Weili Jiang

Extensive transmission of isoniazid resistant M. tuberculosis and its association with increased multidrug-resistant TB in two rural counties of eastern China: a molecular epidemiological study.

BackgroundThe aim of this study was to investigate the molecular characteristics of isoniazid resistant Mycobacterium tuberculosis (MTB), as well as its contribution to the dissemination of multi-drug resistant TB (MDR-TB) in rural areas of eastern China.MethodsA population-based epidemiological study was conducted in two rural counties of eastern China from 2004 to 2005. In total, 131 isoniazid resistant MTB isolates were molecularly characterized by DNA sequencing and genotyped by IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping.ResultsThe katG315Thr mutation was observed in 74 of 131 isoniazid resistant isolates and more likely to be MDR-TB (48.6%) and have mutations in rpoB gene (47.3%). Spoligotyping identified 80.2% of isoniazid resistant MTB isolates as belonging to the Beijing family. Cluster analysis by genotyping based on IS6110 RFLP, showed that 48.1% isoniazid resistant isolates were grouped into 26 clusters and katG315Thr mutants had a significantly higher clustering proportion compared to those with katG wild type (73%.vs.18%; OR, 12.70; 95%CI, 6.357-14.80). Thirty-one of the 53 MDR-TB isolates were observed in 19 clusters. Of these clusters, isoniazid resistance in MDR-TB isolates was all due to the katG315Thr mutation; 18 clusters also contained mono-isoniazid resistant and other isoniazid resistant isolates.ConclusionsThese results highlighted that isoniazid resistant MTB especially with katG315Thr is likely to be clustered in a community, develop extra resistance to rifampicin and become MDR-TB in Chinese rural settings.

Prevalence and Genetic Characterization of Second-Line Drug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in Rural China

ABSTRACT This study aimed to investigate the prevalence of resistance to second-line antituberculosis (anti-TB) drugs and its association with resistance-related mutations in Mycobacterium tuberculosis isolated in China. In the present study, we collected 380 isolates from a population-based study in China and tested the drug susceptibility to first- and selected second-line drugs. These results were compared with polymorphisms in the DNA sequences of genes associated with drug resistance and MIC values of the studied second-line drugs. Of 43 multidrug-resistant M. tuberculosis isolates, 13 showed resistance to fluoroquinolones or injectable second-line drugs (preextensively drug-resistant TB [pre-XDR-TB]), and 4 were resistant to both and thus defined as extensively drug-resistant TB (XDR-TB). Age and previous TB therapy, including use of second-line drugs, were two independent factors associated with increased resistance to both first- and second-line drugs. Molecular analysis identified the most frequent mutations in the resistance-associated genes: D94G in gyrA (29.1%) and A1401G in rrs (30.8%). Meanwhile, all 4 XDR-TB isolates had a mutation in gyrA, and 3 of them carried the A1401G mutation in rrs. Mutations in gyrA and rrs were associated with high-level resistance to fluoroquinolones and the second-line injectable drugs. In addition to the identification of resistance-associated mutations and development of a rapid molecular test to diagnose the second-line drug resistance, it should be a priority to strictly regulate the administration of second-line drugs to maintain their efficacy to treat multidrug-resistant TB.

Screening toll-like receptor markers to predict latent tuberculosis infection and subsequent tuberculosis disease in a Chinese population

