Synovitis of the wrist caused by Mycobacterium florentinum.

Mycobacterium florentinum is a newly identified, rare, slow-growing species of nontuberculous mycobacteria (NTM). Here, we report a case of M. florentinum-induced synovitis of the wrist in an immunocompromised Japanese patient. M. florentinum was identified by sequence analysis of the rpoB, hsp65, and 16S rRNA genes. The M. florentinum strain in this study could not be differentiated from certain M. triplex strains by the hsp65 or 16S rRNA sequences alone, because they occasionally shared more than 99 % sequence identity. The isolated M. florentinum strain was only susceptible to clarithromycin and amikacin. Initially, the patient was treated with clarithromycin, levofloxacin, and ethambutol, and then with clarithromycin, levofloxacin, and rifampicin. To our knowledge, M. florentinum-induced synovitis has not been previously reported. Our results suggest that, in addition to other well-known pathogenic NTM, the recently identified M. florentinum strain should be considered as a possible cause of synovitis. Moreover, we should be cautious when identifying M. florentinum because this strain closely resembles M. triplex in genotype.

Isolation of novel mycobacteria contaminating an aquarium fish tank in a Japanese university hospital.

AIMS: To better understand nontuberculous mycobacteria (NTM) contamination in a hospital setting, six freshwater fish gut homogenates and water in an aquarium fish tank placed on the reception counter of a nursing station were cultured for mycobacteria. METHODS AND RESULTS: By direct sequencing of 16s rRNA, rpoB and hsp65, scotochromogenic and nonchromogenic Mycobacterium szulgai isolates containing hsp65 type II (GenBank accession nos. FJ384762 and FJ384764, respectively), Mycobacterium gordonae isolates containing rpoB clusters B and E (GenBank accession no. FJ384766), and Mycobacterium kansasii isolates containing hsp65 type VI were collected from the gut homogenates and water from the fish tank. However, no isolates were obtained from the tap water used to refill the fish tank. A randomly amplified polymorphic DNA (RAPD) analysis using a 10-mer primer (5'-TGGTCGCGGC) showed that some NTM from the fish tank water were identical to those obtained from the gut homogenates. CONCLUSIONS: Fish and water in the tank were contaminated by the novel NTM. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings could help to elucidate infection routes and contamination sources of novel NTM from water sources.

Descriptive analysis of the prevalence and the molecular epidemiology of Mycobacterium avium complex-infected pigs that were slaughtered on the main island of Okinawa.

Recent genetic studies have revealed that several epidemiological factors affect Mycobacterium avium complex (MAC) infection in pig populations. However, mechanisms underlying the spread of MAC infection among hog farms have not been clarified. In consideration of this situation, we cross-sectionally investigated the mechanisms underlying the spread of MAC on the island of Okinawa. Pigs slaughtered (n=706,763) and 331 hog farms on Okinawa were surveyed during the years 2002-2004. Two outbreaks of MAC infection were occurred in several farms during survey period. Bacteria were isolated from randomly selected pigs and genotype of isolates was determined by using genetic finger printing methods with the insertion sequence (IS) 1245 restriction fragment length polymorphism (RFLP). Most isolates had large numbers of IS1245 copies, while strains with low copy numbers of IS1245 and isolates without IS1245 were seen in few farms. MACs strains were repeatedly isolated from pigs of the affected farms during the survey period. Those farms with an identical pig rearing systems showed synchronic changes in the prevalence of MAC infection. An industrial farm without an outbreak had an independent pig flow, but maintained distinct MAC strains. Multivariate analysis did not reveal independent factors for the prevalence of the MAC infection. These findings suggest that there were three clusters distinguished genetically in the main island of Okinawa, which were potentially spread by common pig flow. However, the outbreaks occurred because of unspecified conditions on each farm environment.

Intractable ulcer caused by Mycobacterium shinshuense: successful identification of mycobacterium strain by 16S ribosomal RNA 3'-end sequencing.

An extremely rare case of intractable ulcer caused by Mycobacterium shinshuense is described. A 59-year-old Japanese woman developed an ulcerated subcutaneous induration on the upper arm. Ziehl-Neelsen staining revealed positive bacilli. Tissue culture isolated Mycobacterium species, but standard identification techniques (including molecular biological approaches such as DNA-DNA hybridization) could not distinguish the precise causative pathogen, although it was narrowed down to three possibilities: Mycobacterium marinum, Mycobacterium ulcerans and M. shinshuense. Finally, a novel 16S rRNA sequencing method enabled the diagnosis of M. shinshuense infection. The epidemiology of the cutaneous infection caused by this mycobacterium has yet to be elucidated, but a review of reported cases indicated that ulcers having some resemblance to those caused by M. ulcerans infection were found in nonendemic areas and that M. shinshuense could be considered as the cause. The approach introduced in this report could provide a powerful tool for the identification of this organism.

