鸟分枝杆菌复合群对16种抗感染药物药敏试验的分析

目的 通过比较3种新型喹诺酮类药物(西他沙星、加替沙星和莫西沙星)与其他13种抗感染药物对鸟分枝杆菌复合群(MAC)分离株的体外活性,初步探讨喹诺酮类药物用于治疗MAC疾病的可能性.方法 琼脂梯度稀释法测定上述16种抗感染药物对MAC分离株的最低抑菌浓度(MIC),比较其能抑制90%菌株生长的MIC(MIC90).结果 与胞内分枝杆菌相比,鸟分枝杆菌菌株的MIC范围分布更广,且MIC90多高于胞内分枝杆菌.4种大环内酯类药物中,克拉霉素对鸟分枝杆菌和胞内分枝杆菌的MIC90最低,分别为32和16 mg/L;4种利福霉素类化合物中利福拉齐对鸟分枝杆菌和胞内分枝杆菌的MIC90最低,分别为0.5和0.25 mg/L;5种喹诺酮类药物中西他沙星对鸟分枝杆菌和胞内分枝杆菌的MIC90最低,均为4 mg/L,加替沙星和莫西沙星均为8 mg/L.2株克拉霉素敏感株(MIC=0.5 mg/L)对其他抗感染药物均接近或达到MIC范围的下限,3株克拉霉素不敏感株(MIC=64 mg/L)对除喹诺酮类以外的抗感染药物均接近MIC范围的上限.结论 利福拉齐、西他沙星、加替沙星和莫西沙星对MAC分离株具有较强的体外活性.     

The curious characteristics of pyrazinamide: a review.

Pyrazinamide (PZA) is an important sterilising tuberculosis drug that helps to shorten the duration of current chemotherapy regimens for tuberculosis. When first discovered, it had activity in murine tuberculosis but no apparent in vitro activity, and its subsequent use in treatment depended largely on classic experiments at Cornell University, which showed its requirement for an acid pH for activity and its sterilising activity in the mouse. Recent studies have shown that PZA enters Mycobacterium tuberculosis by passive diffusion, is converted to pyrazinoic acid (POA) by nicotinamidase/pyrazinamidase (PZase) and is then excreted by a weak efflux pump. Protonated POA (HPOA) is reabsorbed into the bacilli under acid conditions and accumulates because the efflux pump is inefficient, causing cellular damage. Unlike other antibacterials, PZA has no defined target of action. PZA is more active against old than against actively growing cultures, probably because the energy production and efflux pump would be slowed down by low bacterial metabolism. This review deals with the activity of PZA in vitro, in macrophages and in animal models. It describes the evidence from clinical trials that it is an effective sterilising drug that acts synergistically with rifampicin. The highly diverse mutations in the PZase gene (pncA) that lead to loss of PZase activity cause PZA resistance. Methods for susceptibility determination either as tests against PZA or nicotinamide in liquid and solid media, as tests for PZase activity or for mutations in pncA, are reviewed.     

Effect of potent antiretroviral therapy on immune responses to Mycobacterium avium in human immunodeficiency virus-infected subjects

To characterize the influence of highly active antiretroviral therapy (HAART) on cell-mediated immunity (CMI) to Mycobacterium avium complex (MAC), we measured immune responses to M. avium in human immunodeficiency virus (HIV)-infected individuals before and during HAART, in subjects with a history of disseminated MAC (DMAC), and in HIV-uninfected control subjects. Forty-seven percent of untreated HIV-infected patients and 78% of control subjects exhibited in vitro proliferative responses to M. avium (P=.03). Proliferative responses to M. avium increased after HAART for 3 months and were present in 77% of subjects after 6 months. Mean interferon-gamma production increased from 199 to 1156 pg/mL after HAART (P=.06). Proliferative responses to M. avium occurred in 76% of DMAC subjects receiving HAART. CD4 and CD8 but not gammadelta T cells expanded in response to M. avium. CMI to M. avium reconstitutes rapidly after HAART and appears sustained even with partial viral suppression.     

