Mycobacterium Tuberculosis In China Research Articles

Transmission dynamics and phylogeography of Mycobacterium tuberculosis in China based on whole-genome phylogenetic analysis

ObjectivesThis study aimed to describe the lineage-specific transmissibility and epidemiological migration of Mycobacterium tuberculosis in China. MethodsWe curated a large set of whole-genome sequences from 3204 M. tuberculosis isolates, including thousands of newly sequenced genomes, and applied a series of metrics to compare the transmissibility of M. tuberculosis strains between lineages and sublineages. The countrywide transmission patterns of major lineages were explored. ResultsWe found that lineage 2 (L2) was the most prevalent lineage in China (85.7%), with the major sublineage 2.2.1 (80.9%), followed by lineage 4 (L4) (13.8%), which comprises major sublineages 4.2 (1.5%), 4.4 (6.2%) and 4.5 (5.8%). We showed evidence for frequent cross-regional spread and large cluster formation of L2.2.1 strains, whereas L4 strains were relatively geographically restricted in China. Next, we applied a series of genomic indices to evaluate M. tuberculosis strain transmissibility and uncovered higher transmissibility of L2.2.1 compared with the L2.2.2 and L4 sublineages. Phylogeographic analysis showed that southern, eastern, and northern China were highly connected regions for countrywide L2.2.1 strain spread. ConclusionsThe present study provides insights into the different transmission and migration patterns of the major M. tuberculosis lineages in China and highlights that transmissible L2.2.1 is a threat to tuberculosis control.

Insight into the drug-resistant characteristics and genetic diversity of multidrug-resistant Mycobacterium tuberculosis in China.

Multidrug-resistant tuberculosis (MDR-TB) has a severe impact on public health. To investigate the drug-resistant profile, compensatory mutations and genetic variations among MDR-TB isolates, a total of 546 MDR-TB isolates from China underwent drug-susceptibility testing and whole genome sequencing for further analysis. The results showed that our isolates have a high rate of fluoroquinolone resistance (45.60%, 249/546) and a low proportion of conferring resistance to bedaquiline, clofazimine, linezolid, and delamanid. The majority of MDR-TB isolates (77.66%, 424/546) belong to Lineage 2.2.1, followed by Lineage 4.5 (6.41%, 35/546), and the Lineage 2 isolates have a strong association with pre-XDR/XDR-TB (P < 0.05) in our study. Epidemic success analysis using time-scaled haplotypic density (THD) showed that clustered isolates outperformed non-clustered isolates. Compensatory mutations happened in rpoA, rpoC, and non-RRDR of rpoB genes, which were found more frequently in clusters and were associated with the increase of THD index, suggesting that increased bacterial fitness was associated with MDR-TB transmission. In addition, the variants in resistance associated genes in MDR isolates are mainly focused on single nucleotide polymorphism mutations, and only a few genes have indel variants, such as katG, ethA. We also found some genes underwent indel variation correlated with the lineage and sub-lineage of isolates, suggesting the selective evolution of different lineage isolates. Thus, this analysis of the characterization and genetic diversity of MDR isolates would be helpful in developing effective strategies for treatment regimens and tailoring public interventions. IMPORTANCE Multidrug-resistant tuberculosis (MDR-TB) is a serious obstacle to tuberculosis prevention and control in China. This study provides insight into the drug-resistant characteristics of MDR combined with phenotypic drug-susceptibility testing and whole genome sequencing. The compensatory mutations and epidemic success analysis were analyzed by time-scaled haplotypic density (THD) method, suggesting clustered isolates and compensatory mutations are associated with MDR-TB transmission. In addition, the insertion and deletion variants happened in some genes, which are associated with the lineage and sub-lineage of isolates, such as the mpt64 gene. This study offered a valuable reference and increased understanding of MDR-TB in China, which could be crucial for achieving the objective of precision medicine in the prevention and treatment of MDR-TB.

Novel oxazolidinones harbor potent in vitro activity against the clinical isolates of multidrug-resistant Mycobacterium tuberculosis in China.