Role of tumor necrosis factor-alpha in Mycobacterium-induced granuloma formation in tumor necrosis factor-alpha-deficient mice.

To study the role of TNF-alpha in mycobacterial infection, we generated TNF-alpha-knockout (KO) mice, in which the third and fourth exons of the TNF-alpha gene were disrupted. The C57BL/6 KO mice were injected with virulent Mycobacterium tuberculosis strain Kurono or avirulent bacillus Calmette-Guérin (BCG) Pasteur (10(6) colony-forming units), through the tail veins. The major organs were removed at weekly intervals, and morphologic observation, assays of IL-1, IL-12, IFN-gamma, and inducible nitric oxide synthase mRNA expression, and colony counts in the lungs and spleen were performed. Peritoneal macrophages from BCG- and H37Rv strain-treated mice produced significant levels of nitric oxide after stimulation in vitro. Formation of abscesses was seen only in the Kurono-treated groups, and these abscesses contained large numbers of mycobacteria. The administration of recombinant TNF-alpha significantly ameliorated the mycobacterial lesions. IFN-gamma mRNA was expressed significantly in virulent H37Rv-treated groups with time, and the number of mycobacterial colonies per unit weight increased remarkably with time. Nitric oxide production was not observed in H37Rv-treated groups but was seen in BCG-treated groups. We concluded that TNF-alpha played an important role in protective immunity against virulent mycobacteria. Because avirulent mycobacteria did not induce granulomas in TNF-alpha-KO mice, TNF-alpha played an indirect role in granuloma formation.

Induction of granulomas in interferon-gamma gene-disrupted mice by avirulent but not by virulent strains of Mycobacterium tuberculosis.

To gain a better understanding of the pathological role of interferon-gamma (IFN-gamma) in specific granuloma formation, IFN-gamma gene-deficient mice (BALB/c and C57BL/6) were produced. The IFN-gamma gene in embryonic stem (ES) cells was disrupted by inserting the beta-galactosidase gene (lacZ) and the neomycin resistance gene (neo) at the translation initiation site in exon 1 by homologous recombination. Six-week-old IFN-gamma-deficient and wild-type mice were inoculated with 10(3)-10(7) bacilli of various strains of Mycobacterium tuberculosis (Kurono, H37Rv, H37Ra and BCG Pasteur) through their tail veins. The mice were examined 7 weeks later for granuloma formation. The avirulent BCG Pasteur and H37Ra strains (10(3)-10(4) bacilli/ml) induced granulomas in the spleen, liver and lungs of IFN-gamma-deficient mice. The granulomas consisted of epithelioid macrophages and Langhans multinucleate giant cells, but lacked caseous necrosis. The virulent Kurono and H37Rv strains induced disseminated abscesses but not granulomas in various organs of IFN-gamma-deficient mice and Mac-3-positive macrophages were not detected in the abscess lesions. These results suggest that IFN-gamma may be primarily responsible for macrophage activation and that other factor(s) may be involved in the granuloma formation mechanism.

[Progressive loss of speech output with festinating speech--a case report].

Primary progressive aphasia is a rare disorder of unknown cause. We report a patient with progressive loss of speech output, a clinical variant of PPA, characterized by festinating speech. A 60-year-old right handed woman was admitted to our hospital, because of progressive deterioration of her speech. On admission, she was alert and orientated without dementia. A severe impairment of her articulation was observed: her speech rate was so fast that her speech became almost intelligible. The orofacial apraxia and difficulty in tapping were also present. The other neurological findings were normal. Neuroradiological studies showed the left perisylvian atrophy. Festinating speech has not been previously reported in patients with PPA; patient with PPA usually show a slow speech rate with effortful expression. Since festinating speech is occasionally present in the extrapyramidal disorders, such as Parkinson's disease, progressive supranuclear palsy, or pure akinesia, it appears likely that the combined lesions of the perisylvian region and the basal ganglia are responsible for her characteristic speech disorder with festinating speech.

Mutation in pncA is a major mechanism of pyrazinamide resistance in Mycobacterium tuberculosis.