The paradox of pyrazinamide: an update on the molecular mechanisms of pyrazinamide resistance in Mycobacteria

Pyrazinamide (PZA) is an important front line anti-tuberculosis drug because of its sterilizing activity against semi-dormant tubercle bacilli. In spite of its remarkable role in shortening the treatment duration from 9 months to 6 months when used in combination with Rifampicin and Isoniazid, PZA remains a difficult paradox because of its incompletely understood mode of action and mechanism of resistance. PZA is a nicotinamide analog prodrug which is converted into the active bactericidal form pyrazinoic acid by the bacterial enzyme pyrazinamidase (PZase). PZA does not appear to have a specific cellular target and instead, exerts its bactericidal effect by disrupting the membrane energetics and acidification of cytoplasm. Majority (72-97%) of PZA-resistant isolates of M. tuberculosis exhibit mutations in their pncA gene or upstream area leading to loss of PZase activity. A wide diversity of pncA mutations scattered along the entire length of pncA gene is unique to PZA resistance. However, PZA resistant isolates with normal PZase activity and wild type pncA sequences have also been reported in several studies which indicate that alternate mechanisms of PZA resistance exist. Investigations into these mechanisms would be useful in developing alternative diagnostic/therapeutic measures. This review presents the update of various mechanisms of PZA resistance in different mycobacteria with special emphasis on mode of action of PZA and mechanisms of resistance in Mycobacterium tuberculosis.     

Therapeutic effects of benzoxazinorifamycin KRM-1648 administered alone or in combination with glycyrrhizin against Mycobacterium avium complex infection in mice]

We previously examined the effects of a Chinese medicine "Mao-Bushi-Saishin-To" (MBST) which has anti-inflammatory activity on the therapeutic efficacies of a benzoxazinorifamycin, KRM-1648 (KRM), against, Mycobacterium avium complex (MAC) infection induced in mice. MBST potentiated the therapeutic activity of KRM against MAC infection. In the present study, we examined the effects of another anti-inflammatory drug Glycyrrhizin, which is effective for chronic hepatitis, on the therapeutic efficacy of KRM against MAC infection induced in mice. First, KRM significantly inhibited the bacterial growth in the lungs and spleen of MAC-infected mice. Glycyrrhizin exhibited no therapeutic activity against MAC infection and did not affect the expression of the therapeutic efficacy of KRM. Secondly, treatment of murine peritoneal macrophages (M phi s) with Glycyrrhizin caused no significant changes in the M phi anti-MAC activity.     