To investigate the in vitro activities of five oxazolidinones in parallel against the reference strains of different mycobacterial species and clinical isolates of Mycobacterium tuberculosis (Mtb), and shed light on the differences in the efficacy of these homolog drugs. The minimum inhibitory concentrations (MICs) of linezolid, tedizolid, sutezolid, delpazolid, and contezolid against 16 mycobacterial reference strains and 69 M. tuberculosis clinical isolates, including 17 drug-susceptible isolates and 52 multidrug-resistant (MDR) isolates, were determined by microplate alamarBlue assay (MABA). The intracellular killing activities of contezolid and linezolid against Mtb H37Rv were compared. In addition, mutations in the linezolid resistance-related genes (rplC, rplD, and 23S rRNA) of the Mtb clinical isolates were also analyzed. Tedizolid exhibited the strongest inhibitory activities against the reference strains of both rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM), among the tested oxazolidinones. In contrast, sutezolid only manifested potent activity against reference strains of SGM. Linezolid, delpazolid, and contezolid were less active against the non-tuberculous mycobacterial references. For the Mtb clinical isolates, the antimicrobial action was ranked as: sutezolid > tedizolid > contezolid and linezolid > delpazolid, whereas no difference between drug-sensitive and multiple drug-resistant isolates was observed. Notably, contezolid demonstrated obviously superior intracellular antimicrobial activity than linezolid. Few strains harbored mutations in rrl gene or rplD genes, although these strains had drug susceptible profiles to linezolid. Different oxazolidinones can have discrepant antimicrobial activity against different mycobacterial species, or have different manifestations out of cell or in cell. Understanding these differences would be helpful in choosing the appropriate drug in clinical practice.

Analysis on drug resistance-associated mutations of multi-drug resistant Mycobacterium tuberculosis based on whole-genome sequencing in China

Objective: To analyze the resistance mutational profiles of multi-drug resistant Mycobacterium tuberculosis in China and the correlation between major mutation types and genotypes based on the whole-genome sequencing data. Methods: Search and download of the genome-wide sequencing data of M. tuberculosis published in China by August 2019 on NCBI database were conducted. Mutation frequency of drug resistance-related gene loci based on whole-genome sequencing was used to predict the molecular susceptibility of strains, and the correlation between mutation types and genotypes was analyzed. Results: According to the results of molecular resistance and susceptibility profiles, 1 024 MDR strains were identified from 2 019 M. tuberculosis strains. The major mutation types of resistance-related genes to common drugs were katG S315T (73.2%, isoniazid), rpoB S450L (63.1%, rifampicin), rpsL K43R (70.0%, streptomycin), embB M306V (37.4%, ethambutol), pncA_promoter T (-11)C (7.9%, pyrazinamide), gyrA A90V (32.3%, fluoroquinolones), rrs A1401G (67.7%, second-line injection drugs), fabG1_promoter C (-15) T (87.0%, Ethionamide), folC I43T (30.4%, P-aminosalicylic acid). Among them, the frequencies of katG S315T, embB M306V, rpsL K43R, gyrA A90V in lineage 2 were significantly higher than those in lineage 4, and folC I43T was only found in lineage 2. The proportion of katG S315T was significantly higher in the ancient Beijing genotype compared to the modern genotype, in contrast, the proportion of rpsL K43R was significantly higher in modern Beijing genotype, the differences were significant (all P<0.05). Conclusions: The results showed the main mutation types of resistance-related genes of MDR strains to many commonly used anti-tuberculosis drugs in China based on whole-genome sequencing, providing a basis for the development of sensitive and specific rapid molecular detection methods. At the same time, it was also found that the major mutation types of MDR-related genes were related to the genotype of the strains.

Increased prevalence of levofloxacin-resistant Mycobacterium tuberculosis in China is associated with specific mutations within the gyrA gene

ObjectivesTo compare the prevalence of levofloxacin (LFX) resistance and the population structure of Mycobacterium tuberculosis (MTB) with different mutations conferring LFX resistance between 2005 and 2015. MethodsA total 542 MTB isolates were randomly selected from pulmonary tuberculosis (TB) patients in 2005 and 2015 and analyzed regarding minimum inhibitory concentrations (MICs) and quinolone resistance-determining regions (QRDR). ResultsOne hundred and eleven of the 542 MTB isolates analyzed (20.5%) were resistant to LFX. There were 42 and 69 LFX-resistant isolates from 2005 and 2015, respectively, and MIC high-level LFX resistance was significantly higher in 2015 (40.6%, 28/69) than in 2005 (16.7%, 7/42) (p = 0.02). There were 87 (78.4%) mutations of these 111 LFX-resistant isolates. In addition, a significant difference in proportion was observed in the isolates with mutations in codon 90 of the gyrA gene between 2005 and 2015 (11.9% in 2005 versus 29.0% in 2015, p = 0.04). ConclusionsThere was an alarming increase in prevalence of LFX-resistant TB in China between 2005 and 2015. This dynamic change is mostly attributed to the increase in high-level LFX resistance. Moreover, a significant difference was noted in the proportion of LFX-resistant isolates harboring specific mutations within the gyrA gene between 2005 and 2015.

