Discrepancies between the genotypes and phenotypes of clarithromycin-resistant Mycobacterium abscessus complex.

SETTING: Macrolides are a key drug class used for the treatment of Mycobacterium abscessus complex disease. OBJECTIVE: To verify the relationship between phenotypic susceptibility and genotypic resistance to clarithromycin (CLM). DESIGN: Subspecies of M. abscessus complex from 145 consecutive patients were identified using hsp65 and rpoB gene sequencing, and tested for CLM susceptibility, classification into the erm(41) sequevars responsible for inducible resistance and the presence of rrl mutations associated with acquired resistance. RESULTS: The isolates comprised 74 M. abscessus subsp. abscessus, 69 M. abscessus subsp. massiliense and two M. abscessus subsp. bolletii. M. abscessus subsp. abscessus isolates comprised 15 sequevars, with the majority corresponding to sequevar 1 (n = 24), sequevar 6 (n = 13) and sequevar 2 (n = 8). Interestingly, seven M. abscessus subsp. abscessus isolates (9.5%) presented genetically functional, but not phenotypic, inducible resistance. Moreover, rrl was mutated in only 14.3% (1/7) of acquired resistance isolates. However, M. abscessus subsp. massiliense and M. abscessus subsp. bolletii isolates with acquired resistance at day 3 showed mutations at positions 2057-2059 (P < 0.05). CONCLUSIONS: Our study indicates that genotypic inducible and acquired resistance in M. abscessus subsp. abscessus does not always coincide with phenotypic susceptibility. Rigorous phenotypic evaluation is thus important because of the considerable impact on patients.

Mycobacterium marinum infection in Japanese forest green tree frogs (Rhacophorus arboreus).

Four Japanese forest green tree frogs (Rhacophorus arboreus) were presented with emaciation, abdominal distention and ulcerative and nodular cutaneous lesions affecting the brisket, limbs, digits and ventral abdomen. Another three frogs had been found dead in the same tank 1 year previously. Necropsy examination of these seven frogs revealed splenomegaly and hepatomegaly, with multiple tan-yellow nodular foci present in the liver, spleen, heart, lungs, ovaries and kidneys. Microscopically, five frogs had necrosis and surrounding granulomatous inflammation in the liver, spleen, kidneys, lungs, intestine and ovaries, with numerous acid-fast bacilli in the areas of necrosis. Two frogs had granulomatous lesions in the lungs, liver, spleen, heart, coelomic membrane, stomach and intestinal wall. These lesions had no or minimal necrosis and few acid-fast bacilli. Mycobacterium spp. was cultured from three frogs and identified as Mycobacterium marinum by colony growth rate and photochromogenicity and DNA sequencing. This is the first report of M. marinum infection in Japanese forest green tree frogs.

Effect of oestrogen on Mycobacterium avium complex pulmonary infection in mice.

The purpose of the present study was to elucidate the role of oestrogen in the pathogenesis of Mycobacterium avium complex (MAC) pulmonary disease, which occurs most frequently in postmenopausal women. The study was carried out in a murine infectious model using ovariectomized DBA/2 female mice. Infection with MAC was established by intratracheal administration of bacilli. In some experiments, ovariectomized mice were treated with exogenous 17 beta-estradiol (E2). The number of bacilli in the lungs of infected mice which received ovariectomy was significantly larger than that in the lungs of sham-operated control mice, and treatment of ovariectomized mice with exogenous E2 restored the burden of bacilli to the same level as that in the sham-operated control mice. We next examined the effect of E2 in vitro using bone marrow-derived macrophages obtained from DBA/2 female mice. The macrophages showed bacteriostatic activity against MAC after treatment with interferon-gamma (IFN-gamma) and this activity was further enhanced by the exogenous addition of E2 to the culture medium. In parallel with these findings, E2 augmented the production of reactive nitrogen intermediates (RNI) by macrophages pretreated with IFN-gamma and stimulated with MAC, as shown by evaluating nitrite production and inducible nitric oxide synthase mRNA expression. These findings taken together suggest that absence of endogenous oestrogen appears to be responsible for the development of MAC pulmonary disease in this mouse model and that the enhancement by E2 of anti-MAC activity of murine macrophages induced through increased RNI production may play some role in resistance to MAC infection.

