Guanglu Jiang

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.).

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.

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.

Rifampin Stability in 7H9 Broth and Löwenstein-Jensen Medium

ABSTRACT The objectives of this study were to monitor the stability of rifampin (RIF) in Löwenstein-Jensen medium (L-J medium) and 7H9 broth, which are the media commonly used for drug susceptibility testing (DST) of Mycobacterium tuberculosis. Rifampin degradation in stock solution, 7H9 broth, and L-J medium and during the inspissation process for L-J medium preparation was serially monitored by high-performance liquid chromatography (HPLC). L-J medium-based DST was conducted to examine the effect of L-J medium storage on the DST outcome. The RIF stock solution was stable for at least 3 months when kept at either 4°C or −20°C; RIF in 7H9 broth and L-J medium was almost 50% decayed after 1 week of storage at 37°C, and rifampin could not be detected in 7H9 or L-J medium after 3 weeks or 6 weeks of storage at 37°C. Approximately half of the drug was decomposed after 4 months of storage at 4°C for both media, and after 6 months of storage at 4°C, RIF in L-J medium was undetectable, while 38% of RIF remained in 7H9 medium. Approximately 21, 24, 29, and 35% RIF degradations were detected when the L-J medium was coagulated at 75°C, 80°C, 85°C, and 90°C, respectively. The DST outcomes when using L-J medium stored for different periods of time were consistent with the RIF concentration monitoring data. Rifampin in stock solution is stable for at least 3 months at a reduced storage temperature. Media containing RIF should be prepared strictly according to validated standard operating procedures. RIF degradation is a possible reason for false resistance categorizations of M. tuberculosis isolates in the clinical laboratory.

Prevalence and drug resistance of nontuberculous mycobacteria, northern China, 2008-2011.

To the Editor: Nontuberculous mycobacteria (NTM), defined as members of Mycobacterium species other than those in the M. tuberculosis complex or M. leprae, are mostly considered to be opportunistic pathogens (1). However, many NTM can and do cause disease in immune-competent hosts. Pulmonary infection by NTM can be a source of diagnostic uncertainty, especially in locations such as in China, where acid-fast staining of sputum samples is the mainstay of diagnosis for tuberculosis (2). NTM are also relatively resistant to many of the first- and second-line drugs used to treat tuberculosis, thus making accurate diagnosis and drug-susceptibility testing critical to clinical management of NTM infections (3). The medical and public health communities have been concerned about increasing prevalence of NTM infection in China, and 2 recent surveys, 1 from Shanghai and another from a rural population in Shandong Province, gave somewhat conflicting reports of the prevalence of these infections (4,5). We therefore decided to conduct a survey of NTM isolates in Beijing from the National Tuberculosis Clinical Laboratory of the Beijing Chest Hospital. We also tested isolates from specimens collected in this laboratory against an extended drug susceptibility panel to determine which drug regimens would be most useful in therapy for various NTM infections. During January 2008–December 2011, sputum samples collected from 3,714 patients attending the Beijing Chest Hospital with suspected pulmonary tuberculosis were positive for mycobacterial spp. Among the surveillance population, 92% were from northern China, including 13 provinces and the 2 major urban conurbations of Beijing and Tianjin. From our survey, the Han ethnic group accounted for 82% of patients, and 61% of total patients were from urban, rather than rural, areas. Most (59%) of the patients were male, and 40% were attending the hospital for re-treatment of pulmonary tuberculosis; mean age was 51 ± 20 years. Of these mycobacterial isolates, 95 (2.6%) were positive for NTM; NTM were identified during initial screening for resistance to p-nitrobenzoic acid. We identified the strains to species level by sequencing the internal transcribed spacer region of the 16S-23S rRNA and 16S rRNA genes (6), which is able to discriminate between even closely related species such as M. chelonae and M. abscessus (7). Of the 95 NTM isolates, 38 (40%) were M. intracellulare and 28 (29%) were M. abscessus (Table). Five additional species were also identified: M. fortuitum (8%), M. gordonae (8%), M. kansasii (7%), M. avium (5%), and M. parascrofulaceum (1%). A survey performed recently in Shandong Province also identified M. intracellulare as the most common isolate (4), but in that study, it represented 52 (81%) of 64 cases. By contrast, 2 previous surveys found M. chelonae to be the most commonly isolated species (20% and 27% of isolates) (5,8). However, none of the isolates from our study were M. chelonae. Differences in isolates may represent the representative patient population from which they were derived; M. chelonae was most commonly isolated from hospitals in southern China (5,8). The most common NTM species found in eastern Asia was M. avium complex, in keeping with findings from our study (9). Documenting another trend, the International Union Against Tuberculosis and Lung Disease reported that M. fortuitum was the most frequently encountered species in Turkey (33.9%), the Czech Republic (17.5%), Portugal (16.5%), and other countries in Europe (10). Table Species and drug-resistance profiles of 95 nontuberculous mycobacteria strains, northern China, 2008–2011* Drug susceptibility testing (DST) was performed by the proportion method according to the WHO Guidelines for the Programmatic Management of Drug-resistant Tuberculosis, 2011 Update [cited 2014 May 12] http://whqlibdoc.who.int/publications/2011/9789241501583_eng.pdf. We tested 3 first-line anti-tuberculosis drugs (rifampin, isoniazid, and ethambutol) and 7 second-line agents (streptomycin, capreomycin, amikacin, protionamide, para-amino salicylic acid, ofloxacin, and levofloxacin) (Table). If a patient had multiple positive NTM isolates, DST was performed on the last isolate. In agreement with other studies (4,5), ethambutol remained the most useful agent against NTM; its overall resistance rate among isolates tested was 42%. Ranking of second or third agents, however, should be guided by species identification and DST. For example, levofloxacin appears to be a good choice for M. kansasii, M. gordonae, or M. fortiutum infections (overall resistance rate 22%), but a poor choice against M. avium complex infections (overall resistance rate 95%). The second most prevalent species in our study (28% of isolates), M. abscessus, was resistant to the test drugs in >90% of cases, highlighting the difficulties associated with treatment for some NTM infections. Our study suggests that there has been no substantial increase in the prevalence of NTM in respiratory isolates from persons in northern China. Most of the isolates show substantial and extensive drug resistance, providing major therapeutic challenges for clinicians, especially if patients are treated as they would be for drug susceptible tuberculosis. To guide therapy, both species-level identification and DST of NTM isolates should be performed. Our data suggest that testing the efficacy of some second-line agents, in particular, fluoroquinolones, may be beneficial in identifying further options for therapy.