BackgroundWe investigated whether polymorphisms in the toll-like receptor genes or gene–gene interactions are associated with susceptibility to latent tuberculosis infection (LTBI) or subsequent pulmonary tuberculosis (PTB) in a Chinese population.MethodsTwo matched case–control studies were undertaken. Previously reported polymorphisms in the toll-like receptors (TLRs) were compared between 422 healthy controls (HC) and 205 LTBI patients and between 205 LTBI patients and 109 PTB patients, to assess whether these polymorphisms and their interactions are associated with LTBI or PTB. A PCR-based restriction fragment length polymorphism analysis was used to detect genetic polymorphisms in the TLR genes. Nonparametric multifactor dimensionality reduction (MDR) was used to analyze the effects of interactions between complex disease genes and other genes or environmental factors.ResultsSixteen markers in TLR1, TLR2, TLR4, TLR6, TLR8, TLR9, and TIRAP were detected. In TLR2, the frequencies of the CC genotype (OR = 2.262; 95% CI: 1.433–3.570) and C allele (OR = 1.566; 95% CI: 1.223–1.900) in single-nucleotide polymorphism (SNP) rs3804100 were significantly higher in the LTBI group than in the HC group, whereas the GA genotype of SNP rs5743708 was associated with PTB (OR = 6.087; 95% CI: 1.687–21.968). The frequencies of the GG genotype of SNP rs7873784 in TLR4 (OR = 2.136; 95% CI: 1.312–3.478) and the CC genotype of rs3764879 in TLR8 (OR = 1.982; 95% CI: 1.292-3.042) were also significantly higher in the PTB group than in the HC group. The TC genotype frequency of SNP rs5743836 in TLR9 was significantly higher in the LTBI group than in the HC group (OR = 1.664; 95% CI: 1.201–2.306). An MDR analysis of gene–gene and gene–environment interactions identified three SNPs (rs10759932, rs7873784, and rs10759931) that predicted LTBI with 84% accuracy (p = 0.0004) and three SNPs (rs3804100, rs1898830, and rs10759931) that predicted PTB with 80% accuracy (p = 0.0001).ConclusionsOur results suggest that genetic variation in TLR2, 4, 8 and 9, implicating TLR-related pathways affecting the innate immunity response, modulate LTBI and PTB susceptibility in Chinese.

Association between cytokine gene polymorphisms and tuberculosis in a Chinese population in Shanghai: a case–control study

BackgroundPolymorphisms in cytokine genes are known to influence cytokine levels, which may influence susceptibility to tuberculosis (TB) infection and disease. Differences in cytokine expression probably determine whether TB progresses, resolves, or becomes latent. In particular, the balance between the Th1 and Th2 cytokine responses influences the expression of disease in individuals with pulmonary TB (PTB). We performed a case–control study of 120 patients diagnosed with PTB, 240 with latent TB infection (LTBI), and 480 healthy controls (HC), to explore the association between polymorphisms in cytokine genes and a predisposition to Mycobacterium tuberculosis infection and TB disease.ResultsA single-gene analysis showed a dominant association between the AA genotype or A allele at nucleotide −874 of the interferon γ (IFN-γ) gene and LTBI. The A allele at nucleotide −1082 of the interleukin 10 (IL-10) gene was significantly more common in PTB patients than in LTBI subjects. Moreover, the polymorphisms at IFN-γ −874 and IL10 − 1082 were associated with protein levels of IFN-γ and IL-10, respectively, in the PTB group. The genotype frequencies of other polymorphisms did not differ between the PTB patients, LTBI and HC subjects. Furthermore, combinations of polymorphisms with IFN-γ −874 were associated with LTBI, whereas combinations with IL10 − 1082 were more likely associated with PTB.ConclusionsThere are positive associations between the IFN-γ −874 polymorphism and TB and between the IL10 − 1082 polymorphism and LTBI. Our data provide genetic evidence of the multiple disease hypothesis that many cytokine genes are involved in TB susceptibility.

A major subgroup of Beijing family Mycobacterium tuberculosis is associated with multidrug resistance and increased transmissibility.

This study investigated further the association between the Beijing family Mycobacterium tuberculosis circulating in rural China and anti-tuberculosis (TB) drug resistance. In total, 351 M. tuberculosis isolates were collected through a population-based epidemiological study, 223 (63·5%) of which were resistant to at least one anti-TB drug, including 53 (15·1%) multidrug-resistant (MDR) isolates. Spoligotyping found 243 isolates (69·2%) that belonged to the Beijing family. A major subgroup of the Beijing family identified by mycobacterial interspersed repetitive unit (MIRU) genotyping (223325173533), showed significantly higher frequencies of MDR (44·7% vs. 13·7%, OR 6·18, 95% CI 2·68-14·23), katG and rpoB mutations (31·6% vs. 9·3%, OR 4·27, 95% CI 1·86-9·80), and being clustered by IS6110 RFLP genotyping (60·5% vs. 21·0%, OR 6·14, 95% CI 2·82-13·37) in comparison with other Beijing family isolates. Our data suggest that MIRU genotype 223325173533 of the Beijing family is associated with MDR and increased transmissibility.

Population-based investigation of fluoroquinolones resistant tuberculosis in rural eastern China.

