A descriptive study on isoniazid resistance-associated mutations, clustering and treatment outcomes of drug-resistant tuberculosis in a high burden country.

PURPOSE: To describe katG and inhA mutations, clinical characteristics, treatment outcomes and clustering of drug-resistant tuberculosis (TB) in the State of São Paulo, southeast Brazil. METHODS: Mycobacterium tuberculosis isolates from patients diagnosed with drug-resistant TB were screened for mutations in katG and inhA genes by line probe assay and Sanger sequencing, and typed by IS6110-restriction fragment-length polymorphism for clustering assessment. Clinical, epidemiological and demographic data were obtained from surveillance information systems for TB. RESULTS: Among the 298 isolates studied, 127 (42.6%) were isoniazid-monoresistant, 36 (12.1%) polydrug-resistant, 93 (31.2%) MDR, 16 (5.4%) pre-extensively drug-resistant (pre-XDR), 9 (3%) extensively drug-resistant (XDR) and 17 (5.7%) susceptible after isoniazid retesting. The frequency of katG 315 mutations alone was higher in MDR isolates, while inhA promoter mutations alone were more common in isoniazid-monoresistant isolates. Twenty-six isolates phenotypically resistant to isoniazid had no mutations either in katG or inhA genes. The isolates with inhA mutations were found more frequently in clusters (75%) when compared to the isolates with katG 315 mutations (59.8%, p = 0.04). In our population, being 35-64 years old, presenting MDR-, pre-XDR- or XDR-TB and being a retreatment case were associated with unfavourable TB treatment outcomes. CONCLUSION: We found that katG and inhA mutations were not equally distributed between isoniazid-monoresistant and MDR isolates. In our population, clustering was higher for isolates with inhA mutations. Finally, unfavourable TB outcomes were associated with specific factors.

Correlating genetic mutations with isoniazid phenotypic levels of resistance in Mycobacterium tuberculosis isolates from patients with drug-resistant tuberculosis in a high burden setting.

We analysed mutations in katG, inhA and rpoB genes, and isoniazid phenotypic resistance levels in Mycobacterium tuberculosis isolates from drug-resistant TB patients from São Paulo state, Brazil. Isolates resistant to the critical concentration of isoniazid in MGIT (0.1 µg/mL) were screened for mutations in katG 315 codon, inhA promoter region and rpoB RRDR by MTBDRplus assay and subjected to determination of isoniazid resistance levels by MGIT 960. Discordances were resolved by Sanger sequencing. Among the 203 isolates studied, 109 (54%) were isoniazid-monoresistant, 47 (23%) MDR, 29 (14%) polydrug-resistant, 12 (6%) pre-XDR and 6 (3%) XDR. MTBDRplus detected isoniazid mutations in 75% (153/203) of the isolates. Sequencing of the entire katG and inhA genes revealed mutations in 18/50 wild-type isolates by MTBDRplus (10 with novel mutations), resulting in a total of 32/203 (16%) isolates with no mutations detected. 81/83 (98%) isolates with katG 315 mutations alone had intermediate resistance. Of the 66 isolates with inhA C-15T mutation alone, 51 (77%) showed low-level, 14 (21%) intermediate and 1 (2%) high-level resistance. 5/6 (83%) isolates with mutations in both katG and inhA had high-level resistance. Inferred mutations corresponded to 22% (16/73) of all mutations found in rpoB. Mutations detected in katG regions other than codon 315 in this study might be potential new isoniazid resistance markers and could explain phenotypic resistance in some isolates without katG and inhA classic mutations. In our setting, 16% of isoniazid-resistant isolates, some with high-level resistance, presented no mutations either in katG or inhA.

Growth characteristics of liquid cultures increase the reliability of presumptive identification of Mycobacterium tuberculosis complex.

We evaluated the microscopic and macroscopic characteristics of mycobacteria growth indicator tube (MGIT) cultures for the presumptive identification of the Mycobacterium tuberculosis complex (MTBC) and assessed the reliability of this strategy for correctly directing isolates to drug susceptibility testing (DST) or species identification. A total of 1526 isolates of mycobacteria received at the Instituto Adolfo Lutz were prospectively subjected to presumptive identification by the observation of growth characteristics along with cord formation detection via microscopy. The presumptive identification showed a sensitivity, specificity and accuracy of 98.8, 92.5 and 97.9 %, respectively. Macroscopic analysis of MTBC isolates that would have been erroneously classified as non-tuberculous mycobacteria based solely on microscopic morphology enabled us to direct them rapidly to DST, representing a substantial gain to patients. In conclusion, the growth characteristics of mycobacteria in MGIT, when considered along with cord formation, increased the reliability of the presumptive identification, which has a great impact on the laboratory budget and turnaround times.

Evaluation of the BACTEC MGIT 960 system and the resazurin microtiter assay for susceptibility testing of Mycobacterium tuberculosis to second-line drugs.

Drug resistance in tuberculosis is a major threat to public health and control of the disease worldwide. Given the need of a rapid and accurate detection of Mycobacterium tuberculosis resistance to second-line drugs, this study evaluated the performance of the BACTEC MGIT 960 for second-line, drug susceptibility testing in comparison with the resazurin microtiter assay (REMA), in order to implement the automated methodology in the diagnostic routine of a reference laboratory. Drug susceptibility testing (DST) for second-line drugs of 151 MDR M. tuberculosis clinical isolates was performed by both BACTEC MGIT 960 and REMA, and a panel of 26 M. tuberculosis reference isolates from a proficiency test was tested by the BACTEC MGIT 960. DST for second-line drugs by the BACTEC MGIT 960 system was more rapid, highly reproducible and showed 100% of proficiency. After these results, this methodology was successfully implemented in our diagnostic routine for all MDR-TB patients.