Non-tuberculous mycobacteriosis. What has been coming out

Diagnosis of non-tuberculous mycobacteriosis is relatively easy, because of recent technological advances (HRCT, MGIT, PCR, DDH etc). Although many reports of this disease have been published, there are many problems to resolve. (1) Prevalence of non-tuberculous mycobacteriosis: Shigeki SATO (Department of Medical Oncology and Immunology, Nagoya City University Graduate School of Medical Sciences) Questionnaire surveys to determine the prevalence of nontuberculous mycobacterial (NTM) disease were carried out in 2001, 2007, and 2009. The NTM disease rate was estimated at 5.9/100,000, confirming that Japan has one of the world's highest NTM disease rates. Examination of the proportions of M. avium and M. intracellulare disease in Japan by region revealed that the M. avium/M. intracellulare disease ratio increased in different regions since past reports. In the 2007 survey, the M. avium disease rate had increased over the 2001 level. M. kansasii had a high disease rate in the Kinki and Kanto regions. Disease rates tended to be high in regions that have a metropolis. However, the disease rate was low in Aichi Prefecture, so that the presence in a region of a metropolis is probably not of itself a factor causing a high disease rate. The distributions of the bacteria causing NTM thus vary among different countries and regions. (2) Polyclonal infection of Mycobacterium avium using variable numbers of tandem repeats (VNTR) analysis: Tomoshige MATSUMOTO (Department of Clinical Research and Development, Center for Infectious Diseases, Osaka Prefectural Hospital Organization, Osaka Prefectural Medical Center for Respiratory and Allergic Diseases) Mycobacterium avium complex (MAC) is refractory to therapy, containing rifampicin (RFP), ethambutol (EB), and clarithromycin (CAM). It was widely accepted that therapeutic difficulties of pulmonary MAC treatment was caused by highly resistance to antibiotics or repeated re-infection from environment. Variable number of tandem repeats (VNTR) analysis of MAC is available. So, we studied the MAC-VNTR of clinical isolates from 29 patients with pulmonary MAC, refractory to the therapy. Compared the clinical isolates before with after each therapy, clinical isolates derived from the all except one patient showed the same VNTR patterns, before and after. According to MAC-VNTR analysis of the clinical isolates we studied, frequency of polyclonal infection was low (1/29). We concluded that the highly resistance to antibiotics or the repeated same VNTR type infection from environment made refractory pulmonary MAC. (3) An approach to identify susceptibility genes in patients with non-HIV-related pulmonary Mycobaterium avium complex (MAC) infection: Naoto KEICHO (Department of Respiratory Diseases, Research Institute, National Center for Global Health and Medicine) Mycobacterium avium complex causes human pulmonary disease. Th1 T cells play a role in protective immunity from mycobacterial infection. Genetic defect of Interferon-gamma/ Interleukin-12 axis is known to cause familial non-tuberculous mycobacterial infection. On the other hand, non-mendelian type of genetic abnormalities such as polymorphisms of HLA, CFTR and SLC11A1 (NRAMP1) genes has also been investigated as disease susceptibility genes. Recently our group has reported disease association with MHC-class I related chain-A molecule (MICA), comparing 300 sporadic cases with 300 healthy controls. (4) Genetic feature of Mycobacterium avium complex: Taku NAKAGAWA, Kenji OGAWA (Department of Pulmonary Medicine, National Hospital Organization Higashinagoya National Hospital) The bacterial factors contributing to the pathogenesis of M. avium complex infection and diversity of disease progression remain unclear. MATR-VNTR typing is inexpensive and easy to perform and has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. MATR-VNTR typing revealed that M. avium isolates from HIV-positive patients are analogous to the isolates from pig enterically-transmitted rather than those from HIV-negative patients with pulmonary diseases. M. avium comprises four subspecies. We performed genetic analysis by using Insertion Sequence (IS) for 114 clinical isolates of M. avium. All clinical isolates were identified as M. avium subsp. hominissuis by sequence analysis of hsp65. PCR detection rate of IS901 was about 70%, while detection rate in Europe and America was 0-8%. Compared with the original IS901, 60 point mutations were found in the sequence of the insertion sequence detected from all PCR-positive clinical isolates. This new insertion sequence was designated ISMav6. It became clear that M. avium strains in Japan are distinct from strains in Western countries in terms of the prevalence of ISMav6. We conducted genetic analysis for M. avium isolates collected from 11 hospitals all over Japan, but MATR-VNTR typing failed to show that distinct clusters correlate with disease progression or region. Genetic typing for M. intracellulare using VNTR has not yet been developed. We identified VNTR loci in the genome of M. intracellulare ATCC1395 and applied them as a molecular epidemiological tool to clinical isolates. (5) Infection source of pulmonary Mycobactrium avium complex (MAC) disease: Yukiko NISHIUCHI (Toneyama Institute for Tuberculosis Research Osaka City University Medical School), Ryoji MAEKURA (National Hospital Organization Toneyama National Hospital) Pulmonary MAC disease is characterized as the polyclonal infection and the recurrence, which suggest the presence of polyclonal niche of MAC in environment surrounding patients. We revealed that MAC was recovered from bathrooms but not from other sites of residences. The bathtub inlet was the niche with polyclonal colonization of MAC in the bathrooms of MAC patients. The identical/related genotypic profiles with isolates from patients were revealed by pulsed field gel electrophoresis. These results implied that the residential bathroom might be one of the infectious sources of pulmonary MAC disease.     

