Shengfen Wang

National Survey of Drug-Resistant Tuberculosis in China

BACKGROUND The available information on the epidemic of drug-resistant tuberculosis in China is based on local or regional surveys. In 2007, we carried out a national survey of drug-resistant tuberculosis in China. METHODS We estimated the proportion of tuberculosis cases in China that were resistant to drugs by means of cluster-randomized sampling of tuberculosis cases in the public health system and testing for resistance to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and streptomycin and the second-line drugs ofloxacin and kanamycin. We used the results from this survey and published estimates of the incidence of tuberculosis to estimate the incidence of drug-resistant tuberculosis. Information from patient interviews was used to identify factors linked to drug resistance. RESULTS Among 3037 patients with new cases of tuberculosis and 892 with previously treated cases, 5.7% (95% confidence interval [CI], 4.5 to 7.0) and 25.6% (95% CI, 21.5 to 29.8), respectively, had multidrug-resistant (MDR) tuberculosis (defined as disease that was resistant to at least isoniazid and rifampin). Among all patients with tuberculosis, approximately 1 of 4 had disease that was resistant to isoniazid, rifampin, or both, and 1 of 10 had MDR tuberculosis. Approximately 8% of the patients with MDR tuberculosis had extensively drug-resistant (XDR) tuberculosis (defined as disease that was resistant to at least isoniazid, rifampin, ofloxacin, and kanamycin). In 2007, there were 110,000 incident cases (95% CI, 97,000 to 130,000) of MDR tuberculosis and 8200 incident cases (95% CI, 7200 to 9700) of XDR tuberculosis. Most cases of MDR and XDR tuberculosis resulted from primary transmission. Patients with multiple previous treatments who had received their last treatment in a tuberculosis hospital had the highest risk of MDR tuberculosis (adjusted odds ratio, 13.3; 95% CI, 3.9 to 46.0). Among 226 previously treated patients with MDR tuberculosis, 43.8% had not completed their last treatment; most had been treated in the hospital system. Among those who had completed treatment, tuberculosis developed again in most of the patients after their treatment in the public health system. CONCLUSIONS China has a serious epidemic of drug-resistant tuberculosis. MDR tuberculosis is linked to inadequate treatment in both the public health system and the hospital system, especially tuberculosis hospitals; however, primary transmission accounts for most cases. (Funded by the Chinese Ministry of Health.).

Tuberculosis prevalence in China, 1990–2010; a longitudinal analysis of national survey data

BACKGROUND China scaled up a tuberculosis control programme (based on the directly observed treatment, short-course [DOTS] strategy) to cover half the population during the 1990s, and to the entire population after 2000. We assessed the effect of the programme. METHODS In this longitudinal analysis, we compared data from three national tuberculosis prevalence surveys done in 1990, 2000, and 2010. The 2010 survey screened 252,940 eligible individuals aged 15 years and older at 176 investigation points, chosen by stratified random sampling from all 31 mainland provinces. All individuals had chest radiographs taken. Those with abnormal radiographs, persistent cough, or both, were classified as having suspected tuberculosis. Tuberculosis was diagnosed by chest radiograph, sputum-smear microscopy, and culture. Trained staff interviewed each patient with tuberculosis. The 1990 and 2000 surveys were reanalysed and compared with the 2010 survey. FINDINGS From 1990 to 2010, the prevalence of smear-positive tuberculosis decreased from 170 cases (95% CI 166-174) to 59 cases (49-72) per 100,000 population. During the 1990s, smear-positive prevalence fell only in the provinces with the DOTS programme; after 2000, prevalence decreased in all provinces. The percentage reduction in smear-positive prevalence was greater for the decade after 2000 than the decade before (57% vs 19%; p<0.0001). 70% of the total reduction in smear-positive prevalence (78 of 111 cases per 100,000 population) occurred after 2000. Of these cases, 68 (87%) were in known cases-ie, cases diagnosed with tuberculosis before the survey. Of the known cases, the proportion treated by the public health system (using the DOTS strategy) increased from 59 (15%) of 370 cases in 2000 to 79 (66%) of 123 cases in 2010, contributing to reduced proportions of treatment default (from 163 [43%] of 370 cases to 35 [22%] of 123 cases) and retreatment cases (from 312 [84%] of 374 cases to 48 [31%] of 137 cases; both p<0.0001). INTERPRETATION In 20 years, China more than halved its tuberculosis prevalence. Marked improvement in tuberculosis treatment, driven by a major shift in treatment from hospitals to the public health centres (that implemented the DOTS strategy) was largely responsible for this epidemiological effect. FUNDING Chinese Ministry of Health.