Modified protocol for drug susceptibility testing of MGIT cultures of Mycobacterium tuberculosis by the MGIT 960.

A rapid detection of resistance in Mycobacterium tuberculosis is crucial for management and control of tuberculosis. This study evaluated a more rapid and cost-effective drug susceptibility testing (DST) protocol using primary isolates of M. tuberculosis in mycobacteria growth indicator tube (MGIT). Ninety-four M. tuberculosis isolates in MGIT were subjected to DST by the manufacturer's method, i.e., primary isolates were subcultured and DST was performed from positive cultures for a maximum of 5days; and by our modified method, i.e., DST was performed directly from primary MGIT cultures positive for more than 5days. Results were concordant for 76 (81%) isolates. Agreement between both methods was 92.0%, 98.9%, 97.7%, and 95.5% for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. Six isolates failed to grow on the recommended method, including 3 resistant isolates. Not performing subculture of primary M. tuberculosis isolates yields reliable results, decreasing the turnaround time and the cost of the test.

Absence of mycobacterial DNA in peripheral blood and artery specimens in patients with Takayasu arteritis.

The objective of this study was to demonstrate the presence of mycobacterial nucleic acid sequences in peripheral blood and arteries from patients with Takayasu arteritis (TA). Polymerase chain reaction was performed to detect mycobacterial DNA from three different nucleic acid sequences including the insertion sequence (IS) 6110, the 65-kDa heat shock protein gene (HSP65), and the 16S ribosomal RNA (rRNA) gene in peripheral blood from 32 TA patients and in arterial specimens from 10 TA patients. Twenty-eight HIV-negative patients with pulmonary tuberculosis prior to therapy were tested for IS6110 in peripheral blood as positive controls, and 24 blood donors were evaluated as healthy controls (HC). All TA patients were negative for the insertion sequence IS6110 and for HSP65 and 16S rRNA genes in blood samples and in arterial specimens. IS6110 sequence was found in peripheral blood from 22 (78.5 %) patients with pulmonary tuberculosis but not in HC. In conclusion, the strategy of mycobacterial-specific nucleic acid amplification in the peripheral blood and arterial specimens of TA patients was unable to lend support to the association between TA and tuberculosis long suggested in the literature.

Natural occurrence of horizontal transfer of Mycobacterium avium- specific insertion sequence IS1245 to Mycobacterium kansasii.

Mycobacterium kansasii carrying IS1245, a highly prevalent insertion sequence among Mycobacterium avium isolates, was detected in a mixed culture of M. avium and M. kansasii. The insertion sequence was stable and able to transpose by a replicative mechanism in M. kansasii. These findings may have significant implications for molecular diagnosis and treatment outcome.

Maintenance of Mycobacterium tuberculosis on glass beads.

The intent of this study was to estimate the shelf life of Mycobacterium tuberculosis strains, and to observe the loss of viability in some of them from year to year. From 2000 to 2004, 10,015 cultures of M. tuberculosis were preserved by freezing on glass beads at -70 degrees C. With the expectation that the loss of viability might be around 5-10%/yr of storage, 730 strains were analyzed in order to establish the prevalence of recovery within a 5% margin of error. This study shows that 94% of the strains preserved at -70 degrees C on glass beads could be recovered within 30 days. The recovery rates for drug-susceptible and drug-resistant strains showed no significant differences. The growth rates and the number of strains that showed abundant growth before the 30th day of incubation represent important features, since the subculture of a strain preserved for future use ought to quickly produce abundant growth in order to avoid misinterpretation of the tests. Our experience indicates that storage of M. tuberculosis on glass beads at -70 degrees C is a suitable procedure for an active culture collection in a public health laboratory like ours, where maintenance of M. tuberculosis cultures is a complementary activity and must be quick, practical, effective, and economical.

Detection of mixed infections with Mycobacterium lentiflavum and Mycobacterium avium by molecular genotyping methods.

Three mycobacterial isolates, one from the blood of an HIV-infected patient and two consecutive isolates from a woman with unknown HIV status, had been identified as belonging to the Mycobacterium avium complex by conventional procedures. In both patients, using genetic analysis procedures such as PCR-restriction enzyme analysis (PRA) of the hsp65 gene, a commercially available reverse hybridization-based assay (INNO-LiPA mycobacteria) and/or sequencing analysis of the 16S-23S internal transcribed spacer (ITS), the presence of Mycobacterium lentiflavum was also demonstrated. At the time of detection, both cases were also infected with M. avium, suggesting an underestimation of infection with M. lentiflavum and co-infection with different Mycobacterium species.

[Distribution of PRA patterns of clinical isolates of the Mycobacterium avium complex from Spain and South America]. / Distribución de patrones PRA en aislamientos clínicos del complejo Mycobacterium avium procedentes de España y Suramérica.

Mycobacterium avium complex (MAC) infections are the most frequent systemic infections associated with advanced AIDS. DNA probes for accurate identification of mycobacteria are available but are very expensive in many Latin American settings. Consequently, most Latin American diagnostic laboratories employ inaccurate and outdated tests for mycobacteria identification. Therefore, PCR restriction analysis (PRA) of the hsp65 gene was evaluated for the identification of 163 MAC human isolates originated from Spain and South America. The predominant PRA type in each country was: M. avium type I in Argentina (23/42, 55%) and Brazil (48/72, 67%), M. avium type II in Spain (18/26, 69%) and M. avium type III in Colombia (10/23, 43%). The Colombia frequency is noteworthy, since the PRA type III was quite infrequent in the other three countries. Furthermore, its presence has not been reported outside the Americas. The advantages and disadvantages of PRA in diagnostic mycobacteriology are discussed.