Epidemiology of isoniazid resistance mutations and their effect on tuberculosis treatment outcomes.

Isoniazid resistance is highly prevalent in Vietnam. We investigated the molecular and epidemiological characteristics and the association with first-line treatment outcomes of the main isoniazid resistance mutations in Mycobacterium tuberculosis in codon 315 of the katG and in the promoter region of the inhA gene. Mycobacterium tuberculosis strains with phenotypic resistance to isoniazid from consecutively diagnosed smear-positive tuberculosis patients in rural Vietnam were subjected to Genotype MTBDRplus testing to identify katG and inhA mutations. Treatment failure and relapse were determined by sputum culture. In total, 227 of 251 isoniazid-resistant strains (90.4%) had detectable mutations: 75.3% in katG codon 315 (katG315) and 28.2% in the inhA promoter region. katG315 mutations were significantly associated with pretreatment resistance to streptomycin, rifampin, and ethambutol but not with the Beijing genotype and predicted both unfavorable treatment outcome (treatment failure or death) and relapse; inhA promoter region mutations were only associated with resistance to streptomycin and relapse. In tuberculosis patients, M. tuberculosis katG315 mutations but not inhA mutations are associated with unfavorable treatment outcome. inhA mutations do, however, increase the risk of relapse, at least with treatment regimens that contain only isoniazid and ethambutol in the continuation phase.

Clustering of Beijing genotype Mycobacterium tuberculosis isolates from the Mekong delta in Vietnam on the basis of variable number of tandem repeat versus restriction fragment length polymorphism typing.

BACKGROUND: In comparison to restriction fragment length polymorphism (RFLP) typing, variable number of tandem repeat (VNTR) typing is easier to perform, faster and yields results in a simple, numerical format. Therefore, this technique has gained recognition as the new international gold standard in typing of Mycobacterium tuberculosis. However, some reports indicated that VNTR typing may be less suitable for Beijing genotype isolates. We therefore compared the performance of internationally standardized RFLP and 24 loci VNTR typing to discriminate among 100 Beijing genotype isolates from the Southern Vietnam. METHODS: Hundred Beijing genotype strains defined by spoligotyping were randomly selected and typed by RFLP and VNTR typing. The discriminatory power of VNTR and RFLP typing was compared using the Bionumerics software. RESULTS: Among 95 Beijing strains available for analysis, 14 clusters were identified comprising 34 strains and 61 unique profiles in 24 loci VNTR typing ((Hunter Gaston Discrimination Index (HGDI = 0.994)). 13 clusters containing 31 strains and 64 unique patterns in RFLP typing (HGDI = 0.994) were found. Nine RFLP clusters were subdivided by VNTR typing and 12 VNTR clusters were split by RFLP. Five isolates (5%) revealing double alleles or no signal in two or more loci in VNTR typing could not be analyzed. CONCLUSIONS: Overall, 24 loci VNTR typing and RFLP typing had similar high-level of discrimination among 95 Beijing strains from Southern Vietnam. However, loci VNTR 154, VNTR 2461 and VNTR 3171 had hardly added any value to the level of discrimination.

Tuberculosis relapse in Vietnam is significantly associated with Mycobacterium tuberculosis Beijing genotype infections.

BACKGROUND: In Vietnam, the Mycobacterium tuberculosis Beijing genotype is associated with multi-drug resistance and is emerging. A possible explanation for this genotype's success is an increased rate of relapse. METHODS: In a prospective cohort study, isolates from patients with smear-positive tuberculosis were subjected to drug susceptibility testing and to spoligotyping and variable number of tandem repeats typing before treatment and after recurrence of tuberculosis. RESULTS: Among 1068 patients who were actively followed up over 18 months for recurrence, 23 relapse cases occurred (1.39 cases/100 person-years). After adjustment for genotype, tuberculosis treatment history, and drug resistance, relapse was significantly associated with the Beijing genotype (adjusted hazard ratio [aHR], 5.48; 95% confidence interval [CI], 2.06-14.55) and isoniazid resistance (aHR, 5.91; 95% CI, 2.16-16.16). CONCLUSIONS: The strongly increased relapse rate in tuberculosis cases caused by Beijing strains probably contributes to the successful spread of this genotype in Vietnam and elsewhere.