Spoligotyping and Drug Resistance Analysis of Mycobacterium tuberculosis Strains from National Survey in China

Background Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC), is one of the major causes of death in the world today. Although China has the second largest global case rate of tuberculosis, a systematic study of TB prevalence in China has not been completed. From 2006 to 2007, the base line surveillance of tuberculosis was carried out by Ministry of Health, and more than 4000 representative strains were selected from 31 provinces in China. Methodology/Principal Findings The aim of the present research was to survey the genotypes of representative Mycobacterium tuberculosis (M. tuberculosis) strains from China using spacer oligonucleotide typing (spoligotyping), and to analyze the relationship between genotype and drug resistance for the first time. A total of 4017 clinical isolates were collected from 2007 to 2008 throughout China. Among those M. tuberculosis isolates, 2500 (62.2%) isolates were Beijing genotypes. The percentage of Beijing genotypes in northern China was higher than in southern China (76.5% vs. 53.2%). Additionally, the frequencies of rifampin-resistant, ofloxacin-resistant and multidrug-resistant isolates were significantly higher in Beijing genotype strains than non-Beijing strains. Furthermore, a novel genotype named “China Southern genotype (CS)” was only isolated from Fujian and Guangdong provinces. Hence, it is very practical to uncover the reason for prevalence of the CS type in southern China. Conclusions/Significance In conclusion, Beijing family genotypes were still the predominant genotype throughout China, which exhibited a greater correlation with rifampin-resistance, ofloxacin-resistance and MDR phenotypes than other TB spoligotypes, and some regions of China showed several unique characters in the distribution of M. tuberculosis genotypes. Our research represents an important contribution for the TB control and research community, which completes broad pictures on drug resistance levels and distribution of M. tuberculosis strain types over China.

Study of the Rifampin Monoresistance Mechanism in Mycobacterium tuberculosis

ABSTRACT Rifampin (RIF) susceptibility is a key factor in determining the treatment effectiveness of the standardized treatment regimens. In Mycobacterium tuberculosis, both target gene mutation and the efflux pump play major roles in the resistance to antituberculosis drugs. By eliminating RIF-resistant strains with rpoB mutation, the choice of RIF-monoresistant strains may allow us to identify the RIF-specific efflux pump genes. This study explored the RIF monoresistance mechanism in M. tuberculosis. Data from DNA sequencing and MIC measurements revealed that specific mutations, including Ser531Leu and His526Asp in RpoB, show high-level drug resistance. Three-dimensional structure modeling provided further evidence that the affinity between RIF and RpoB mutants was in accordance with the drug resistance level of the corresponding isolates. Furthermore, transcription-level analysis among the nonmutated isolates indicated that three efflux pumps (Rv0783, Rv2936, and Rv0933) might be involved in exporting RIF from the cell. Compared to 8 μg/ml for wild-type Escherichia coli, the MICs for the transgenic E. coli strains with either Rv0783 or Rv2936 were 32 and 16 μg/ml, respectively. In conclusion, our study indicated that several RpoB mutant types, including Ser531Leu and His526Asp, show high-level RIF resistance attributed to low affinity between RpoB mutant proteins and RIF. In addition, this work demonstrates that Rv2936 and Rv0783 may be responsible for low-level resistance to RIF by exporting RIF from cells. The predicted structure of RpoB and the newly identified efflux pumps in this study will provide a novel approach to design new drugs and develop novel diagnosis technologies.

A novel method based on high resolution melting (HRM) analysis for MIRU-VNTR genotyping of Mycobacterium tuberculosis.

The mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) method is one of the most important methods that have been used in recent years for genotyping Mycobacterium tuberculosis. Agarose gel electrophoresis and capillary electrophoresis have been used to determine the size of amplicons, however, both of these methods have shortcomings. Here, we develop and evaluate a novel method for MIRU-VNTR typing based on high resolution melting (HRM) analysis. The MIRU40 locus was selected to evaluate different real-time PCR machines and the accuracy of our method; the Roche LightCycler 480 provided greatest consistency between the T(m) value and repeat number and was used in subsequent evaluations. Our method gives greater accuracy in comparison with conventional agarose gel electrophoresis (98.9% vs. 90.9%, p=0.017), and, with the help of fitting formulae, can be used to obtain the number of MIRU tandem repeats from the T(m) value. To validate our method we analyzed 12 classical MIRU loci to genotype 88 clinical isolates. The number of MIRU tandem repeats was determined accurately, quickly and conveniently.

Diagnostic accuracy of the PURE-LAMP test for pulmonary tuberculosis at the county-level laboratory in China.

Background Early and effective detection of Mycobacterium tuberculosis (MTB), particularly in smear-negative tuberculosis (TB), is a priority for global TB control. Loop-mediated isothermal amplification with a procedure for ultra rapid DNA extraction (PURE-LAMP) can detect TB in sputum samples rapidly and with high sensitivity and specificity. However, the PURE-LAMP test has not been effectively evaluated, especially in resource-limited laboratories. In this study, we evaluated the performance of the PURE-LAMP test for TB detection in TB suspects from two county-level TB dispensaries in China. Methodology/Principal Findings From April 2011 to February 2012, patients with suspected TB were continuously enrolled from two county-level TB laboratories in China. Three sputum samples (spot, night, and morning sputum) were collected from each recruited patient. Detection of MTB by PURE-LAMP was compared to a reference standard L-J culture. The results showed that the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection was 70.67%, while the sensitivity of the PURE-LAMP test based on spot sputum for MTB detection in smear positive and culture positive patients and smear negative and culture positive patients was 92.12% and 53.81%, respectively. The specificity of PURE-LAMP based on spot sputum for MTB detection was 98.32%. The sensitivity and specificity of the PURE-LAMP test based on three sputa combination for MTB detection was 88.80% and 96.86%, respectively. The results also showed that the PURE-LAMP test had a significantly lower contamination rate than did solid culture. Conclusions/Significance The study suggested that, in peripheral-level TB laboratories in China, the PURE-LAMP test showed high sensitivity and specificity for TB detection in TB suspects, making it a more effective, rapid, and safe method worthy of broader use in the future.

