Comprehensive multicenter evaluation of a new line probe assay kit for identification of Mycobacterium species and detection of drug-resistant Mycobacterium tuberculosis.

We evaluated a new line probe assay (LiPA) kit to identify Mycobacterium species and to detect mutations related to drug resistance in Mycobacterium tuberculosis. A total of 554 clinical isolates of Mycobacterium tuberculosis (n = 316), Mycobacterium avium (n = 71), Mycobacterium intracellulare (n = 51), Mycobacterium kansasii (n = 54), and other Mycobacterium species (n = 62) were tested with the LiPA kit in six hospitals. The LiPA kit was also used to directly test 163 sputum specimens. The results of LiPA identification of Mycobacterium species in clinical isolates were almost identical to those of conventional methods. Compared with standard drug susceptibility testing results for the clinical isolates, LiPA showed a sensitivity and specificity of 98.9% and 97.3%, respectively, for detecting rifampin (RIF)-resistant clinical isolates; 90.6% and 100%, respectively, for isoniazid (INH) resistance; 89.7% and 96.0%, respectively, for pyrazinamide (PZA) resistance; and 93.0% and 100%, respectively, for levofloxacin (LVX) resistance. The LiPA kit could detect target species directly in sputum specimens, with a sensitivity of 85.6%. Its sensitivity and specificity for detecting RIF-, PZA-, and LVX-resistant isolates in the sputum specimens were both 100%, and those for detecting INH-resistant isolates were 75.0% and 92.9%, respectively. The kit was able to identify mycobacterial bacilli at the species level, as well as drug-resistant phenotypes, with a high sensitivity and specificity.

[Treatment outcome of patients with multidrug-resistant pulmonary tuberculosis during pregnancy].

PURPOSE: To know the treatment outcome of patients with multidrug-resistant tuberculosis (MDR-TB) during gestation. METHOD: Retrospective study of 3 cases of pregnant women, who were treated for MDR-TB with a regimen including pyrazinamide, ethambutol, para-aminosalicylic acid, cycloserine and amoxicillin-clavulanic acid. RESULT: All patients showed a good response to anti-tuberculosis chemotherapy without any serious adverse effect, and were culture-negative at the time of delivery. Two patients delivered vaginally at weeks 40, and one patient delivered surgically at weeks 38. All newborns were healthy, and their tuberculin skin tests and placental tissue examinations were negative for tuberculosis. CONCLUSION: MDR-TB can be successfully treated during pregnancy by using a regimen including effective second-line anti-tuberculosis drugs.

[Resistance-conferring mutations of Mycobacterium tuberculosis strains with low level resistance to isoniazid].

OBJECTIVE: We investigated the prevalence of isoniazid (INH) resistance-conferring mutations in the INH-indeterminate Mycobacterium tuberculosis (MTB) strains. MATERIALS AND METHODS: We initially selected a sample of 47 clinical isolates of MTB from patients, who visited the Osaka Prefectural Medical Center for Respiratory and Allergic Diseases from 2000 to 2005. Strains resistant to the concentration of 1 - 2 microg/ml were defined as "indeterminate". INH resistance-conferring mutations were determined by DNA microarray. RESULTS: Of 47 INH-indeterminate strains, only 13 (27.7%) were found to have no resistance mutations, 23 (48.9%) had mutation within the inhA regulatory region at -15 C to T, and 2 (4.3%) had mutation within the inhA regulatory region at -8 T to A, 6 (12.8%) had mutation within the katG gene at 1778 G to A, and 3 (6.4%) had mutations within the katG gene both at 1778 G to A and at 982 T to G. CONCLUSIONS: We showed that the majority of INH-indeterminate strains have resistance-conferring mutations, which were mainly detected within the inhA regulatory region.

[Molecular epidemiology of tuberculosis by the use of IS6110 restriction fragment length polymorphism: a study from 2001 to 2003].

