment. This is first reported case in Japan in which PCR of the otorrhea is positive. Key words:Middle ear tuberculosis, Aural tuberculosis, Tuberculous otitis media, Polymerase chain reaction *From the Department of Respiratory Medicine, National Minami-Kyoto Hospital, 11 Naka-ashihara, Joyo Kyoto 610-0113 Japan. (Received 30 Oct. 1998/Accepted 1 Dec. 1998) <6> Kekkaku Vol.74, No.5: 457-461, 1999 THE COMBINATION THERAPY OF CLARITHROMYCIN AND SPARFLOXACIN FOR PULMONARY MYCOBACTERIUM GORDONAE INFECTION Yumiko TOMIYAMA*, Shigefumi MAESAKI, Bin YONG, Yasuhito HIGASHIYAMA, Yoshitsugu MIYAZAKI, Kazunori TOMONO, Takayoshi TASHIRO, and Shigeru KOHNO Seventy years old woman had fever and hemosputum at May 1997. She was diagnosed as mycobacteriosis because of the positive acid fast bacilli smear from sputum. Mycobac- terium gordonae was isolated from sputum, gastric juice, and bronchial aspirate. The combination therapy of isoniazid, rifampicin, ethambutol, and clarithromycin was admin- istrated;however, M. gordonae was not eradicated from sputum. Sparfloxacin was ad- ministered instead of isoniazid based on the result of drug susceptibility test. The smear became negative and M.gordonae was eradicated from sputum one month after the initiation of treatment with the combination of clarithromycin and sparfloxacin. Key words:Mycobacterium gordonae, Non-tuberculous mycobacteriosis, Clarithromycin, Sparfloxacin *From the Second Department of Internal Medicine, Nagasaki University School of Medicine, Sakamoto 1-7-1, Nagasaki 852-8501 Japan. (Received 9 Oct. 1998/Accepted 14 Dec. 1998)