Co-occurrence of amikacin-resistant and -susceptible Mycobacterium tuberculosis isolates in clinical samples from Beijing, China

OBJECTIVES This study examined the phenomenon of heteroresistance in Mycobacterium tuberculosis clinical isolates obtained from retreated patients in Beijing, China between 2006 and 2011. METHODS The iPLEX Gold assay platform was used to determine the prevalence of heteroresistance to injectable second-line drugs (amikacin, kanamycin and capreomycin) in resistant isolates. RESULTS Heteroresistance was identified in 10.9% of 220 phenotypic amikacin-resistant isolates. CONCLUSIONS Heteroresistance was related mainly to the short duration and repeated use of amikacin and capreomycin during retreatment. These findings further our understanding of the evolution of resistance to injectable drugs used for tuberculosis treatment and help guide the rational use of injectable drugs during therapy.

Pyrazinamide resistance among multidrug-resistant tuberculosis clinical isolates in a national referral center of China and its correlations with pncA, rpsA, and panD gene mutations

Our study was aimed to identify the phenotypic and genotypic pyrazinamide (PZA) resistance features among multidrug-resistant (MDR) isolates in a national tuberculosis (TB) referral center of China. PZA susceptibility test was performed for a total of 142 MDR-TB clinical isolates using the MGIT 960 PZA kits, and the pncA, rpsA, and panD genes were sequenced. Extensively drug-resistant (XDR) and pre-XDR strains had higher PZA resistance rate than that of MDR strains which were still sensitive to fluoroquinolone and aminoglycoside (42.9%, 24/56) (χ(2)=8.922, P=0.012). No panD mutation was detected among the PZA resistant strains with wild-type pncA and rpsA genes. Our study indicates that PZA-resistant frequency increases with TB drug resistance level; pncA, rpsA, and panD mutations had strong, low, and no correlation with PZA resistance, and rapid molecular assay will facilitate the timely identification of the PZA-sensitive MDR-TB.

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.

Sensititre® MYCOTB MIC plate for drug susceptibility testing of Mycobacterium tuberculosis complex isolates.

SETTING National Tuberculosis Clinical Laboratory, China. OBJECTIVE To evaluate the accuracy and feasibility of the MYCOTB MIC plate in anti-tuberculosis drug susceptibility testing. DESIGN MYCOTB testing of Mycobacterium tuberculosis isolates, the Löwenstein-Jensen (LJ) proportion method and the resurazine microtitre assay (REMA), which is extensively used for in-house assay for minimal inhibition concentration (MIC) testing, were performed simultaneously for comparison. A total of 126 clinical isolates were tested using both MYCOTB and the LJ proportion method against 12 anti-tuberculosis drugs; 80 were also tested using REMA. RESULTS Categorical agreement between MYCOTB and the LJ proportion method was 99.2% for rifampicin, ofloxacin, amikacin, kanamycin and cycloserine, and 98.4% for isoniazid and para-aminosalicylic acid; ethambutol (EMB) had the lowest agreement (86.5%). The overall categorical agreement between MYCOTB and REMA ranged between 98.8% and 100%. MYCOTB outcomes, interpreted on day 10 and 21, were stable for all drugs except EMB. CONCLUSION MYCOTB is a rapid, convenient, quantitative and accurate method for testing both first- and second-line anti-tuberculosis drugs.

Pulmonary tuberculosis caused by Mycobacterium bovis in China.

The epidemiology of Mycobacterium bovis infection in humans in China is unknown. In this study, pulmonary tuberculosis caused by M. bovis in China was studied. A total of 4069 clinical strains isolated from sputa during the 2007–2009 nationwide surveillance of drug-resistant tuberculosis in China were analyzed. M. bovis was identified by para-nitrobenzoic acid and thiophen-2-carboxylic acid hydrazide growth tests, spoligotyping and multiplex PCR amplification. In addition, a total of 1828 clinical specimens were recruited from Beijing Chest Hospital (Beijing, China) for Löwenstein-Jensen (LJ) culture, both on standard LJ medium and LJ medium containing 4.5 mg/ml(W/V) sodium pyruvate, the latter being the preferred medium for M. bovis growth. The isolates which demonstrated more vigorous on pyruvate containing medium than on standard LJ medium were then identified by multiplex PCR amplification. Only 1 isolate from the nationwide surveillance was confirmed as M. bovis-BCG. The isolate belonged to a predominant spoligotype SB0120 (ST482). In addition, no M. bovis isolate was acquired by the continuous screening step in Beijing Chest Hospital. M. bovis has a negligible contribution to pulmonary tuberculosis in China, so neither laboratory identification nor clinical treatment of M. bovis infection need be considered in routine work.