Value of pyrazinamide for composition of new treatment regimens for multidrug-resistant Mycobacterium tuberculosis in China

BackgroundPyrazinamide still may be a useful drug for treatment of rifampin-resistant (RR-TB) or multidrug-resistant tuberculosis (MDR-TB) in China while awaiting scale up of new drugs and regimens including bedaquiline and linezolid. The level of pyrazinamide resistance among MDR-TB patients in China is not well established. Therefore, we assessed pyrazinamide resistance in a representative sample and explored determinants and patterns of pncA mutations.MethodsMDR-TB isolates from the 2007 national drug resistance survey of China were sub-cultured and examined for pyrazinamide susceptibility by BACTEC MGIT 960 method. pncA mutations were identified by sequencing. Characteristics associated with pyrazinamide resistance were analyzed using univariable and multivariable log-binominal regression.ResultsOf 401 MDR-TB isolates, 324 were successfully sub-cultured and underwent drug susceptibility testing. Pyrazinamide resistance was prevalent in 40.7% of samples, similarly among new and previously treated MDR-TB patients. Pyrazinamide resistance in MDR-TB patients was associated with lower age (adjusted OR 0.54; 95% CI, 0.34–0.87 for those aged ≧60 years compared to < 40 years). Pyrazinamide resistance was not associated with gender, residential area, previous treatment history and Beijing genotype. Of 132 patients with pyrazinamide resistant MDR-TB, 97 (73.5%) had a mutation in the pncA gene; with 61 different point mutations causing amino acid change, and 11 frameshifts in the pncA gene. The mutations were scattered throughout the whole pncA gene and no hot spot region was identified.ConclusionsPyrazinamide resistance among MDR-TB patients in China is common, although less so in elderly patients. Therefore, pyrazinamide should only be used for treatment of RR/MDR-TB in China if susceptibility is confirmed. Molecular testing for detection of pyrazinamide resistance only based on pncA mutations has certain value for the rapid detection of pyrazinamide resistance in MDR-TB strains but other gene mutations conferring to pyrazinamide resistance still need to be explored to increase its predictive ability .

External quality control of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China.

The aim of our study was to evaluate the performance of conventional drug susceptibility testing (DST) among the tuberculosis (TB)-specialized hospitals in China. A total of 40 hospitals participated in the external quality assurance program for assessment of DST results from each hospital. The sensitivity, specificity, and accuracy of DST were analyzed. The mean accuracy was 96.5% for isoniazid (INH), 95.8% for rifampin (RIF), 97.0% for ethambutol (EMB), 96.8% for ofloxacin (OFX), 97.1% for kanamycin (KAN), 96.1% for amikacin (AMK), and 93.6% for capreomycin (CAP), respectively. Of the 40 participating laboratories, 4 (10.0%) and 6 (15%) failed to achieve 90% accuracy for INH and RIF, respectively. In addition, six hospitals (15%) were confirmed as certified to provide reliable DST results for both first-line and second-line drugs. The certified proportion for DST dropped from 73.9% in the non-western region to 59.2% in the western region. The significant difference was observed in the certified proportion for first-line drugs between the western and non-western region (P = 0.013). Our results demonstrate that the quality of phenotypical DST is frequently unsatisfactory, with approximately one-third of participated laboratories failing to produce certified phenotypical DST results. In addition, the uncertified laboratories majorly come from the western region in China.