OBJECTIVE: To characterize the correlation of the mutations in the pncA gene encoding pyrazinamidase (PZase) of Mycobacterium tuberculosis to a loss of PZase activity and development of pyrazinamide (PZA) resistance. DESIGN: The association of PZase activity, minimum inhibitory concentrations (MICs), and mutations in the pncA gene of M. tuberculosis isolated in mostly Asian countries was investigated. RESULTS: One hundred thirty-five out of 168 isolates were PZase positive, and 33 were negative. The MICs of PZA at pH 6.0 were over 400 micrograms/ml for all 33 PZase-negative isolates, while those of PZase-positive isolates were equal to or less than 200 micrograms/ml. Among 33 PZase-negative isolates sequenced, 32 (97%) had mutations within the pncA gene. A mutation was seen in various regions throughout the pncA gene. It was surprising that all three strains of in vitro selected PZA resistant mutants were PZase-positive and showed no change in the pncA gene. These results indicate that additional mechanisms may be involved in PZA resistance. No mutations were observed in all of 135 PZase-positive M. tuberculosis isolates tested, indicating that mutations in the pncA gene could be involved in the loss of PZase activity. CONCLUSIONS: Sequencing analysis of the pncA gene should provide rapid diagnosis of PZA resistant clinical isolates of M. tuberculosis.

Resistance to antituberculosis drugs in Japan.

SETTING: Five years after the last survey of drug-resistant tuberculosis in Japan, a serious new phenomenon has gradually begun to appear. A nationwide survey was conducted by the Tuberculosis Research Committee. OBJECTIVE: To determine resistance patterns to five anti-tuberculosis drugs and risk factors. DESIGN: Cultures were obtained from patients hospitalized at 38 hospitals in various districts of Japan throughout 6 months, from 1 June through 30 November in 1992. Drug susceptibility testing was carried out in the national reference laboratory. RESULTS AND CONCLUSIONS: Resistance to one or more drugs was found in 5.6% of new cases and 27.8% of recurrent cases (P < 0.001). About 88% of drug resistant isolates from the new cases were resistant to one drug, while 50.8% of the drug resistant isolates from the recurrent cases had resistance to two or more drugs (P < 0.001). Resistance rates to both isoniazid and rifampin in new cases was very low (only 0.14%). Primary drug resistance rates were higher in age groups less than 60 years old, compared to those of 60 years and over (P = 0.05). Compared with the rate in Japanese patients, foreign-born individuals had a higher resistance rate in the recurrent cases (P = 0.034). This survey indicated a similar trend in resistance rates to five antituberculosis drugs to those of the last survey in 1987.

[A study on prevalence of resistance to antituberculosis drugs in Japan: comparison of results in the local facilities and in the reference laboratory].

During five years since the last survey of drug-resistant tuberculosis in Japan, features of tuberculosis in Japan have been changed. A nationwide survey was conducted by the Tuberculosis Research Committee of Japan. A total of 38 hospitals in various districts of Japan participated in the cooperative study. Each collaborating laboratory sent all mycobacterial cultures isolated during 1 June to 30 November, 1992 to the reference laboratory of the Committee, where species of the isolates were identified and drug susceptibility of Mycobacterium tuberculosis isolates were reexamined. The reference laboratory received a total of 1,236 cultures. Among them, 290 cultures were excluded from further examination by various reasons, such as contamination (52 cultures), non-viability (53), growth of nontuberculous mycobacteria (182) and other reasons (3). Thus, drug susceptibility test results were available for 946 cultures, including 26 cultures from non-Japanese persons. In the local laboratories, two methods, the absolute concentration method using 1% Ogawa egg slant (standard method, 26 hospitals) and its modified method using a microwell plate (microtiter method, 12 hospitals), were used for drug susceptibility testing, and the standard method was used in the reference laboratory. The results in the local laboratories were compared with those in the reference laboratory. The overall coincidence rate between drug susceptibility results reported from the local laboratories and those from the reference laboratory was 92.5%. A high coincidence rate (94.3%) was seen when the standard method was used in both local and reference laboratories. On the other hand, the coincidence rates between the results with the microtiter method in the local laboratory and those with the standard method in the reference laboratory were lower (standard method vs microtiter method; P < 0.01). Out of 19 hospitals, when the isolates were tested by the standard method 17 (89.5%) showed high coincidence rates (over 85%). Three hospitals using the microtiter method showed the coincidence rate over 90%, while other three showed lower rate (less than 80%) with high overestimation rates (over 19%), indicating that there are variations among facilities in performing the microtiter test. A part of the results concerning the resistance patterns to five antituberculosis drugs will be reported elsewhere.

[Epidemiology of Mycobacterium tuberculosis infection based on RFLP analysis].