OBJECTIVE: To analyze the situation of tuberculosis infection by DNA fingerprinting in the middle and eastern part of Osaka, Japan. DESIGN: We performed IS6110 restriction fragment length polymorphism (RFLP) on 1200 isolates from tuberculosis patients who visited our hospital from January 2001 to December 2003. A cluster was defined as a series of isolates with more than 90% similarity by IS6110 RFLP and those with the same drug-susceptibility pattern. The isolates with fewer than six copies of IS6110 were considered to be clustered if the IS6110 RFLP patterns and the variable numbers of tandem repeats with 16 regions of ETR and MIRU "allele profile" were identical. RESULTS: The number of samples in incremental study periods was 422 in 2001, 817 between 2001 and 2002 and 1200 between 2001 and 2003. The percentage of clustered cases was 27.8% in 2001, 19.1% in 2002 and 19.5% in 2003. The cumulative percentage of clustered cases was 27.8% in the first year, 29.7% over two years and 32.6% over three years. The percentage of clustered cases of isolates with a drug resistance was significantly lower (25.0%) than that of drug susceptible isolates (33.7%). Next, we investigated the clustered cases by gender and age. The percentage of clustered cases with isolates from young males and females (0-19 years old) was 23.8%. In contrast, the percentage of clustered cases with isolates from 20-59 year-old females gradually decreased from 14.7% to 4.4%. Conversely, the percentage of clustered cases from young and middle aged male (20-59 years old) was higher (20.2%-32.4%) than that of females. CONCLUSION: The sharp increase in the cumulative cluster formation rate was curbed by the decline in the tuberculosis incidence rate in Osaka, Japan, after the first year of examination. We thought that this phenomenon suggests the success of the anti-tuberculosis measure in Japan.

Turkish and Japanese Mycobacterium tuberculosis sublineages share a remote common ancestor.

Two geographically distant M. tuberculosis sublineages, Tur from Turkey and T3-Osaka from Japan, exhibit partially identical genotypic signatures (identical 12-loci MIRU-VNTR profiles, distinct spoligotyping patterns). We investigated T3-Osaka and Tur sublineages characteristics and potential genetic relatedness, first using MIRU-VNTR locus analysis on 21 and 25 samples of each sublineage respectively, and second comparing Whole Genome Sequences of 8 new samples to public data from 45 samples uncovering human tuberculosis diversity. We then tried to date their Most Recent Common Ancestor (MRCA) using three calibrations of SNP accumulation rate (long-term=0.03SNP/genome/year, derived from a tuberculosis ancestor of around 70,000years old; intermediate=0.2SNP/genome/year derived from a Peruvian mummy; short-term=0.5SNP/genome/year). To disentangle between these scenarios, we confronted the corresponding divergence times with major human history events and knowledge on human genetic divergence. We identified relatively high intrasublineage diversity for both T3-Osaka and Tur. We definitively proved their monophyly; the corresponding super-sublineage (referred to as "T3-Osa-Tur") shares a common ancestor with T3-Ethiopia and Ural sublineages but is only remotely related to other Euro-American sublineages such as X, LAM, Haarlem and S. The evolutionary scenario based on long-term evolution rate being valid until T3-Osa-Tur MRCA was not supported by Japanese fossil data. The evolutionary scenario relying on short-term evolution rate since T3-Osa-Tur MRCA was contradicted by human history and potential traces of past epidemics. T3-Osaka and Tur sublineages were found likely to have diverged between 800y and 2000years ago, potentially at the time of Mongol Empire. Altogether, this study definitively proves a strong genetic link between Turkish and Japanese tuberculosis. It provides a first hypothesis for calibrating TB Euro-American lineage molecular clock; additional studies are needed to reliably date events corresponding to intermediate depths in tuberculosis phylogeny.

IS6110 DNA fingerprinting analysis of individually separated colonies of Mycobacterium tuberculosis.

There are no data so far that show IS6110 restriction fragment length polymorphism (RFLP) patterns of individually separated tuberculosis bacilli from clinical isolates, and their alterations during follow-up surveys. We picked 20-60 tuberculosis clones from clinical isolates under anti-tuberculosis medication, and individually analysed their DNA fingerprinting patterns using IS6110 RFLP as well as spoligotyping as a second typing. The study using cloned bacilli of Mycobacterium tuberculosis showed that clinical isolates contained several clones with different DNA fingerprints and that their band patterns altered weakly but distinctly during follow-up surveys. However, there was no significant difference in the fingerprinting patterns when clinical isolates were to RFLP without separating to subjected/individual colonies. In view of the IS6110 RFLP of individually separated tuberculosis bacilli, we have now speculated several possibilities: (1) that clones with different DNA fingerprints exist in clinical isolates; (2) that IS6110 RFLP patterns of the materials depend on the population of the original clone and the variants having DNA fingerprints different from the original pattern; and (3) that their band patterns are influenced not only by the stability of the original germ having its own fingerprint, but also by the fragility of the new clones.

Genetic and phenotypic characterization of pyrazinamide-resistant mycobacterium tuberculosis complex isolates in Japan.