Empirical use of fluoroquinolones (FQ) to treat a variety of bacterial infections may inadvertently select for FQ-resistant strains of Mycobacterium tuberculosis(MTB), especially in rural China where the use of FQ in treating infections has not been standardized. Here we determine the prevalence and describe the transmission of FQ-resistant MTB in two rural counties in eastern China through a combination of conventional epidemiology with IS6110-based restriction fragment length polymorphism(RFLP) analysis and DNA sequencing of drug-resistance determining regions. Phenotypic FQ resistance was detected in 31 of 351(8.8%) isolates. FQ resistance was equally distributed between patient-isolates deemed drug resistant and drug-susceptible, but mostly observed in those with treatment history of respiratory infection. Mutations in gyrA were found in 54.8% of FQ resistant isolates, and one isolate with a gyrB mutation. Despite predominating in entire bacilli population(69.2%), Beijing family strain had similar proportion of FQ resistance to the other(10.3% vs. 4.7%, p = 0.060). IS6110RFLP identified 2 clusters(4 isolates) among FQ resistant isolates and 3 clusters composed of both 4 FQ resistant isolates and 6 FQ susceptible isolates. Our results indicate that FQ-resistant MTB has emerged among the circulating bacillary population in rural eastern China. The relatively low level of clustering among FQ-resistant strains suggests most are acquired de novo, likely due to widespread FQ use.

Comparison of the socio-demographic and clinical features of pulmonary TB patients infected with sub-lineages within the W-Beijing and non-Beijing Mycobacterium tuberculosis

BACKGROUND Highly lethal outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are increasing. Mycobacterium tuberculosis variant Beijing family and its members is regarded as a successful clone of M. tuberculosis that is associated with drug resistance in China. Understanding the genetic characteristics and molecular mechanism of drug resistant tuberculosis within Beijing family may help to clarify its origin and evolutionary history and the driving forces behind its emergence and current dissemination. METHODS Totally of 1222 Mycobacterium tuberculosis isolates were recovered from patients in six counties of two provinces in eastern China within 2010/2012. Strain lineage and its major subgroups were studied respectively by using Spoligotyping and MIRU-VNTR. The 1st-line drug susceptibility was analyzed by proportional method and 2nd-line drug susceptibility was determined by the HAINs MTBDRsl test. The genetic characterization of drug resistance was analyzed by sequencing the previously reported genes and loci associated with drug resistance together with the multiple genotyping including MIRU-VNTR, Spoligotyping and LSP genotyping. RESULTS Of the 1222 Mtb isolates, 298 (24.4%) were resistant to 1st-line drug and 73 (5.9%) were simultaneously resistant to INH and RIF namely MDR-TB. Respectively 23.8% of 1st-line drug resistant TB and 12.0% of the drug susceptible TB contained the mutation associated with 2nd-line drugs by HAINs test. The Spoligotyping of 1222 Mtb isolates revealed the 967 (79.1%) of the isolates belonged to the W-Beijing family. Within W-Beijing family, 78.8% MDR-TB were observed in the isolates with simultaneous deletion of RD105 and RD207, with sub-lineage 181 accounting for 75% of MDR-TB. Analysis of 24 MIRU-VNTR loci revealed that 88.2% (15/17) of MDR and extensively drug resistant (XDR) clustered isolates were sub-lineage 181. CONCLUSIONS Sublineage 181 might have the capacity to spread throughout the general community in rural China. This is the first report on the extensive association of sub-lineage 181 with MDR TB and possibly pre-XDR TB and XDR TB. It is important to monitor sublineage 181 to verify its heightened transmission and understand its importance in the global MDR-TB and XDR-TB epidemics.

Use of the T-SPOT.TB assay to screen latent tuberculosis infection among the TB contacts in Shanghai, China