Synergistic activities of clofazimine with moxifloxacin or capreomycin against Mycobacterium tuberculosis in China

Clofazimine (CFZ) is a promising candidate drug for use in the management of multidrug-resistant tuberculosis (MDR-TB) patients. In this study, the minimum inhibitory concentration (MIC) method and checkerboard method were used to investigate potential synergies between CFZ and moxifloxacin (MOX) or capreomycin (CAP). Thirty Mycobacterium tuberculosis strains were collected, including 13 MDR strains, 2 extensively drug-resistant (XDR) strains, 3 pan-sensitive strains and 12 strains resistant to other drugs. When the minimum fractional inhibitory concentration indexes (FICIs) were calculated, synergy was found in 21 (70.00%) M. tuberculosis strains against the CFZ/CAP combination and 29 (96.67%) against the CFZ/MOX combination. When the maximum FICIs were calculated, 10 of 15 MDR/XDR strains and 2 of 15 other drug-resistant or pan-sensitive strains showed antagonism against the CFZ/CAP combination, whilst 8 of 15 MDR/XDR strains and 1 of 15 other drug-resistant or pan-sensitive strains showed antagonism against the CFZ/MOX combination, respectively. In conclusion, these findings demonstrate that the combination of CFZ and MOX shows better synergism than the combination of CFZ and CAP. The MDR/XDR isolates are more likely to show antagonism than the other drug-resistant or pan-sensitive strains in both the CFZ/MOX and CFZ/CAP combinations. CFZ in combination with MOX may be a promising drug regimen for the treatment of MDR-TB, particularly for susceptible M. tuberculosis infections.

Multi-center evaluation of GenoType MTBDRsl line probe assay for rapid detection of pre-XDR and XDR Mycobacterium tuberculosis in China

The implementation of rapid and reliable drug susceptibilities diagnosis is fundamental for effective treatment of multidrug-resistant tuberculosis(MDR-TB). The present study aimed to assess the diagnostic performance of the 2nd-version GenoType MTBDRsl kit as well as the impact of its implementation on the turnaround time in a multi-center Chinese study. Totally 353 MDR-TB patient specimens were consecutively tested. The 2nd-version GenoType MTBDRsl assay, drug susceptibility testing with the MGIT 960 system, and sequencing were performed and compared. MTBDRsl testing identified the major genotypes associated with fluoroquinolones resistance, predominated by gyrA MUT3B (Asp94Asn and Asp94Tyr, 26.5%) and MUT3C (Asp94Gly, 19.5%). The genotypes associated with resistance to 2nd-line injectable drugs(SLIDs) were rrsMUT1(A1401G, 64.9%) and absence of WT1(C1402T, 10.5%). The sensitivities for detection of resistance to fluoroquinolones, SLIDs, and their combination (extensively drug resistance, XDR) were 80.5%, 80.7% and 73.5% and specificities were 100.0%, 99.3% and 99.1%, respectively. Implementation of this test significantly reduced the turnaround time between sample collection and result reporting from 45 to 3 days, a reduction by 93.3% (p, 0.001). With a favorable diagnostic performance and short turnaround time, the 2nd-version GenoType MTBDRsl assay proves its value for early diagnosis of resistance to 2nd-line drugs as well as of XDR-TB in China.

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.

A 10-Year Comparative Analysis Shows that Increasing Prevalence of Rifampin-Resistant Mycobacterium tuberculosis in China Is Associated with the Transmission of Strains Harboring Compensatory Mutations.

In this work, we conducted bacterial population profile studies to assess trends of rifampin (RIF) resistance of Mycobacterium tuberculosis isolates collected across China from 2005 to 2015. Totals of 273 and 269 randomly selected M. tuberculosis isolates from 2005 and 2015, respectively, were analyzed. The rates of RIF resistance (36.4%), isoniazid resistance (39.0%), and levofloxacin resistance (25.7%) in 2015 were significantly higher than those in 2005 (28.2%, 30.0%, and 15.4%, respectively; P < 0.05). Genotypic data revealed 256 (95.2%) Beijing-type isolates in 2015, a rate significantly higher than that in 2005 (86.4%) (P < 0.01). A higher proportion of mutations was identified within the rifampin resistance-determining region (RRDR) of rpoB in isolates from 2015 (99.0%) than in 2005 isolates (85.7%, P < 0.01). In addition, a significantly higher proportion of RIF-resistant isolates carrying compensatory mutations was observed in 2015 (31.6%) than in 2005 (7.8%). Notably, the great majority of these compensatory mutations (91.9%) were observed in isolates that harbored a mutation of codon 531 of the rpoB gene. In conclusion, our data demonstrate that resistance to RIF, isoniazid, and levofloxacin has become significantly more prevalent during the past decade. In addition, the prevalence of the Beijing genotype significantly increased from 2005 to 2015. Notably, a significantly increased frequency of strains with mutations in rpoC or rpoA is observed among those that have codon 531 mutations, which suggests that they may be compensatory and may play a role in facilitating transmission.