Increased transmission of Mycobacterium tuberculosis Beijing genotype strains associated with resistance to streptomycin: a population-based study.

BACKGROUND: Studies have shown that the Mycobacterium tuberculosis Beijing genotype is an emerging pathogen that is frequently associated with drug resistance. This suggests that drug resistant Beijing strains have a relatively high transmission fitness compared to other drug-resistant strains. METHODS AND FINDINGS: We studied the relative transmission fitness of the Beijing genotype in relation to anti-tuberculosis drug resistance in a population-based study of smear-positive tuberculosis patients prospectively recruited and studied over a 4-year period in rural Vietnam. Transmission fitness was analyzed by clustering of cases on basis of three DNA typing methods. Of 2531 included patients, 2207 (87%) were eligible for analysis of whom 936 (42%) were in a DNA fingerprint cluster. The clustering rate varied by genotype with 292/786 (37%) for the Beijing genotype, 527/802 (67%) for the East-African Indian (EAI) genotype, and 117/619 (19%) for other genotypes. Clustering was associated with the EAI compared to the Beijing genotype (adjusted odds ratio (OR(adj)) 3.4: 95% CI 2.8-4.4). Patients infected with streptomycin-resistant strains were less frequently clustered than patients infected with streptomycin-susceptible strains when these were of the EAI genotype (OR(adj) 0.6, 95% CI 0.4-0.9), while this pattern was reversed for strains of the Beijing genotype (OR(adj) 1.3, 95% CI 1.0-1.8, p for difference 0.002). The strong association between Beijing and MDR-TB (OR(adj) 7.2; 95% CI 4.2-12.3) existed only if streptomycin resistance was present. CONCLUSIONS: Beijing genotype strains showed less overall transmissibility than EAI strains, but when comparisons were made within genotypes, Beijing strains showed increased transmission fitness when streptomycin-resistant, while the reverse was observed for EAI strains. The association between MDR-TB and Beijing genotype in this population was strongly dependent on resistance to streptomycin. Streptomycin resistance may provide Beijing strains with a fitness advantage over other genotypes and predispose to multidrug resistance in patients infected with Beijing strains.

Mixed tuberculosis infections in rural South Vietnam.

Tuberculosis patients may be infected with or have disease caused by more than one Mycobacterium tuberculosis strain, usually referred to as "mixed infections." These have mainly been observed in settings with a very high tuberculosis incidence and/or high HIV prevalence. We assessed the rate of mixed infections in a population-based study in rural Vietnam, where the prevalences of both HIV and tuberculosis are substantially lower than those in previous studies looking at mixed infections. In total, 1,248 M. tuberculosis isolates from the same number of patients were subjected to IS6110 restriction fragment length polymorphism (RFLP) typing, spoligotyping, and variable-number-tandem-repeat (VNTR) typing. We compared mixed infections identified by the presence of (i) discrepant RFLP and spoligotype patterns in isolates from the same patient and (ii) double alleles at ≥ 2 loci by VNTR typing and assessed epidemiological characteristics of these infections. RFLP/spoligotyping and VNTR typing identified 39 (3.1%) and 60 (4.8%) mixed infections, respectively (Cohen's kappa statistic, 0.57). The number of loci with double alleles in the VNTR pattern was strongly associated with the proportion of isolates with mixed infections according to RFLP/spoligotyping (P < 0.001). Mixed infections occurred more frequently in newly treated than in previously treated patients, were significantly associated with minor X-ray abnormalities, and were almost significantly associated with lower sputum smear grades. Although the infection pressure in our study area is lower than that in previously studied populations, mixed M. tuberculosis infections do occur in rural South Vietnam in at least 3.1% of cases.

The Mycobacterium tuberculosis Beijing genotype does not affect tuberculosis treatment failure in Vietnam.