Familial pulmonary Mycobacterium avium complex disease.

We report two Japanese families affected by pulmonary Mycobacterium avium complex (MAC) disease, involving an older brother and younger sister in one family and two brothers in the second family. We investigated whether defects in the natural resistance-associated macrophage protein gene (NRAMP1) underlay susceptibility to MAC in these cases. All of the patients had computed tomographic findings of peripheral nodules and bronchiectasis. Pulse-field gel electrophoresis patterns of mycobacterial genomic DNA restriction fragments revealed that none of the MAC strains isolated from the patients was epidemiologically related to any of the others. Direct sequencing of the complementary DNA of the patients' NRAMP1 revealed a nonconservative missense mutation at codon 419 in one patient, which was heterozygous and was not seen in his affected sibling. No variations similar to those found in mice that show susceptibility to MAC were found. The results suggest an underlying genetic defect in host defense rather than exposure to an unusually virulent strain of MAC as the pathogenetic factor in MAC disease; however, alterations in the coding region of NRAMP1 do not appear to explain the susceptibility to MAC.

Recurrent hemoptysis in tuberculosis with non-cavitary lung disease as a symptom of mild hemophilia A in a young adult.

Hemoptysis in patients with tuberculosis is usually associated with smear-positive and cavitary lung disease. The present case describes a patient suffering from recurrent hemoptysis associated with tuberculosis who had smear-negative and non-cavitary lung disease, and who was subsequently diagnosed as having mild hemophilia A. Although mild hemophilia A sometimes escapes detection until adolescence, there has been no reported case of mild hemophilia A detected by recurrent hemoptysis due to pulmonary tuberculosis. Here, we report a rare case of recurrent hemoptysis in a patient with tuberculosis who had smear-negative and non-cavitary lung disease and who was finally shown to have hemophilia A.

Effect of proton pump inhibitor alone or in combination with clarithromycin on mycobacterial growth in human alveolar macrophages.

The effect of omeprazole, a clinically used proton pump inhibitor, alone or in combination with clarithromycin was evaluated against Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium tuberculosis, using a human alveolar macrophage model of infection. Omeprazole exhibited no significant effect on the growth of the two M. avium complex strains or on the mycobactericidal activity of clarithromycin against them. In contrast, omeprazole significantly promoted the growth of Mycobacterium tuberculosis and the anti-mycobacterial activity of clarithromycin against it in human alveolar macrophages. It was speculated that intracellular acidic milieu around M. tuberculosis might be one reason for the lower activity of clarithromycin in the treatment of human tuberculosis.

Effect of clarithromycin regimen for Mycobacterium avium complex pulmonary disease.

We have investigated the efficacy of a clarithromycin-containing four-drug regimen for Mycobacterium avium complex (MAC) pulmonary disease in 46 patients without acquired immunodeficiency syndrome (AIDS). The patients were 14 males and 32 females with a mean age of 60.9 +/- 11.5 yr. Patients received 10 mg/kg/d of clarithromycin plus ethambutol, rifampin, and initial kanamycin and subsequent quinolone for 24 mo. Seven patients (15.2%) were dropped in the first 6 mo. Among 39 patients who received more than 6 mo of therapy, 28 patients (71.8%) converted their sputa to negative: 26 of 31 patients (83.9%) infected with clarithromycin-susceptible strains and two of eight patients (25.0%) with resistant or intermediate strains. The timing of sputum conversion was 3.6 +/- 1.9 mo, with a range of 2 to 9 mo. The conversion rate was significantly lower in patients who were infected with clarithromycin-resistant or intermediate strains, who had had prior therapy (55.0% versus 89.5%), or who were acid-fast bacilli (AFB) smear-positive at entry (60.7% versus 100%). The age and sex of patients, the species of pathogen (M. avium or M. intracellulare), type and extent of the disease, and the use of kanamycin did not significantly affect the conversion rate. Although the regimen was efficacious for newly treated patients, frequent adverse reactions and a low conversion rate of sputum in retreated patients are problems that remain to be solved.