BACKGROUND There is limited information on the prevalence of latent tuberculosis infection (LTBI) and the risk factors among contacts of pulmonary TB, which might contribute significantly to TB burden and therefore become the major concern for TB control in Shanghai, China. METHODS A total of 45 diagnosed TB patients from each of 7 Shanghai districts were chosen and approximately 3 contacts per case were randomly selected. A structured questionnaire was used to acquire socio-demographic information and to assess the degree of exposure to index cases. LTBI screening was performed by T-SPOT.TB assay. RESULTS Of 969 enrolled contacts, 39% were men, average age was 45 ± 19.1 years, and 76.3% were household contacts. LTBI was identified in 29.6% of subjects. Those contacting smear-positive TB patients were 2.24 times (95%CI, 1.586-3.162) more likely to develop LTBI than those otherwise; household contact significantly increased the likelihood of LTBI by 1.52 times (95%CI, 1.061-2.180). Furthermore, among household contacts, the risk of LTBI was higher in spouses and first-degree relatives and increased with age and contact duration. CONCLUSIONS A high prevalence of LTBI was observed among the TB contacts in Shanghai, highlighting the need of intervention among the household contacts and those contacting smear-positive TB patients.

Access to Bacteriologic-Based Diagnosis in Smear Positive Retreatment Tuberculosis Patients in Rural China: A Cross-Sectional Study in Three Geographic Varied Provinces

Objective To determine factors influencing the utilization and accessibility to bacteriologic-based tuberculosis (TB) diagnosis among sputum smear positive (SS+) retreatment TB patients, and to develop strategies for improving the case detection rate of MDR-TB in rural China. Study Design and Setting A cross-sectional study of SS+ TB retreatment patients was conducted in eight counties from three provinces with different implementation period and strategy of MDR-TB program in China. Demographic and socioeconomic parameters were collected by self-reporting questionnaires. Sputum samples were collected and cultured by the laboratory of county-designated TB clinics and delivered to prefectural Centers for Disease Prevention and Control (CDC) labs for DST with 4 first-line anti-TB drugs. Results Among the 196 SS+ retreatment patients, 61.22% received culture tests during current treatment. Patients from more developed regions (OR = 24.0 and 3.6, 95% CI: 8.6–67.3 and 1.1–11.6), with better socio-economic status (OR = 3. 8, 95% CI: 1.3–10.7), who had multiple previous anti-TB treatments (OR = 5.0, 95% CI: 1.6–15.9), and who failed in the most recent anti-TB treatment (OR = 2.6, 95% CI: 1.0–6.4) were more likely to receive culture tests. The percentage of isolates resistant to any of first-line anti-TB drugs and MDR-TB were 50.0% (95% CI: 39.8%-60.2%) and 30.4% (95% CI: 21.0%-39.8%) respectively. Conclusions Retreatment SS+ TB patients, high risk MDR-TB population, had poor utilization of access to bacteriologic-based TB diagnosis, which is far from optimal. The next step of anti-TB strategy should be focused on how to make bacteriological-based diagnosis cheaper, safer and more maneuverable, and how to assure the DST-guided treatment for these high-risk TB patients.

The Epidemiological Significance and Temporal Stability of Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats-Based Method Applied to Mycobacterium tuberculosis in China

This study aimed to validate the epidemiological significance and temporal stability of Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) typing in a genetically and geographically diverse set of clinical isolates from patients diagnosed with pulmonary tuberculosis in China. Between 2010 and 2013, a total of 982 Mycobacterium tuberculosis isolates were collected from four population-based investigations in China. Apart from the currently applied 24-locus MIRU-VNTR, six additional hypervariable loci were analyzed in order to validate the MIRU-VNTR combinations in terms of their epidemiological links, clustering time span, and paired geographic distance. In vitro temporal stability was analyzed for both individual MIRU-VNTR loci, and for several combinations of loci. In the present study, four MIRU-VNTR combinations, including the hypervariable loci 3820, 3232, 2163a, and 4120, were evaluated. All of these combinations obtained a Hunter-Gaston discriminatory index (HGDI) value over 0.9900 with a reduced clustering proportion (from 32.0% to 25.6%). By comparing epidemiological links, clustering time span, and paired geographic distance, we found that the performances of the four MIRU-VNTR combinations were comparable to the insertion sequence 6110 restriction fragment length polymorphism (IS6110-RFLP), and significantly better than that of 24-locus MIRU-VNTR genotyping alone. The proportion of temporally stable loci ranged from 90.5% to 92.5% within the combined MIRU-VNTR genotyping, which is higher than IS6110-RFLP (85.4%). By adding four hypervariable loci to the standard 24-locus MIRU-VNTR genotyping, we obtained a high discriminatory power, stability and epidemiological significance. This algorithm could therefore be used to improve tuberculosis transmission surveillance and outbreak investigation in China.