Isoniazid resistance in <i>Mycobacterium tuberculosis</i>

Drug resistant tuberculosis (TB) is increasing worldwide and it is now estimated by the World Health Organisation (WHO) that 7% of TB cases globally are resistant to isoniazid (INH). INH is a key drug for the treatment of TB, alongside rifampicin (RIF). Understanding factors which influence the emergence and propagation of INH resistance and the influence of INH resistance on TB treatment success are vital in improving global TB control efforts. Vietnam is ranked 12th of 22 high burden countries for TB and has a high prevalence of INH resistance (25%) with a relatively modest prevalence of TB resistant to both INH and RIF (multi-drug resistant, MDR TB) (2.7%). The first study in this thesis developed rapid screening tests for both RIF and INH resistance in Mycobacterium tuberculosis isolates which showed high accuracy. The specificity and the sensitivity of the MAS-PCR test for INH resistance compared to the conventional phenotypic DST were 100% [95% CI 92.9-100%] and 90% [95% CI 82.4-95.1 %], respectively. The second study demonstrated that the minimum inhibitory concentration (MIC) for INH is influenced by both the mutation responsible for resistance to INH and the lineage backbone of the M.tuberculosis isolate. A two-way ANOVA of MIC including both strain lineage (p=0.003) and resistance mutation (p<0.001) showed highly significant independent effects of both factors on MIC level. MIC to INH of isolates with a katG315 mutation (2ug/ml) was significantly higher compared to MIC to INH of isolates with an inhA-15 mutation (0.25p.g/ml) and wild-type isolates (0- 0.lug/ml) (p<0.00l). The independent effect of the strain lineage on INH MIC was predicted to correspond to a 2.68-fold (95% Cl 1.52-4.73, p<0.001) increase for Beijing lineage strains and a 1.40-fold (95% Cl 0.72-2.74, p=0.32) increase for EuroAmerican lineage strains in comparison with strains of the Indo-Oceanic lineage. The third study investigated the impact of INH resistance on outcome in patients with HIV-associated TB meningitis (HIV/TBM) and showed that HIV/TBM patients infected with INH resistant M.tuberculosis which remains susceptible to RIF have significantly worse outcomes than patients infected with fully susceptible strains. The adjusted hazard ratio for MDR patients in comparison with fully susceptible or streptomycin (STR)-monoresistant isolates was 5.21 [95% Cl 2.38-11.42], p<0.0001 compared with HR= 1.78 [95% Cl 1.18-2.66], p=0.005 for patients with INH resistant isolates (+/-STR resistance). The final study investigated the distribution of N-acetylator-2 (NAT2) phenotypes for INH metabolism in healthy volunteers from the Vietnamese Kinh population and correlated this with phenotypes using caffeine as a surrogate indicator for INH metabolism. This study showed that characterisation of 3 single nucleotide polymorphisms is sufficient to determine the acetylator status of individuals of Vietnamese Kinh ethnicity and that there is a predominance of fast acetylators (65%) in this popUlation. A simple PCR-RFLP test was developed to enable rapid determination of acetylator status for further studies. Collectively, these results contribute to our understanding of INH resistance in M.tuberculosis and provide molecular tools for further studies of this crucial drug

Isoniazid resistance in Mycobacterium tuberculosis

Drug resistant tuberculosis (TB) is increasing worldwide and it is now estimated by the World Health Organisation (WHO) that 7% of TB cases globally are resistant to isoniazid (INH). INH is a key drug for the treatment of TB, alongside rifampicin (RIF). Understanding factors which influence the emergence and propagation of INH resistance and the influence of INH resistance on TB treatment success are vital in improving global TB control efforts. Vietnam is ranked 12th of 22 high burden countries for TB and has a high prevalence of INH resistance (25%) with a relatively modest prevalence of TB resistant to both INH and RIF (multi-drug resistant, MDR TB) (2.7%) The first study in this thesis developed rapid screening tests for both RIF and INH resistance in Mycobacterium tuberculosis isolates which showed high accuracy. The specificity and the sensitivity of the MAS-PCR test for INH resistance compared to the conventional phenotypic DST were 100% [95% CI 92.9-100%] and 90% [95% CI 82.4-95.1 %], respectively. The second study demonstrated that the minimum inhibitory concentration (MIC) for INH is influenced by both the mutation responsible for resistance to INH and the lineage backbone of the M.tuberculosis isolate. A two-way ANOVA of MIC including both strain lineage (p=0.003) and resistance mutation (p<0.001) showed highly significant independent effects of both factors on MIC level. MIC to INH of isolates with a katG315 mutation (2ug/ml) was significantly higher compared to MIC to INH of isolates with an inhA-15 mutation (0.25p.g/ml) and wild-type isolates (0- 0.lug/ml) (p<0.00l).EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Isoniazid resistance in Mycobacterium tuberculosis

Drug resistant tuberculosis (TB) is increasing worldwide and it is now estimated by the World Health Organisation (WHO) that 7% of TB cases globally are resistant to isoniazid (INH). INH is a key drug for the treatment of TB, alongside rifampicin (RIF). Understanding factors which influence the emergence and propagation of INH resistance and the influence of INH resistance on TB treatment success are vital in improving global TB control efforts. Vietnam is ranked 12th of 22 high burden countries for TB and has a high prevalence of INH resistance (25%) with a relatively modest prevalence of TB resistant to both INH and RIF (multi-drug resistant, MDR TB) (2.7%) The first study in this thesis developed rapid screening tests for both RIF and INH resistance in Mycobacterium tuberculosis isolates which showed high accuracy. The specificity and the sensitivity of the MAS-PCR test for INH resistance compared to the conventional phenotypic DST were 100% [95% CI 92.9-100%] and 90% [95% CI 82.4-95.1 %], respectively. The second study demonstrated that the minimum inhibitory concentration (MIC) for INH is influenced by both the mutation responsible for resistance to INH and the lineage backbone of the M.tuberculosis isolate. A two-way ANOVA of MIC including both strain lineage (p=0.003) and resistance mutation (p<0.001) showed highly significant independent effects of both factors on MIC level. MIC to INH of isolates with a katG315 mutation (2ug/ml) was significantly higher compared to MIC to INH of isolates with an inhA-15 mutation (0.25p.g/ml) and wild-type isolates (0- 0.lug/ml) (p<0.00l).EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Mutations Prevalent among Rifampin- and Isoniazid-Resistant Mycobacterium tuberculosis Isolates from a Hospital in Vietnam