鸟-胞内分枝杆菌复合群鸟和胞内分枝杆菌亚种对20种药物的敏感性特征及亚种间药物敏感性比较

目的 分析鸟-胞内分枝杆菌复合群鸟分枝杆菌亚种和胞内分枝杆菌亚种对20种药物的敏感性特征及比较2个亚种间的药物敏感性差异。方法 用微孔板Alamar blue显色法分别检测97株鸟分枝杆菌和172株胞内分枝杆菌对克拉霉素、卷曲霉素和利福布丁等20种药物的最小抑菌浓度,判断其敏感性,分析2个亚种菌株对20种药物的敏感性特征以及对利福霉素类、氨基糖苷类和大环内酯类内药物的交叉耐药性,并比较2个亚种对20种药物的敏感性差异。结果 对鸟分枝杆菌敏感率高于80%的药物依次是卷曲霉素(100%,97/97)、阿米卡星(97.9%,95/97)、利福布丁(96.9%,94/97)、利福平和卡那霉素(83.5%,81/97)及克拉霉素(82.5%,80/97);对胞内分枝杆菌敏感率高于80%的药物依次是阿米卡星(99.4%,171/172)、卡那霉素(95.9%,165/172)、利福布丁(95.4%,164/172)、克拉霉素(94.2%,162/172)、氯法齐明(87.2%,150/172)、卷曲霉素和罗红霉素(86.1%,148/172)及利福喷丁(82.6%,142/172)。97株鸟分枝杆菌分别有1株、1株和17株而172株胞内分枝杆菌中分别有0株、7株和6株同时对氨基糖苷类、利福霉素类和大环内酯类内各种药物同时耐药或中度敏感。鸟分枝杆菌对利福平和卷曲霉素的敏感率明显高于胞内分枝杆菌。胞内分枝杆菌对利福喷丁、妥布霉素、卡那霉素、乙胺丁醇、莫西沙星、克拉霉素、阿奇霉素和罗红霉素的敏感率均高于鸟分枝杆菌。结论 鸟和胞内分枝杆菌均对卷曲霉素、阿米卡星、利福布丁和克拉霉素敏感性较高,对胞内分枝杆菌敏感的药物多于鸟分枝杆菌,利福喷丁仅对胞内分枝杆菌抗菌性较好,而利福平仅对鸟分枝杆菌抗菌性较好,乙胺丁醇对两个亚种敏感性均较差;对同一种类的药物做药物敏感性试验可为临床医生治疗鸟或胞内分枝杆菌感染选择合适的替代药物做依据。     

Cytokine profiles in immunocompetent persons infected with Mycobacterium avium complex

To evaluate the immunologic factors that contribute to protection against Mycobacterium avium complex (MAC), cytokine production by peripheral blood mononuclear cells (PBMC) from human immunodeficiency virus-negative persons with pulmonary MAC (MAC patients) and healthy control subjects with a delayed hypersensitivity skin test response to M. avium sensitin (MAS-positive control subjects) was measured. In MAC patients, mycobacterium-stimulated PBMC produced higher concentrations of interleukin (IL)-10 but lower concentrations of interferon (IFN)-gamma, IL-12, and tumor necrosis factor (TNF)-alpha, compared with PBMC from MAS-positive control subjects. Immunolabeling for intracellular IL-10 revealed that this cytokine was produced by both monocytes and T cells. Alveolar macrophages produced TNF-alpha and IL-10 in response to MAC, which suggests that these cytokines are produced in the lungs of patients with pulmonary disease caused by this pathogen. Our findings suggest that IFN-gamma, TNF-alpha, and IL-12 contribute to protection against MAC, whereas IL-10 is immunosuppressive.     

Antimycobacterial activity in vivo of LM427 (rifabutin)

The activity of the rifampin derivative rifabutin (ansamycin; LM427) was tested in vivo against experimental infections of mice with members of the Mycobacterium avium complex (MAC) and a highly virulent strain of Mycobacterium tuberculosis. Rifabutin was moderately effective against an environmental serotype 8 strain of MAC and of significant effectiveness against low dose aerogenic infections with two strains of higher virulence. The drug was also highly effective in causing the rapid resolution of an intravenous M. tuberculosis infection. These data thus contest a previous negative report on the activity of rifabutin in mouse models of M. avium complex.     

