Yifeng Ma

Xpert MTB/RIF and GenoType MTBDRplus assays for the rapid diagnosis of bone and joint tuberculosis

BACKGROUND Bone and joint tuberculosis (BJTB) constitutes about 10-20% of the extrapulmonary tuberculosis (EPTB) cases in China. The GenoType MTBDRplus assay (MTBDR) has been endorsed by the World Health Organization (WHO) for the diagnosis of pulmonary TB (PTB), while the Xpert MTB/RIF assay (Xpert) has also been endorsed by the WHO for the diagnosis of both PTB and EPTB. The diagnostic utility of these two techniques for BJTB was investigated prospectively. METHODS Sixty pus specimens were obtained from orthopedic patients. Smear, culture, Xpert, and MTBDR assays were performed for each specimen, and MGIT 960-based drug susceptibility testing (DST) was conducted for all of the isolates recovered. The diagnostic efficiency of Xpert and MTBDR was evaluated on the basis of bacteriological examination and the composite reference standard (CRS). RESULTS Fifty of the 60 patients were considered to have BJTB according to the CRS. The sensitivities of smear, culture, Xpert, and MTBDR were 26% (13/50), 48% (24/50), 82% (41/50), and 72% (36/50) respectively, while the specificities of all of the tests were 100% (10/10). Xpert was 100% concordant with MGIT 960-based DST for the detection of rifampicin resistance. MTBDR had a sensitivity of 83.3% and a specificity of 100% for the detection of rifampicin resistance and a sensitivity of 85.7% and specificity of 100% for the detection of isoniazid resistance. CONCLUSION With their high sensitivities, short turnaround times, and ability to diagnose TB and detect drug resistance simultaneously, both Xpert and MTBDR are feasible as diagnostic tools for BJTB in clinical practice.

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.

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.

In Vitro Drug Susceptibility of Bedaquiline, Delamanid, Linezolid, Clofazimine, Moxifloxacin, and Gatifloxacin against Extensively Drug-Resistant Tuberculosis in Beijing, China

ABSTRACT Extensively drug-resistant tuberculosis (XDR-TB) is a deadly form of TB that can be incurable due to its extreme drug resistance. In this study, we aimed to explore the in vitro susceptibility to bedaquiline (BDQ), delamanid (DMD), linezolid (LZD), clofazimine (CLO), moxifloxacin (MFX), and gatifloxacin (GAT) of 90 XDR-TB strains isolated from patients in China. We also describe the genetic characteristics of XDR-TB isolates with acquired drug resistance. Resistance to MFX, GAT, LZD, CLO, DMD, and BDQ was found in 82 (91.1%), 76 (84.4%), 5 (5.6%), 5 (5.6%), 4 (4.4%), and 3 (3.3%) isolates among the XDR-TB strains, respectively. The most frequent mutations conferring fluoroquinolone resistance occurred in codon 94 of the gyrA gene (57.8%), and the strains with these mutations (69.2%) were associated with high-level MFX resistance compared to strains with mutations in codon 90 (25.0%) (P < 0.01). All 5 CLO-resistant isolates exhibited ≥4-fold upward shifts in the BDQ MIC, which were attributed to mutations of codons 53 (60.0%) and 157 (20.0%) in the Rv0678 gene. Additionally, mutation in codon 318 of the fbiC gene was identified as the sole mutation related to DMD resistance. In conclusion, our data demonstrate that the XDR-TB strains exhibit a strikingly high proportion of resistance to the current anti-TB drugs, whereas BDQ, DMD, LZD, and CLO exhibit excellent in vitro activity against XDR-TB in the National Clinical Center on TB of China. The extensive cross-resistance between OFX and later-generation fluoroquinolones indicates that MFX and GAT may have difficulty in producing the desired effect for XDR-TB patients.

Transmitted Extended-Spectrum Extensively Drug-Resistant Tuberculosis in Beijing, China, with Discordant Whole-Genome Sequencing Analysis Results

ABSTRACT Drug resistance to tuberculosis remains a major public health threat. Here, we report two cases of extended-spectrum extensively drug-resistant (XXDR) tuberculosis showing resistance to most first- and second-line agents. The results of a correlation of whole-genome sequencing (WGS) and phenotypic testing were discordant, suggesting that overreliance on WGS may miss clinically relevant resistance in extensively drug-resistant disease.

The Clinical Features and Bacteriological Characterizations of Bone and Joint Tuberculosis in China.