[Multidrug-resistant tuberculosis. Additional comment: primary multidrug-resistant tuberculosis--diagnosis and treatment].

Primary drug resistance is defined as the presence of resistant strains of Mycobacterium tuberculosis in a patient with no history of prior anti-tuberculosis chemotherapy. In Japan, a recent study shows that the prevalence of primary resistance has been stable for two decades and that primary multidrug-resistant tuberculosis (MDR-TB) is rare, which suggests the effectiveness of tuberculosis control. We presented four cases of primary MDR-TB that we had experienced from 1984 to 1997, and discussed an issue about diagnosis and treatment of primary MDR-TB. Of the four patients, two young men received surgical resection, which has resulted in a favorable outcome. Of the other two patients, one responded to long-term chemotherapy with ethambutol, ofloxacin and enviomycin. There have been no recurrence so far in the three cases. The rest case died due to progression of tuberculosis. Two of the four patients had been in contact with relatives who had died of MDR-TB. In conclusion, all the tuberculosis patients should be suspected to be primary MDR-TB when they had a history of a contact with a tuberculosis patient in whom chemotherapy had not been successful, and once patients are diagnosed as MDR-TB, surgical intervention should be considered as an adjunctive treatment. To prevent the emergence of primary MDR-TB, it is important to treat MDR-TB patients appropriately and to implement the infection control program.

[A case of miliary tuberculosis with brain tuberculoma, following intraocular tuberculosis].

A 52-year-old woman with visual loss of her left eye consulted a ophthalmology clinic. She was conducted left vitrectomy and administered corticosteroid under the diagnosis of uveitis of unknown cause. But her visual acuity was not improved. Although re-surgery was planned, pus retention was found in her left eye. After her left eye was resected, fever and general malaise appeared suddenly. Her chest X-ray film revealed diffuse micronodular opacities. Acid-fast bacilli were detected from her sputum and identified to be Mycobacterium tuberculosis. She was diagnosed miliary tuberculosis, and then antituberculous chemotherapy consisting of 4 drugs was started. Granulomatous inflammation destructing retina and numerous acid-fact bacilli were found in histologic examination of the resected eye. This case was thought to be miliary tuberculosis disseminated from intraocular tuberculosis. After 2 months of therapy, neurologic symptoms which might be caused by brain tuberucloma appeared and deteriorated rapidly. But by adding corticosteroid to antituberculous therapy, symptoms were diminished gradually.

Evaluation of Roche Amplicor PCR assay for Mycobacterium avium complex in bronchial washing.

SETTING: A commercially available polymerase chain reaction (PCR) test (Roche AmplicorTM Mycobacterium avium and M. intracellulare assay-MAC-PCR) designed to detect M. avium complex (MAC) in bronchial washing was evaluated. DESIGN: A total of 141 specimens from 127 patients with various pulmonary conditions were examined. Results were compared with acid-fast smears, cultures with Ogawa egg medium, as is still commonly used in Japan, and final diagnoses. RESULTS AND CONCLUSIONS: A total of 14 bronchial washing specimens yielded MAC. Six smear- and culture-positive specimens were all MAC-PCR positive. In eight smear-negative and culture-positive specimens, six were MAC-PCR positive. The overall sensitivity versus culture was 85.7% (12/14). However, sensitivity might be over-estimated, as there is a lower recovery rate of MAC with egg-based medium compared with liquid media. In 127 patients, 15 were identified as having pulmonary MAC disease, of whom 13 had positive MAC-PCR in bronchial washing. In the remaining 112 patients, MAC-PCR was negative, which suggests that positive MAC-PCR was not a contaminated result. However, in terms of sensitivity and speed, we were unable to show any additional clinical benefit for using MAC-PCR as opposed to liquid media, in which MAC can frequently be detected in 7 to 14 days.