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum

ABSTRACT The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.

A multicenter study of Cross-Priming Amplification for tuberculosis diagnosis at peripheral level in China

Cross-Priming Amplification (CPA) has been shown to rapidly and effectively detect Mycobacterium tuberculosis (MTB) in sputum samples under isothermal conditions. However, no performance data exist from peripheral-level tuberculosis (TB) clinics in tuberculosis-endemic countries. We conducted a clinical trial at four county-level TB clinics in China to evaluate the effectiveness of the CPA assay. TB suspects were continuously enrolled by a clinician at each clinic. Following informed consent, each patient provided two sputum specimens (spot and morning sputum). Sputum samples were tested by smear microscopy, solid culture and CPA. The National TB reference laboratory (NTRL) collected all culture positive strains and performed 16S-23S rDNA internal transcribed spacer (ITS) sequence analysis for strain identification. Solid culture was used as the gold standard to evaluate the effectiveness of CPA in detecting MTB. A total of 2200 TB-suspected patients were enrolled at the four county-level TB clinics. Compared to solid culture, the sensitivity and specificity of the CPA test for MTB detection within this group was 84.1% (95%CI, 79.5-88.6) and 97.8% (95%CI, 97.1-98.5), respectively, and the sensitivity in smear-negative cases was 59.8% (95%CI, 49.8-69.8). The test failure rate of CPA was 0.8% (32/3918), significantly lower than the 1.7% (106/6138) culture contamination rate.

Genotyping and drug resistance patterns of Mycobacterium tuberculosis strains in five provinces of China.

OBJECTIVE To evaluate the distribution of the Mycobacterium tuberculosis Beijing genotype and the association of the genotype with drug-resistant M. tuberculosis strains in five provinces in China. DESIGN M. tuberculosis strains (n = 158) isolated from five provinces of China were subjected to insertion sequence 6110 restriction fragment length polymorphism (RFLP), spoligotyping and mycobacterial interspersed repetitive units (MIRU) analyses. The prevalence of the Beijing genotype strains in each province was determined and compared. The proportion method was used to test the drug susceptibility of all strains. RESULT Of the 158 strains, 123 (77.8%) were identified as the Beijing genotype by RFLP and spoligotyping. Nearly all the strains (n = 152, 96.2%) were grouped into 14 shared spoligotypes. Six other spoligotypes were unique to China. The prevalence of the Beijing genotype was significantly higher in the interior than in coastal areas (P < 0.001, OR 5.4, 95%CI 2.3-12.7). Resistance to rifampicin (RMP) was associated with the Beijing strain (P = 0.05, OR 3.7, 95%CI 1.2-11.1). CONCLUSION The M. tuberculosis Beijing genotype varies in prevalence in different regions of China and is solely associated with RMP resistance.

A feasibility study of the Xpert MTB/RIF test at the peripheral level laboratory in China

OBJECTIVE To evaluate the performance of Xpert MTB/RIF (MTB/RIF) in the county-level tuberculosis (TB) laboratory in China. METHODS From April 2011 to January 2012, patients with suspected multidrug-resistant tuberculosis (MDR-TB) and non-MDR-TB were enrolled consecutively from four county-level TB laboratories. The detection of Mycobacterium tuberculosis (MTB) by MTB/RIF was compared to detection by Löwenstein-Jensen culture. The detection of rifampin resistance was compared to detection by conventional drug-susceptibility testing. The impact of multiple specimens on the performance of MTB/RIF was also evaluated. RESULTS A total of 2142 suspected non-MDR-TB cases and 312 suspected MDR-TB cases were enrolled. For MTB detection in suspected non-MDR-TB cases, the sensitivity and specificity of MTB/RIF were 94.4% and 90.2%, respectively. The sensitivity in smear-negative patients was 88.8%. For the detection of rifampin resistance in suspected non-MDR-TB cases, the sensitivity and specificity of MTB/RIF were 87.1% and 97.9%, respectively. For the detection of rifampin resistance in suspected MDR-TB cases, the sensitivity and specificity of MTB/RIF were 87.1% and 91.0%, respectively. Using multiple sputum specimens had no significant influence on the performance of MTB/RIF for MTB detection. CONCLUSIONS The introduction of MTB/RIF could increase the accuracy of detection of MTB and rifampin resistance in peripheral-level TB laboratories in China. One single specimen is adequate for TB diagnosis by MTB/RIF.