In Japan, the decline of the tuberculosis incidence rates has been slowed down since 1970s. To study factors influencing this slow down of the decline, we have carried out the analysis of RFLP patterns of 941 strains of M. tuberculosis which were isolated at 38 hospitals in various districts of Japan in 1992. The outline of the results is as follows; (1) Distribution of the number of IS6110: The number of the occurring IS elements varies from 1 to 19, and the majority of the isolates have 9 to 14 copies. This finding is identical to the result of the previous investigation carried out in 1987 using 123 strains of M. tuberculosis. There were groups of individuals with identical patterns among those having the same number of copies. The characteristics of the RFLP pattern variety in Japan looks like that of Africa where tuberculosis in highly prevalent. In our country, however, it is considered that the influence of elderly people is very important. Thirty-seven strains contained only one hybridizing band. In 35 of these strains the copy was observed in a 7.9 kbp fragment, and in the other two strains the copy was observed in a 1.5 kbp fragment. Isolates which contain only one or small number of copies could not be differentiated by IS6110, so that other targets for RFLP analysis such as IS1081, DR sequence, and PGRS are to be further investigated. (2) Cluster analysis was shown to be an appropriate epidemiological methodology.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of Mycobacterium tuberculosis in clinical specimens by polymerase chain reaction and Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test.

The polymerase chain reaction (PCR) using oligonucleotides based on the repetitive sequence (IS986) of Mycobacterium tuberculosis as a primer and the Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test (MTD), which combines an M. tuberculosis rRNA amplification method with the hybridization protection assay format, were evaluated for detection of M. tuberculosis in clinical samples. The detection limits of these two assay systems based on nucleic acid amplification for cultured M. tuberculosis were less than 10 cells per reaction. A total of 135 sputum specimens were examined by the two assay systems. The PCR and the MTD systems for detection of M. tuberculosis gave overall positivity rates of 84.2% (32 of 38) and 91.9% (34 of 37), respectively, as compared with 71.9% (23 of 32) by smear and 96.9% (31 of 32) by culture in the liquid medium MB-Check. Procedures for sample preparation used in the two methods were different. Although the sensitivities of the PCR and MTD appeared to be similar to that of culture with the MB-Check system, the two methods based on nucleic acid amplification should be very useful for rapid detection of M. tuberculosis infections without the long time required for culture of M. tuberculosis.

Restriction fragment length polymorphism analysis of epidemiologically related Mycobacterium tuberculosis isolates.

Restriction fragment length polymorphism (RFLP) analysis of a large number of Japanese isolates of Mycobacterium tuberculosis, containing isolates from small outbreaks of M. tuberculosis infection, and clinical isolates of M. bovis BCG, was carried out using a DNA probe derived from the insertion sequence IS986. Clinical isolates of M. tuberculosis had a high degree of RFLP. The occurrences of the IS element varied from 1 to 19, the majority of isolates having 8 to 15 copies. Very similar fingerprints, however, were seen among strains isolated in the Kanto district. In particular, 3 strains were of the same pattern with or without an additional band. Similarity of the banding patterns of strains isolated in the same district was observed in other areas. Six groups of strains, each group arising from a suspected common source of infection, were analyzed. Of these, 5 showed identical fingerprints within each group, but one showed different fingerprints. RFLP patterns of three strains isolated from individuals with lymphadenitis developed about two months after BCG vaccination, and one strain isolated from a bladder cancer patient with BCG instillation therapy were identical to those of BCG-Tokyo which had been used for the vaccination and therapy. These results confirm that RFLP analysis using IS986 is a suitable tool for epidemiology of tuberculosis.

Comparison of MB-Check, BACTEC, and egg-based media for recovery of mycobacteria.

The rate of recovery and time to the detection of mycobacteria from clinical specimens were measured for biphasic (MB-Check; Nippon Roche Co., Ltd., Tokyo, Japan) and radiometric (BACTEC; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) liquid-based culture systems and egg-based media (3% Ogawa and Ogawa K). From the 245 sputum specimens processed, a total of 86 (35.1%) mycobacterial isolates were detected. Of these, 81 (94.2%) and 80 (93.0%) isolates were detected with the MB-Check and BACTEC systems, respectively, and 65 (75.6%) isolates were detected with the 3% Ogawa egg method. The difference in the percentages of positive cultures between the two systems based on liquid media and the 3% Ogawa egg method was significant (P less than 0.01). This difference was even greater among smear-negative specimens. The detection time was shorter with the liquid-based systems. The mean times to the detection of the Mycobacterium tuberculosis complex were 19.1 days with the MB-Check system, 13.4 days with the BACTEC system, and 21.7 days with the 3% Ogawa egg method. These results indicate that both the MB-Check and the BACTEC systems, based on liquid media, are efficient for the recovery of mycobacteria.