Recent transmission of Mycobacterium tuberculosis in China: the implication of molecular epidemiology for tuberculosis control.

Tuberculosis (TB) has remained an ongoing concern in China. The national scale-up of the Directly Observed Treatment, Short Course (DOTS) program has accelerated the fight against TB in China. Nevertheless, many challenges still remain, including the spread of drug-resistant strains, high disease burden in rural areas, and enormous rural-to-urban migrations. Whether incident active TB represents recent transmission or endogenous reactivation has helped to prioritize the strategies for TB control. Evidence from molecular epidemiology studies has delineated the recent transmission of Mycobacterium tuberculosis (M. tuberculosis) strains in many settings. However, the transmission patterns of TB in most areas of China are still not clear. Studies carried out to date could not capture the real burden of recent transmission of the disease in China because of the retrospective study design, incomplete sampling, and use of low-resolution genotyping methods. We reviewed the implementations of molecular epidemiology of TB in China, the estimated disease burden due to recent transmission of M. tuberculosis strains, the primary transmission of drug-resistant TB, and the evaluation of a feasible genotyping method of M. tuberculosis strains in circulation.

Associations between Mycobacterium tuberculosis Beijing genotype and drug resistance to four first-line drugs: a survey in China.

Investigations on the genetic diversity of Mycobacterium tuberculosis in China have shown that Beijing genotype strains play a dominant role. To study the association between the M. tuberculosis Beijing genotype and the drug-resistance phenotype, 1286 M. tuberculosis clinical isolates together with epidemiological and clinical information of patients were collected from the center for tuberculosis (TB) prevention and control or TB hospitals in Beijing municipality and nine provinces or autonomous regions in China. Drug resistance testing was conducted on all the isolates to the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, and ethambutol). A total of 585 strains were found to be resistant to at least one of the four anti-TB drugs. The Beijing family strains consisted of 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains, whereas the non-Beijing family strains comprised 202 (58.05%) drug-sensitive strains and 146 (41.95%) drug-resistant strains. No significant difference was observed in prevalence (χ2= 2.41, P > 0.05) between the drug-resistant and drugsensitive strains among the Beijing family strains. Analysis of monoresistance, multidrug-resistant TB, and geographic distribution of drug resistance did not find any relationships between the M. tuberculosis Beijing genotype and drug-resistance phenotype in China. Results confirmed that the Beijing genotype, the predominant M. tuberculosis genotype in China, was not associated with drug resistance.

Comparison of antibody responses against Mycobacterium tuberculosis antigen Rv0679c in tuberculosis patients from the endemic and non-endemic regions of the Beijing genotype: a case control study

BackgroundStrains of the Beijing genotype of Mycobacterium tuberculosis (MTB) are reportedly associated with the virulence of tuberculosis (TB) infection, unfavorable outcomes of anti-TB treatment, and the global TB pandemic. Rv0679c, a hypothetical membrane protein related to host cell invasion, has a Beijing genotype-specific mutation at residue 142 (Asn142Lys). Antigenicity differences between Rv0679c-Asn142 (N-type) and Rv0679c-Lys142 (K-type) have been previously observed in mice antigen-antibody responses. However, the immune response to Rv0679c in humans remains unknown. Therefore, we aimed to investigate the anti-Rv0679c immune response in TB patients from the endemic and non-endemic regions of the Beijing MTB genotype.MethodsWe analyzed the Rv0679c-specific antibody responses in 84 subjects from the endemic region of the Beijing genotype MTB in China, including 45 pulmonary TB patients (C-PTB) and 39 healthy controls (C-HC), and 81 subjects from the Philippines (the endemic region of the non-Beijing genotype), including 51 pulmonary TB patients (P-PTB) and 30 healthy controls (P-HC). Anti-tuberculous-glycolipid (TBGL) antigen was used as the control antibody.ResultsTBGL IgG titers were higher in both C-PTB and P-PTB than those in their corresponding HC (C-PTB median 4.2, P-PTB median 11.2; C-PTB vs. P-PTB, p > 0.05), suggesting immune response comparability in PTB from two different countries. C-PTB showed a higher response compared to C-HC for anti-K-type IgG (53.3%) than anti-N-type IgG (6.67%); this response was not observed in P-PTB (both N-type and K-type 9.80%).ConclusionDimorphic antigen Rv0679c was found to be associated with distinct immune response patterns, indicating the role of Beijing/non-Beijing genotype of MTB in stimulating specific responses in TB patients from the endemic region of Beijing MTB. Meanwhile, reactions to Rv0679c in patients and HC from non-endemic regions of the Beijing MTB may be caused by the response to the common epitope of Rv0679c N/K-type.