Polypoid bronchial lesions due to Scedosporium apiospermum in a patient with Mycobacterium avium complex pulmonary disease.

A 69 yr old female was hospitalized for further examination of abnormal shadows on chest radiographs. She had a history of tuberculous pleurisy, rheumatoid arthritis and gold-induced interstitial pneumonia. On admission she still suffered from rheumatoid arthritis. A chest computed tomography scan on admission showed clusters of small nodules in subpleural regions of both lungs combined with bronchiectasis. Mycobacterium avium complex was cultured repeatedly from the sputum. Bronchoscopic examination disclosed white-yellow polypoid lesions in the orifice of the left B4 bronchus. Cultures of the brushing specimen of the polypoid lesions and bronchial aspirates from the B4 bronchus yielded smoky-grey mycelial colonies that were later identified as Scedosporium apiospermum. It was concluded that the polypoid bronchial lesions due to Scedosporium apiospermum were formed in the preexisting dilated bronchus caused by Mycobacterium avium complex pulmonary disease.

Modification of results of drug susceptibility tests by coexistence of Mycobacterium avium complex with Mycobacterium tuberculosis in a sputum sample: case report and experimental considerations.

We report on a patient whose sputum contained both Mycobacterium tuberculosis and Mycobacterium avium complex (MAC). The MAC failed to be detected by the PCR-based AMPLICOR test. The unrecognized coexistence of MAC in the sample modified the results of drug susceptibility tests. Experiments revealed that the presence of both M. tuberculosis and MAC was not detected by the AMPLICOR test under certain conditions.

A resected case of Mycobacterium szulgai pulmonary disease.

We present the first reported case of Mycobacterium szulgai pulmonary disease that needed surgical resection due to unsuccessful antimycobacterial chemotherapy. The patient was a non-immunocompromised 48-year-old male who presented with hemoptysis and whose sputum cultures repeatedly yielded M. szulgai. Antimycobacterial chemotherapy with isoniazid (INH) and rifampin (RMP)/ethambutol (EMB) for three years had been unsuccessful, and subsequent chemotherapy with RMP, EMB, ethionamide and kanamycin had to be discontinued due to liver dysfunction. Surgical resection was finally performed, and resulted in a favorable outcome. Although M. szulgai pulmonary disease is usually well controlled by antimycobacterial chemotherapy alone, surgical treatment may be necessary in some cases.

Interleukin-12 gene expression in human monocyte-derived macrophages stimulated with Mycobacterium bovis BCG: cytokine regulation and effect of NK cells.

Macrophage-derived interleukin-12 (IL-12) is essential for the activation of a protective immune response against intracellular pathogens. In this study, we examined the regulation of IL-12 mRNA expression by monocyte-derived macrophages (MDM) in response to Mycobacterium bovis BCG stimulation. A reverse transcription-PCR assay detected p40 mRNA of IL-12 at 3 h and showed a peak at 6 to 12 h with a subsequent decline. Semiquantitation of mRNA levels by competitive PCR revealed that pretreatment with gamma interferon (IFN-gamma) amplified the expression approximately 100-fold, while pretreatment with tumor necrosis factor alpha (TNF-alpha) or granulocyte-macrophage colony-stimulating factor augmented this expression about 10-fold. In contrast, pretreatment with IL-10 and IL-4 inhibited IL-12 mRNA expression. These results were further confirmed by measuring the p70 bioactive protein level in each conditioned medium by an enzyme-linked immunosorbent assay. Since IL-12 mRNA expression was weak without cytokine pretreatment and IFN-gamma strongly augmented production, we speculated that IFN-gamma might have a role in BCG stimulation of IL-12 mRNA expression. Unexpectedly, the addition of three different kinds of anti-IFN-gamma antibodies and anti-IFN-gamma receptor antibody and the coaddition of anti-TNF-alpha antibody with anti-IFN-gamma receptor antibody all failed to inhibit IL-12 mRNA expression. However, the MiniMACS method used to remove NK cells from a mononuclear cell suspension inhibited the expression of p40 mRNA but not the expression of mRNA of TNF-alpha or IL-1beta. We concluded that the coexistence of NK cells was essential for the induction of IL-12 in MDM stimulated with BCG rather than through the secretion of IFN-gamma.