BACKGROUND: Studies have suggested that the Mycobacterium tuberculosis Beijing genotype causes more severe clinical disease and higher treatment failure rates with standard regimens, possibly in association with an increased risk of acquiring drug resistance. We studied the effect of genotype on treatment failure in a rural area in Vietnam where multidrug resistance is strongly associated with the Beijing genotype. METHODS: In a population-based prospective cohort study, patients with smear-positive tuberculosis were tested before and after treatment by spoligotyping and drug susceptibility analysis. Reinfections were excluded by DNA fingerprinting. The outcome was treatment failure based on culture. RESULTS: Of 1106 patients eligible for analysis, 33 experienced treatment failure (3.0%; 95% confidence interval [CI], 2.1%-4.1%). The proportion of failure was 5.3% (95% CI, 0.3%-7.9%) among 380 patients with Beijing genotype infections. Multidrug-resistant tuberculosis strongly predicted failure (odds ratio [OR], 114; 95% CI, 30-430). After adjusting for multidrug-resistant tuberculosis, treatment failure was not associated with the Beijing genotype (adjusted OR, 0.7; 95% CI, 0.3-2.0). Amplification of drug resistance occurred in 3 patients (0.3%; 95% CI, 0.1%-0.7%) and was associated with multidrug resistance at baseline (P = .004) but not with the Beijing genotype. No multidrug resistance was created. CONCLUSION: The Beijing genotype was not associated with treatment failure in Vietnam; apparent associations were explained by the strong association of this genotype with multidrug resistance. Amplification of resistance in this patient population was rare.

Tuberculosis acquired outside of households, rural Vietnam.

Using population-based data from rural Vietnam, we assessed tuberculosis (TB) transmission within and outside of households. Eighty-three percent of persons with recent household TB were infected by different strains of Mycobacterium tuberculosis than were their household members. This result argues against the effectiveness of active TB case finding among household members.

Validation of the GenoType MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam.

BACKGROUND: To control multidrug resistant tuberculosis (MDR-TB), the drug susceptibility profile is needed to guide therapy. Classical drug susceptibility testing (DST) may take up to 2 to 4 months. The GenoType MTBDRplus test is a commercially available line-probe assay that rapidly detects Mycobacterium tuberculosis (MTB) complex, as well as the most common mutations associated with rifampin and isoniazid resistance.We assessed sensitivity and specificity of the assay by using a geographically representative set of MTB isolates from the South of Vietnam. METHODS: We re-cultured 111 MTB isolates that were MDR, rifampin-resistant or pan-susceptible according to conventional DST and tested these with the GenoType MTBDRplus test. RESULTS: By conventional DST, 55 strains were classified as MDR-TB, four strains were rifampicin mono-resistant and 52 strains were susceptible to all first-line drugs. The sensitivity of the GenoType MTBDRplus was 93.1% for rifampicin, 92.6% for isoniazid and 88.9% for the combination of both; its specificity was 100%. The positive predictive value of the GenoType MTBDRplus test for MDR-TB was 100% and the negative predictive value 90.3%. CONCLUSIONS: We found a high specificity and positive predictive value of the GenoType MTBDRplus test for MDR-TB which merits its use in the MDR-TB treatment program in Vietnam.

Mycobacterium tuberculosis genotype and case notification rates, rural Vietnam, 2003-2006.

Tuberculosis case notification rates (CNRs) for young adults in Vietnam are increasing. To determine whether this finding could reflect emergence of Mycobacterium tuberculosis Beijing genotype, we studied all new sputum smear-positive pulmonary tuberculosis patients registered for treatment in 3 rural districts in Vietnam during 2003-2006. Beijing strain infections were more frequent in younger patients (15-24 years of age, 53%) than in older patients (31%; p<0.001). The increase in CNRs for youngest patients was larger for disease caused by the Beijing genotype than by other genotypes, but the difference was not significant. For patients 15-24 years of age, 85% of fluctuations in CNRs between years was caused by fluctuations in Beijing genotype infections compared with 53% and 23% in the groups 25-64 and >or=65 years of age, respectively (p<0.001). These findings suggest that young adults may be responsible for introducing Beijing strains into rural Vietnam.