Identification of Mycobacterium avium and Mycobacterium intracellulare using three DNA probe tests, and their distributions in Japan]

Identification of Mycobacterium avium complex (MAC) was made using three DNA probe tests for MAC: Gen-Probe Rapid Diagnostic System for the MAC (Gen-Probe Inc., San Diego, U.S.A.), AccuProbe MAC Culture Identification or Confirmation Test (Gen-Probe Inc.); and SNAP Culture Identification Diagnostic Kit (MAC) (Syngene Inc., San Diego, U.S.A.). Various strains of MAC belonging to serovars 21 to 28 were identified by the DNA probe tests and showed the following. First, Serovar 21 and 25 belonged to M. avium and M. intracellulare, respectively. Each of them reacted with species-specific probes used in the three DNA probe tests [i.e., either M. avium-probe (in SNAP test; Probe A) or M. intracellulare-probe (in SNAP test; Probe I)]. Second, serovars 22-24 and 26-28 consisted of M. intracellulare, MAC strains that reacted with Probe X of SNAP test but lacked the reactivity with M. avium- and M. intracellulare probes of all the DNA probe tests, M. scrofulaceum that showed no reactivity with M. avium- or M. intracellulare-probe or Probe X, and M. scrofulaceum that had only the reactivity with Probe X. When the disease-associated MAC strains (35 strains), isolated in the Kanto to Kyushu areas in Japan, were identified using AccuProbe test, both the M. avium and M. intracellulare strains identified by the Gen-Probe test reacted with the MAC-probe but not with the M. tuberculosis complex (MTC)-probe.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of adjuvant granulocyte-macrophage colony-stimulating factor in HIV-related disseminated atypical mycobacterial infection

Mycobacterium avium complex (MAC) continues to be a challenging problem in some patients with advanced human immunodeficiency virus (HIV) infection despite the availability of potent chemotherapeutic agents. The use of adjunctive immunomodulatory therapy has shown promise in vitro, but there is currently limited supportive clinical evidence [Kemper CA, Bermudez LE, Derenski SC. Immunomodulatory treatment of Mycobacterium avium complex bacteraemia in patients with AIDS by use of recombinant granulocyte-macrophage colony-stimulating factor. J Infect Dis 1998;177:914-20; Kedzierska K, Johnson M, Mijch A, Cooke I, Rainbird M, Roberts S, et al. Granulocyte-macrophage colony-stimulating factor augments phagocytosis of Mycobacterium avium complex by human immunodeficiency virus type-1 infected monocytes/macrophages in vitro and in vivo. J Infect Dis 2000;181:390-94]. We report the resolution of MAC disease resistant to traditional therapy, in an HIV-infected individual, following the addition of granulocyte-macrophage colony-stimulating factor (GM-CSF).     

In vitro susceptibilities of Mycobacterium avium and Mycobacterium intracellulare to various drugs

Mycobacterium avium and M. intracellulare isolated from patients infected with M. avium complex (MAC), which were identified by Gen-Probe Rapid Diagnostic System for the MAC, were studied for susceptibility to various antimicrobial agents, including rifampicin, rifabutin, kanamycin, streptomycin, amikacin, ethambutol, clofazimine, isoniazid, ofloxacin, ciprofloxacin and cycloserine. Ratio of resistant strains to test strains to a given agent at prescribed concentration in cases of M. avium and M. intracellulare was compared with each other. Test strains of M. avium were more resistant to rifampicin, rifabutin, kanamycin, streptomycin, amikacin, ethambutol and clofazimine than test strains of M. intracellulare. Conversely, the M. avium strains were more susceptible to ofloxacin, ciprofloxacin and cycloserine than M. intracellulare strains. The difference in the drug susceptibility between M. avium and M. intracellulare was statistically significant by chi 2-test (P less than 0.005-0.05). There was no statistically significant difference between the two MAC species with respect to the susceptibility to isoniazid.     

Comparative antigenic analysis of Mycobacterium avium complex (MAC) isolates from AIDS patients.

Sonicates of several Mycobacterium avium complex (MAC) strains isolated from acquired immunodeficiency syndrome (AIDS) patients were characterized in order to study the prominent antigens of these strains. Sonicates of 6-week-old cultures were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. A major 12 kDa glycoprotein antigen was observed in all the sonicates along with other proteins ranging up to 100 kDa. Western blotting, using the 12 kDa M. leprae 'specific' murine monoclonal antibody (MAb) MLO6, indicated the presence of a determinant in the 12 kDa antigen (in all the MAC isolates studied) which was immunologically cross-reactive with the 12 kDa antigen of M. leprae. The transparent variant of MAC 101 also demonstrated MLO6 reactivity while the opaque variant did not. Polyclonal antiserum raised against MAC 101 sonicate reacted with all the MAC isolates in immunodiffusion. These observations point to the cross-reactivity between these strains and suggest that they possess a M. leprae 'specific' determinant on a cross-reacting component which could be involved in virulence.     

pncA mutations in clinical Mycobacterium tuberculosis isolates from Korea

Background  

Pyrazinamide (PZA) is among the first-line drugs for the treatment of tuberculosis. In vitro, it kills semidormant mycobacteria only at low pH. The purpose of this study was to compare PZA resistance with pyrazinamidase (PZase) activity and the genotype to better understand the molecular basis of PZA resistance and to expand the profile of pncA mutations worldwide.     