[Evaluation of mycobacteria growth indicator tube (MGIT) for drug susceptibility testing of Mycobacterium tuberculosis isolates].

Fifty six clinical isolates of Mycobacterium tuberculosis were tested for drug susceptibility in Mycobacteria Growth Indicator Tube (MGIT) containing 0.1 microgram/ml of INH, 1.0 microgram/ml of RFP, 3.5 micrograms/ml of EB and 0.8 microgram/ml of SM. These results were compared with those obtained by testing the same M.tuberculosis isolates by the absolute concentration method using 1% Ogawa egg slant containing 0.1 microgram/ml of INH, 10 micrograms/ml of REP, 2.5 micrograms/ml of EB and 20 micrograms/ml of SM. Fifty six isolates consisted of 18 pansensitive strains, 27 multidrug resistant strains and 11 single drug resistant strains. The results of individual drugs showed excellent agreement between the MGIT and the Ogawa methods, and overall agreement rate of the two methods were 96.4%. The results were just the same for all drugs in 48 out of 56 strains studied. The drug resistance could be observed much earlier by the MGIT method (mean 5.9 days) than by the Ogawa method (more than 21 days). In conclusion, the MGIT system could be a promising new drug susceptibility test which might become available in Japan replacing the Ogawa method.

[2 cases of lung disease caused by Mycobacterium avium complex occurred in middle-aged women without underlying disorders, which we observed for more than 30 years].

We reported 2 cases of Mycobacterium avium complex lung disease occurred in middle-aged women without underlying disorders, which we could observe for more than 30 years. One case was a 42-year-old woman started with bloody sputum, and the other was a 43-year-old woman with cough and sputum. In both cases, chest X ray films were normal on their first visit. More than 15 years after their first visit, Mycobacterium avium complex was isolated from their sputum or bronchial washing. During the observation, a cluster of small nodules in the periphery of the lung and bronchiectasis appeared and deteriorated, and excretion of the bacilli increased gradually. Their past history and family history were normal. Since lung disease caused by Mycobacterium avium complex progresses very slowly, long-time observation would be necessary to consider its pathogenesis.

Activity of KRM 1648 or rifabutin alone or in combination with clarithromycin against Mycobacterium avium complex in human alveolar macrophages.

SETTING: The activity of KRM 1648 (KRM), a new benzoxazinorifamycin, and rifabutin (RBT), alone or in combination with clarithromycin (CLA), was evaluated against Mycobacterium avium complex (MAC) that multiplied in human alveolar macrophages (AM). DESIGN: AM were recovered by bronchoalveolar lavage, incubated in RPMI 1640 medium with 10% human AB serum, infected with four strains of MAC (of non-acquired immune deficiency syndrome [AIDS] origin), and then treated with each drug alone or in combination. After incubation for 7 days, colony forming units in each well were counted on 7H10 agar. RESULTS: Although concentrations between 0.2 microgram/ml and 20 micrograms/ml of both rifamycins showed clear dose-dependent activities against all MAC strains tested, only 20 micrograms/ml of each drug had modest bactericidal effect. In combination with 2.0 micrograms/ml of CLA, however, 0.2 microgram/ml of both drugs caused a bactericidal response against two of the four MAC strains examined. CONCLUSION: According to this human alveolar macrophage model of MAC infection, KRM and RBT in combination with CLA was found to be a promising candidate against human pulmonary MAC infection, and deserves clinical evaluation.