Vietnam is ranked 13th among the WHO list of 22 high-burden countries, based upon estimated total number of tuberculosis cases. Despite having a model national tuberculosis program, consistently achieving and exceeding WHO targets for detection and cure, drug-resistant and multidrug-resistant tuberculosis cases continue to rise. Rapid multidrug-resistant tests applicable in this setting, coupled with effective treatment regimens, would be a useful tool in reversing this trend, allowing early identification of patients with multidrug-resistant tuberculosis and avoiding resistance-amplifying regimens. Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and −15C→T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB. The most prevalent rifampin resistance-conferring mutations, as in other countries, were in rpoB codons 531 (43%), 526 (31%), and 516 (15%). However, a hybridization-based resistance test with probes targeting the 5 most common mutations would only detect 78% of rifampin-resistant isolates. Overall, these data suggest that rifampin resistance may be used as a surrogate marker for multidrug-resistant tuberculosis and that a sensitivity of between 70 to 80% may be possible for rapid molecular detection of multidrug-resistant tuberculosis in this setting

PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis▿

PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates

Relationship between Mycobacterium tuberculosis Genotype and the Clinical Phenotype of Pulmonary and Meningeal Tuberculosis ▿

We used large sequence polymorphisms to determine the genotypes of 397 isolates of Mycobacterium tuberculosis from human immunodeficiency virus-uninfected Vietnamese adults with pulmonary (n = 235) or meningeal (n = 162) tuberculosis. We compared the pretreatment radiographic appearances of pulmonary tuberculosis and the presentation, response to treatment, and outcome of tuberculous meningitis between the genotypes. Multivariate analysis identified variables independently associated with genotype and outcome. A higher proportion of adults with pulmonary tuberculosis caused by the Euro-American genotype had consolidation on chest X-ray than was the case with disease caused by other genotypes (P = 0.006). Multivariate analysis revealed that meningitis caused by the East Asian/Beijing genotype was independently associated with a shorter duration of illness before presentation and fewer cerebrospinal fluid (CSF) leukocytes. Older age, fewer CSF leukocytes, and the presence of hemiplegia (but not strain lineage) were independently associated with death or severe disability, although the East Asian/Beijing genotype was strongly associated with drug-resistant tuberculosis. The genotype of M. tuberculosis influenced the presenting features of pulmonary and meningeal tuberculosis. The association between the East Asian/Beijing lineage and disease progression and CSF leukocyte count suggests the lineage may alter the presentation of meningitis by influencing the intracerebral inflammatory response. In addition, increased drug resistance among bacteria of the East Asian/Beijing lineage might influence the response to treatment. This study suggests the genetic diversity of M. tuberculosis has important clinical consequences

The influence of host and bacterial genotype on the development of disseminated disease with Mycobacterium tuberculosis

The factors that govern the development of tuberculosis disease are incompletely understood. We hypothesized that some strains of Mycobacterium tuberculosis (M. tuberculosis) are more capable of causing disseminated disease than others and may be associated with polymorphisms in host genes responsible for the innate immune response to infection. We compared the host and bacterial genotype in 187 Vietnamese adults with tuberculous meningitis (TBM) and 237 Vietnamese adults with uncomplicated pulmonary tuberculosis. The host genotype of tuberculosis cases was also compared with the genotype of 392 cord blood controls from the same population. Isolates of M. tuberculosis were genotyped by large sequence polymorphisms. The hosts were defined by polymorphisms in genes encoding Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and Toll-like receptor-2 (TLR-2). We found a significant protective association between the Euro-American lineage of M. tuberculosis and pulmonary rather than meningeal tuberculosis (Odds ratio (OR) for causing TBM 0.395, 95% confidence intervals (C.I.) 0.193–0.806, P = 0.009), suggesting these strains are less capable of extra-pulmonary dissemination than others in the study population. We also found that individuals with the C allele of TLR-2 T597C allele were more likely to have tuberculosis caused by the East-Asian/Beijing genotype (OR = 1.57 [95% C.I. 1.15–2.15]) than other individuals. The study provides evidence that M. tuberculosis genotype influences clinical disease phenotype and demonstrates, for the first time, a significant interaction between host and bacterial genotypes and the development of tuberculosis

Demographic data for cases of TBM and pulmonary tuberculosis recruited to the study.

a<p>Defined as the main place of residence on entry to the study. Urban addresses were those within the central districts of Ho Chi Minh City (HCMC); sub-urban addresses were those within the outer districts of HCMC; rural (HCMC surrounds) addresses were in the immediate surrounding rural districts of HCMC; the other rural addresses were defined by whether they were south east or south west of HCMC.</p

<i>TLR</i>2 T597C SNP allele and bacterial genotype frequencies: comparison with host genotype distribution in the cord blood control group.

a<p>OR was calculated comparing each group to the genotype/allele distribution in the cord blood controls.</p