A case of environmental infection with pulmonary Mycobacterium avium complex disease from a residential bathroom of a patient suggested by variable-number tandem-repeat typing of Mycobacterium avium tandem repeat loci

A 63-year-old woman was referred to our hospital because of bilateral infiltrations and nodular opacities in her chest radiograph taken in the mass radiography screening in September 2010. The chest computed tomography showed patchy infiltrations with bronchiectasis in the lower lung fields on both sides. She was diagnosed with pulmonary Mycobacterium avium complex (MAC) disease based on the bacteria recovered from the sputum and the bronchoalveolar lavage fluid. To elucidate an environmental MAC source, we investigated her home, and isolated M. avium and M. gordonae from the bathtub and shower tap, respectively, in her residential bathroom. Analysis of the hsp65-PRA variants digested with BamHI and some insertion sequences showed that the clinical strains recovered from sputum and strains from the bathtub were M. avium subsp. hominissuis. A dendrogram of the Mycobacterium avium tandem repeat loci variable-number tandem-repeat (MATR-VNTR) analysis of the MAC strains showed that the bathtub strains formed a polyclonal colonization, and that 1 of the 5 MATR-VNTR patterns was identical to the corresponding pattern of the sputum strain from the patient. In conclusion, we believe that the residential bathroom of the patient was the environmental source of her pulmonary MAC disease, as has been previously reported.     

Use of DNA probes to detect Mycobacterium intracellulare and "X" mycobacteria among clinical isolates of Mycobacterium avium complex.

A mycobacterial DNA probe (designated X) was recently developed to help identify Mycobacterium avium complex (MAC) isolates that are nonreactive with probes specific for M. avium or Mycobacterium intracellulare. The prevalence of X probe-positive mycobacteria in clinical specimens and their role in causing disease is unknown. Using a DNA probe kit that includes the X probe, we characterized 100 consecutive clinical MAC isolates as M. avium, M. intracellulare, or X. Lysates from 81 of the isolates reacted with the M. avium probe, 13 with the M. intracellulare probe, 3 with the X probe, and 3 failed to hybridize with any of the probes. All three X-positive isolates were recovered from sputa of patients who were recent immigrants to the United States and who presented with hemoptysis. One isolate was from a Hispanic man infected with human immunodeficiency virus type 1 (HIV-1) and the other 2 were from Filipino patients with no HIV-1 risk factors. This study also showed a higher than expected number of M. intracellulare isolates from blood and cerebrospinal fluid of HIV-1-infected patients.     