Bone and Joint tuberculosis (BJTB) constitutes about 10% of total extra-pulmonary TB cases. Since the BJTB is a paucibacillary condition, there has been no systematic study on the bacterial characterization, especially the epidemiological feature. Here we collected the mycobacterial clinical isolates, analyzed the clinical features and the bacteriological characteristics from 113 BJTB cases reported in China. The mean age of the cases was 40.33 years while most of the patients fell into the 20–29 year age group; local pain was the most common onset symptom of BJTB cases; mean time from symptom onset to BJTB diagnosis was 13.16 months. 31 isolates were defined as drug resistant, including 15 multidrug resistant (MDR) and 2 extensively drug resistant (XDR) isolates according to the drug susceptibility test outcomes; after spoligotyping, 87.6% (99/113) isolates were categorized as Beijing family. In contrast to the isolates from pulmonary tuberculosis patients, here the MIRU-VNTR assay did not find anything significant. A prolonged time span for BJTB diagnosis highlights the requirement of paying further attention to BJTB infection in China. This study provides essential insights into the demographic and microbial characteristics of BJTB cases in China.

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.

Bead capture increases the sensitivity of sputum microscopy for the diagnosis of tuberculosis in Beijing, China

BACKGROUND A review of the scientific literature concluded that indirect smear can improve detection of TB in sputum compared to direct smear. However few laboratories have access to centrifugation in order to perform indirect smear. This study investigated whether an alternative method of magnetic bead concentration could enhance diagnosis of TB in China in laboratories which only perform direct smear microscopy. METHODS A total of 129 sputum samples were investigated by direct smear microscopy, microscopy after TB-Bead extraction and by solid and liquid culture. RESULTS Direct smear had a sensitivity of 40% by Ziehl-Neelsen (ZN) and 45% by auramine compared to combined culture results. After TB-Bead extraction, this increased to 65% for ZN and 70% for auramine. CONCLUSION Magnetic bead concentration of mycobacteria from sputum led to a significant improvement (p<0.05) in the sensitivity of microscopy compared with direct smear.

Rifabutin Resistance Associated with Double Mutations in rpoB Gene in Mycobacterium tuberculosis Isolates

The objective of this study was to investigate the cross-resistance between rifampin (RIF) and rifabutin (RFB) among clinical Mycobacterium tuberculosis (MTB) isolates, and the correlations between specific rpoB mutations and the minimum inhibitory concentrations (MICs) of RIF and RFB. A total of 256 RIF-resistant isolates were included from the National Tuberculosis Clinical Laboratory in China. The MICs of MTB isolates against RIF and RFB were determined by using a microplate alamarBlue assay. In addition, all the MTB isolates were sequenced for mutations in rpoB gene. 204 out of 256 isolates (79.7%) were resistant to RFB, whereas 52 (20.3%) were susceptible to RFB. RIF-resistant/INH-susceptible (RR) group had a significant lower proportion of RFB-resistance than MDR- (P = 0.04) and XDR-TB group (P < 0.01). DNA sequencing revealed that there were 218 isolates (85.2%) with a single mutation, 26 (10.1%) with double mutations, and 12 (4.7%) without mutation in rpoB gene. Notably, although the single substitution of Leu511Pro, Asp516Gly, and His526Asn did not result in RFB resistance, 77.8% (7/9) of the MTB isolates with these double mutations became resistant to RFB. Compared with RR group (38.9%, 7/18), MDR-TB (63.5%, 106/167) had significantly higher proportion of isolates with mutations in codon 531 of rpoB gene (P = 0.04). Our data demonstrate that various rpoB mutations are associated with differential resistance to RIF and RFB. A single specific mutation in codons 511, 516, 526, and 533 was linked to the susceptibility to RFB for MTB, while the strains with these double mutations irrelevantly conferring RFB resistance produced RFB-resistant phenotype.

Detection of pyrazinamide resistance of Mycobacterium tuberculosis using nicotinamide as a surrogate

OBJECTIVES Despite the importance of pyrazinamide (PZA) in tuberculosis treatment, PZA susceptibility testing is not routinely performed because of its acid pH requirement. We evaluated the Microplate Alamar Blue assay (MABA) to detect resistance to PZA using nicotinamide (NIC) as a surrogate in neutral pH and identify the appropriate cutoff point for the assay. METHODS The NIC minimal inhibition concentrations (MICs) for 125 Mycobacterium tuberculosis clinical isolates were tested by MABA at nine different concentrations (8-2000 μg/mL). The PZA susceptibility testing by the BACTEC MGIT 960 system was used as a reference method. The pncA gene and its promoter region were sequenced for all the recruited strains. RESULTS A total of 64 of 125 clinical isolates were identified as resistant by MGIT 960. Using a minimum inhibitory concentration (MIC) of >500 μg/mL as the cutoff concentration to define resistance presented the best fit of the MABA assay with the MGIT 960 outcomes. MABA demonstrated sensitivity of 100% (95% confidence interval, 92.6-100), specificity of 95.2% (95% confidence interval, 86.0-98.8) and an accuracy of 97.6% compared to the MGIT 960 method. Nine PZA susceptible strains defined by MGIT 960 also had pncA mutations; however, three of them were defined as PZA resistant by NIC MABA with MIC ≥2000 μg/mL. CONCLUSIONS The NIC substitution method for PZA susceptibility test is reliable, cheap, rapid and easy, which makes it promising for use in clinical laboratories.