Analysis on the polymorphisms of human B cell epitopes of 5 specificity antigens in Mycobacterium tuberculosis in China

To study the diversities of human B cell epitopes of five proteins (Mpt83, Mpt70, LpqH, PstS3, GroES) in Mycobacterium tuberculosis isolates from China. We selected 179 strains isolated from patients with tuberculosis in different regions of China, and cultured these strains by L-J medium. The gene sequences of these 5 proteins were amplified and sequenced by PCR. The variations (single nucleotide polymorphisms, SNP) of the DNA sequences were compared and analyzed according to the immune epitope database (IEDB). The synonymous mutation rate (dS), non-synonymous mutation rate (dN) and dN/dS values were calculated with MEGA6 software. Among the 179 clinical MTBC isolates, 7 SNP mutations were found in the mpt83gene. The dN/dS value of the whole mpt83 gene was 0.88. One SNP mutation was found in the mpt70 gene. The dN/dS values of the whole pstS3 gene and non-B epitopes areas were 1.74 and 1.40 respectively. But no mutation was found in the groES gene. The mpt83, lpqH and pstS3 genes of 179 MTBC strains contain gene SNPs and human B cell epitopes diversities, while the coding gene and human B cell epitopes of protein mpt70 and groES were conservative.

Mycobacterial Interspersed Repetitive Unit Can Predict Drug Resistance of Mycobacterium tuberculosis in China

Background: Recently, Mycobacterial Interspersed Repetitive Unit (MIRU) was supposed to be associated with drug resistance in Mycobacterium tuberculosis (M. tuberculosis), but whether the association exists actually in local strains in China was still unknown. This research was conducted to explore that association and the predictability of MIRU to drug resistance of Tuberculosis (TB).Methods: The clinical isolates were collected and the susceptibility test were conducted with Lowenstein–Jensen (LJ) medium for five anti-TB drug. Based on PCR of MIRU-VNTR (Variable Number of Tandem Repeat) genotyping, we tested the number of the repeat unite of MIRU. Then, we used logistic regression to evaluate the association between 15 MIRU and drug resistance. In addition, we explored the most suitable MIRU locus of identified MIRU loci for drug resistance by multivariate logistic regression.Results: Of the 102 strains, one isolate was resistant to rifampicin and one isolate was resistant to streptomycin. Among these fifteen MIRU, there was a association between MIRU loci polymorphism and anti-tuberculosis drug resistance, ETRB (P = 0.03, OR = 0.19, 95% CI 0.05–0.81) and ETRC (P = 0.01, OR = 0.14, 95% CI 0.03–0.64) were negatively related to isoniazid resistance; MIRU20 (P = 0.05, OR = 2.87, 95% CI 1.01–8.12) was positively associated with ethambutol resistance; and QUB11a (P = 0.02, OR = 0.79, 95% CI 0.65–0.96) was a negative association factor of p-aminosalicylic acid resistance.Conclusion: Our research showed that MIRU loci may predict drug resistance of tuberculosis in China. However, the mechanism still needs further exploration.

Prevalence and transmission of pyrazinamide resistant Mycobacterium tuberculosis in China

Pyrazinamide (PZA) is an important first-line anti-tuberculosis drug, however, there are relatively few available data on PZA resistant (PZA-R) rate in China. From June 2009 to June 2012, we selected 493 isolates from five field settings in China to investigate PZA-R by pncA gene sequencing. The result showed that PZA-R rate was 1.0% (2/196) among pan-susceptible isolates, 3.1% (4/130) among isoniazid (INH) mono-resistant isolates, 14.0% (6/43) among rifampin (RIF) mono-resistant isolates and 43.5% (54/124) among multidrug resistant (MDR) isolates. MDR tuberculosis (TB), RIF mono-resistance, and retreatment were found to be risk factors for PZA-R. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA mutations indicate that transmission of PZA-R isolates plays an important role in emergence of PZA-R TB. The results suggest that, it is necessary to conduct PZA susceptibility test among MDR isolates and modify the treatment regimens accordingly.