Genetic research about Mycobacterium avium complex

A variable-number tandem-repeat (VNTR) typing method using the Mycobacterium avium tandem repeat (MATR) loci (MATR-VNTR) is employed in Japan for epidemiological studies using clinical isolates of M. avium. In this study, the usefulness of this MATR-VNTR typing method was compared with that of the IS1245-restriction fragment length polymorphism (IS1245-RFLP) typing method and a mycobacterial interspersed repetitive-unit (MIRU)-VNTR typing method reported previously (Boschiroli, C. Hubbans, P. Overduin, K. Stevenson, M. C. Gutierrez, P. Supply, and F. Biet, Clin Microbiol.) Seventy clinical isolates identified as M. avium from human immunodeficiency virus-negative patients with MAC infections were used. MATR-VNTR typing using 15 loci distinguished 56 patterns of different allele profiles, yielding a Hunter-Gaston discriminatory index (HGDI) of 0.990. However, IS1245-RFLP and MIRU-VNTR typing yielded HGDIs of 0.960 and 0.949, respectively, indicating that MATR-VNTR has an excellent discriminatory power compared with MIRU-VNTR and IS1245-RFLP typing. Moreover, concomitant use of the MATR-VNTR method and IS1245-RFLP typing increased the HGDI to 0.999. MATR-VNTR typing is inexpensive and easy to perform and could thus be useful in establishing a digital multifacility database that will greatly contribute to the clarification of the transmission route and pathophysiology of M. avium infections. Mycobacterium avium (n=81) from patients with pulmonary infections who were HIV-negative and isolates (n=33) from HIV-positive patients were subjected to genetic analysis by PCR detection of three M.avium-specific insertion sequences (IS901, IS1245 and IS1311) and nucleotide sequencing of the heat-shock protein 65 (hsp65) gene. All clinical isolates were identified as 'M. avium subspecies hominissuis' by sequence analysis of hsp65. Compared with clinical isolates of M. avium reported elsewhere, IS1245 was found less frequently in Japanese isolates (96/114 isolates, 84%) and IS901 was detected more frequently (76/114 isolates, 67%). One isolate was found to lack IS1311, which has not been reported previously for 'M. avium subsp. hominissuis.' Nucleotide sequence analysis of the PCR products for IS901 revealed that all clinical isolates had the same new insertion sequence, designated ISMav6, which had 60 point mutation compared with the nucleotide sequence of the original IS901. These results suggest that 'M. avium subsp. hominissuis' with ISMav6 is prevalent in Japan. ISMav6 may have implications for the virulence of M. avium and contribute to an increase of M. avium infections in this country. Clarithromycin (CAM) is the key drug in the various treatment regimens of Mycobacterium avium complex (MAC) diseases and the only drug for which drug susceptibility has been shown to correlate with a clinical response in these diseases. A point mutation at position 2058 or 2059 of the 23S rRNA gene has been reported to occur in high-level CAM-resistant isolates. This study examined a correlation between the results from a drug susceptibility test and the mutation of genes involved in drug resistance in MAC. Furthermore, we adapted a rapid detection method using amplification refractory mutation system (ARMS)-PCR to identify a mutation in 23S rRNA gene in MAC isolates. Using MICs of CAM, drug susceptibility was tested for 253 clinically-isolated MAC strains. Of these, 28 CAM-sensitive strains and 26 CAM-resistant strains were analysed by sequence analysis and ARMS-PCR. The drug susceptibility test showed that a bimodal distribution was associated with 227 CAM-sensitive strains and 26 CAM-resistant strains. Sequence analysis revealed that all 28 CAM-sensitive strains tested were wild type, whereas 24 of the 26 CAM-resistant strains were mutant type. The sensitivity of the sequence and ARMS-PCR analyses were 92.3% and 84.6%, respectively, and specificity of both was 100%. We found a correlation between drug susceptibility testing and the mutation of drug-resistant genes in this study. ARMS-PCR allowed rapid determination of CAM resistance, even when samples consisted of the mixed type of CAM-sensitive and CAM-resistant strains. MAC is divided into Mycobacterium avium, and the less-epidemiologically studied Mycobacterium intracellulare. Genetic typing for M. intracellulare using variable number of tandem repeats (VNTR) has not yet been developed. The aim of this study was to identify VNTR loci in the genome of M. intracellulare and apply them as an epidemiological tool to clinical isolates. Here, we identified 25 VNTR loci on the M. intracellulare genome, which 16 showed variations among clinical isolates in the number of tandem repeat motifs. Among the 74 M. intracellulare isolates, 49 genotypes were distinguished using the 16 VNTR loci, resulting in a Hunter-Gaston discriminatory index of 0.988. Moreover, all 16 VNTR loci were stable in different sets of isolates recovered within time intervals ranging from 2 to 1551 days from 14 separate patients. These results indicate that for use as epidemiological markers of M. intracellulare, the loci in this VNTR assay highly discriminating and stable over time.     