The pncA gene mutations associated with pyrazinamide (PZA) resistance in Mycobacterium tuberculosis complex were determined in 26 PZA-resistant isolates in Japan. Of the 26 PZA-resistant isolates included, 21 were negative for pyrazinamidase (PZase). Of these, 20 isolates had various pncA mutations, resulting in alteration of primary amino acid sequence. However, 1 PZase-negative isolate did not have any mutation on pncA gene. The remaining 5 PZA-resistant isolates were positive for PZase and had identical pncA alleles with PZA-susceptible isolates. IS6110 RFLP analysis demonstrated various distinct IS6110 types and 5 pairs of isolates were very close to each other (>90% identical pattern). This study demonstrates that most of the PZA resistance is a result of various mutations on pncA resulting in loss of PZase activity. Further investigation, particularly on PZase-positive but PZA-resistant isolates and a PZase-negative isolate with no mutation on pncA, should be urgently done.

[Molecular epidemiologic study of tuberculosis--a study on isolates of M. tuberculosis in southern half of Osaka Prefecture].

Restriction fragment length polymorphism, RFLP or DNA fingerprinting technique provides a very useful tool for the study of epidemiology of tuberculosis transmission in human. We performed RFLP analysis with the IS6110 insertion sequence of the organisms isolated from culture-positive patients who visited our Hospital during the period from January to December 2001. Our Hospital covers patients living in southern half of Osaka Prefecture including a part of Osaka City, which is the highest TB prevalence area in Japan. The number of copies of IS6110 per isolate ranged from 1 to 21. Most isolates (67%) carried 10 to 15 copies. Of 410 available isolates during the year of 2001, 131 (32%) belonged to a cluster and 279 (68%) did not. The clusters comprised one matching isolate in minimum to 13 isolates in maximum and had a total of 49 distinct RFLP patterns. The average age of the clustered cases was 52.1 years and 64% cases belonged to patients with ages younger than 60 years. Above findings suggest that many cases of tuberculosis in southern part of Osaka Prefecture result from recent transmission. It remains to be elucidated, however, how and where these recent infections occurred in these clustered cases.

[A study of INH 0.1 microgram/ml resistant M. tuberculosis strains assessed by BrothMIC MTB-1 method].

In the antimycobacterial susceptibility test for INH using the egg-based Ogawa media, 3 concentrations (0.1, 1, or 5 micrograms/ml) of INH were used, and 1 microgram/ml was used as a critical concentration for INH resistance. However, it was controversial whether INH 0.1 microgram/ml resistant M. tuberculosis was clinically significant or not. We investigated the MIC values of INH 0.1 microgram/ml resistant strains by using BrothMIC MTB-1 method, and 115 strains of M. tuberculosis confirmed by DNA-prove test were used. The distribution of MIC values of 115 strains determined by Ogawa INH susceptibility test was shown in figure. By BrothMIC MTB-1 method, they were classified into 3 groups; susceptible, low resistant and high resistant groups. The mean MIC value of INH 0.1 microgram/ml resistant M. tuberculosis was estimated to be 4.53 micrograms/ml with its 95% confidence interval 3.21-5.85 micrograms/ml, and they were determined as "resistant" in BrothMIC MTB-1 method. These results supported the idea that patients with INH 0.1 microgram/ml resistant M. tuberculosis strains should be regarded as clinically "resistant".

[Fluctuation in the ratio between drug-resistant and -susceptible M. tuberculosis bacilli which were isolated from patients with prolonged anti-tuberculosis treatment].

PURPOSE: In this study, we examined the fluctuation in the ratio between strains of tuberculosis bacilli resistant and susceptible to the anti-tuberculosis drugs. MATERIAL & METHOD: We selected the cases in which the bacilli acquired drug resistance during the therapy and the cases in which drug resistance was fluctuating during prolonged anti-tuberculosis treatment. We selected the isolates throughout the medication period, and the selected isolates were separated into respective single colonies. Then we measured the minimal inhibitory concentration (MIC) for each colony by microdilution test for M. tuberculosis complex, BrothMIC MTB-1 (Kyokuto Pharmaceutical Inc., Tokyo). RESULTS: Five patients were eligible for analysis because their medical histories were clearly recorded and drug resistance changed during the course. The MIC of 19 isolates, 202 colonies were measured. Isolates with coexisting drug-resistant and -susceptible colonies were detected in 2 patients. From these results, we considered that the fluctuation in the ratio between drug-resistant and -susceptible bacilli changes with the progression of anti-tuberculosis medication. CONCLUSION: We showed in this article that the drug-resistant bacilli increased rapidly when the strains acquired the drug resistance during medication, and when the administration of particular anti-tuberculosis drug was stopped, the susceptible bacilli seemed to increase gradually. However, the strain immediately became fully drug-resistant when the particular antibacterial drug was readministered.