Prevalence and risk factors of mixed Mycobacterium tuberculosis complex infections in China

Mixed infections have been considered as a potential obstacle for tuberculosis treatment and control. To date, few studies have been done to determine the rate of mixed infections of Mycobacterium tuberculosis in China. In this study, we used the standard 24-loci MIRU-VNTR method to genotype the representative M. tuberculosis isolates from the national drug-resistant survey conducted in China. A total of 3248 M. tuberculosis complex (MTBC) strains had complete 24-loci MIRU-VNTR results and available for the analyses. Overall, MIRU-VNTR typing identified 115 (3.5%) isolates as being mixed MTBC infections in China. Statistical analysis revealed that mixed infections were significantly more likely to occur in men than women. Compared with the percentage of mixed infection from patients aged 45-56 years, the percentages of mixed infections were higher among patients aged 25-44 years [OR (95% CI): 1.844(1.129-3.014)] and old patients [older than 65 years OR (95% CI): 1.908(1.097-3.319)]. In addition, significantly higher frequencies of hemoptysis (P=0.022) and chest pain (P=0.012) were observed among mixed infections, using patients infected with a single strain as a reference. In conclusion, this study has provided the first comprehensive understanding of mixed MTBC infections in China, which will be essential to generate the effective TB control strategies.

Development of a Hierarchical Variable-Number Tandem Repeat Typing Scheme for Mycobacterium tuberculosis in China

Molecular typing based on variable-number tandem repeats (VNTR) analysis is a promising tool for identifying transmission of Mycobacterium tuberculosis. However, the currently proposed 15- and 24-locus VNTR sets (VNTR-15/24) only have limited resolution and contain too many loci for large-scale typing in high burden countries. To develop an optimal typing scheme in China, we evaluated the resolution and robustness of 25 VNTR loci, using population-based collections of 1362 clinical isolates from six provinces across the country. The resolution of most loci showed considerable variations among regions. By calculating the average resolution of all possible combinations of 20 robust loci, we identified an optimal locus set with a minimum of 9 loci (VNTR-9) that could achieve comparable resolution of the standard VNTR-15. The VNTR-9 had consistently high resolutions in all six regions, and it was highly concordant with VNTR-15 for defining both clustered and unique genotypes. Furthermore, VNTR-9 was phylogenetically informative for classifying lineages/sublineages of M. tuberculosis. Three hypervariable loci (HV-3), VNTR 3232, VNTR 3820 and VNTR 4120, were proved important for further differentiating unrelated clustered strains based on VNTR-9. We propose the optimized VNTR-9 as first-line method and the HV-3 as second-line method for molecular typing of M. tuberculosis in China and surrounding countries. The development of hierarchical VNTR typing methods that can achieve high resolution with a small number of loci could be suitable for molecular epidemiology study in other high burden countries.

Multicenter Research on the BACTEC MGIT 960 system for the second-line drugs susceptibility testing of Mycobacterium tuberculosis in China

The reliability of the BACTEC MGIT 960 system for the second-line drugs (capreomycin [CPM], kanamycin [KAN], ofloxacin [OFX] and ethionamide [ETH]) susceptibility testing (DST) of Mycobacterium tuberculosis (M. tuberculosis) was compared to that of traditional Lowenstein-Jensen (L-J) proportion method (PM) among four different sites in China. After resolution of discrepant results by retesting the strains using both methods in the National Reference Laboratory of tuberculosis, the overall concordance values between the 2 systems were 99.7% (kappa value: 0.97) for CPM, 99.7% (kappa value: 0.97) for KAN, 100.0% (kappa value: 1.00) for OFX, and 98.6% (kappa value: 0.95) for ETH. The average turnaround time with BACTEC MGIT 960 system among four sites was 8.9 ± 1.7 days, significantly shorter than 28 days with the traditional L-J PM. Therefore, the BACTEC MGIT 960 system is a reliable and rapid method for the second-line drug susceptibility testing of tuberculosis in China. Notably, a stricter quality control program should be routinely carried out when clinical laboratories perform the second-line DST with BACTEC MGIT 960 system.