Reactivities of various mycobacteria species against DNA probes (Gen-Probe Rapid Diagnostic System) specific to Mycobacterium tuberculosis complex, Mycobacterium avium and Mycobacterium intracellulare

Various mycobacterial species (22 species, 178 strains) were studied for their reactivity to DNA probe specific for Mycobacterium tuberculosis complex (MTC), M. avium or M. intracellulare, using Gen-Probe Rapid Diagnostic System (Gen-Probe Inc., San Diego, Calif., U.S.A.). All the MTC strains, including M. tuberculosis, M. africanum, M. bovis and M. microti reacted with MTC-DNA probe at the % hybridization value of 42.8-51.9% (values higher than 10% are regarded as positive), but their reactivity to MAC-DNA probes (0.8-2.5%) was under the cut off value (10%). The test strains (28 strains) of M. avium complex (MAC) segregated into two groups on the basis of reactivity to DNA probes specific for M. avium and M. intracellulare, that is, one group (16 strains) positively reacted with M. avium-probe but not with M. intracellulare-probe, and the other group (12 strains) showed the converse reactivity. The two groups did not show a reactivity with MTC-probe higher than the cut off value. Nontuberculous mycobacteria other than MAC, including M. kansasii, M. marinum, M. simiae, M. asiaticum, M. scrofulaceum, M. gordonae, M. szulgai, M. malmoense, M. xenopi, M. gastri, M. nonchromogenicum, M. terrae, M. triviale, M. fortuitum, and M. chelonae (subsp. abscessus and chelonae) reacted with neither MTC- nor MAC-probe and values for % hybridization (0.6-3.6%) were lower than the cut off value. These findings indicate extremely superior specificity of the DNA probes (Gen-Probe) for MTC, M. avium and M. intracellulare, thereby indicating the usefulness of Gen-Probe Rapid Diagnostic System for the MTC and MAC in clinical use.     

Identification of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium tuberculosis complex by Gen-Probe Rapid Diagnostic System

DNA probe testing for Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis complex (MTC) was performed using Gen-Probe Rapid Diagnostic System (Gen-Probe Inc., San Diego, Calif., U.S.A.). By DNA probe test carried out blindfold for 48 mycobacterial strains with code numbers obtained from Kyoto University (Prof. F. Kuze), 13, 7, and 5 strains were identified as to be M. avium, M. intracellulare, and MTC, respectively. The diagnostic specificity and sensitivity of this testing were 100%. In this experiment, % hybridization of M. avium complex (MAC) and MTC were 25-55% and 45-52%, respectively. DNA probe test for 54 MTC strains including M. tuberculosis, M. bovis, M. africanum and M. microti revealed that 53 strains, except for one strain donated as a niacin-negative M. tuberculosis, reacted with MTC probe but not with MAC-probes. The one exceptional strain reacted with both the MTC- and M. avium-probes. However, when ten colonies randomly isolated from this strain on 7H11 agar plate were subjected to the DNA probe test again, all of these colonies reacted with M. avium probe, but not with MTC probe. Moreover, one representative colony was found to have alpha-antigen specific for the MAC.     

Sources of disseminated Mycobacterium avium infection in AIDS

OBJECTIVES: To identify the sources of disseminated Mycobacterium avium complex (MAC) infection in AIDS. METHODS: HIV positive subjects with CD4 counts <100/mm(3) in Atlanta, Boston, New Hampshire and Finland were entered in a prospective cohort study. Subjects were interviewed about potential MAC exposures, had phlebotomy performed for determination of antibody to mycobacterial lipoarabinomannin and for culture. Patient-directed water samples were collected from places of residence, work and recreation. Patients were followed for the development of disseminated MAC. Univariate and multivariate risk factors for MAC were analyzed. RESULTS: Disseminated MAC was identified in 31 (9%) subjects. Significant risks in univariate analysis included prior Pneumocystis carinii pneumonia (PCP) (hazard ratio 1.821), consumption of spring water (4.909), consumption of raw seafood (34.3), gastrointestinal endoscopy (2.894), and showering outside the home (0.388). PCP, showering and endoscopy remained significant in a Cox proportional hazards model. There was no association between M. avium colonization of home water and risk of MAC. In patients with CD4<25, median OD antibody levels to lipoarabinomannin at baseline were 0.054 among patients who did not develop MAC and 0.021 among patients who did develop MAC (P=0.077). CONCLUSIONS: MAC infection results from diverse and likely undetectable environmental and nosocomial exposures. Mycobacterial infection before HIV infection may confer protection against disseminated MAC in advanced AIDS.