Naihui Chu

Species identification of Mycobacterium abscessus subsp. abscessus and Mycobacterium abscessus subsp. bolletii using rpoB and hsp65, and susceptibility testing to eight antibiotics.

OBJECTIVES To separate Mycobacterium abscessus subsp. bolletii from Mycobacterium abscessus subsp. abscessus using species identification, and to investigate the in vitro activity of amikacin, cefoxitin, imipenem, levofloxacin, moxifloxacin, clarithromycin, azithromycin, and linezolid against Mycobacterium abscessus. METHODS Seventy M. abscessus isolates, previously identified by 16S rRNA sequencing, were further identified by comparative sequence analysis of rpoB and hsp65. Drug susceptibility testing was conducted using the microplate Alamar Blue assay in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines and interpreted using CLSI breakpoints. RESULTS Of the 70 strains, 45 (64%) were M. abscessus subsp. abscessus and 25 (36%) were M. abscessus subsp. bolletii. The majority of M. abscessus isolates were susceptible to azithromycin, amikacin, linezolid, and imipenem (M. abscessus subsp. abscessus: 93%, 98%, 93%, and 73%, respectively; M. abscessus subsp. bolletii: 96%, 96%, 80%, and 68%, respectively). Approximately half of the M. abscessus isolates were moderately susceptible to cefoxitin and moxifloxacin (M. abscessus subsp. abscessus 53% and 49%; M. abscessus subsp. bolletii 72% and 68%). Nearly all the M. abscessus isolates were resistant to levofloxacin (M. abscessus subsp. abscessus 96%, M. abscessus subsp. bolletii 100%). Inducible clarithromycin resistance was found in M. abscessus. After 14 days of incubation, 83% M. abscessus subsp. abscessus and 36% M. abscessus subsp. bolletii were resistant to clarithromycin. CONCLUSIONS Using rpoB and hsp65, M. abscessus subsp. bolletii could be distinguished from M. abscessus subsp. abscessus. Amikacin and azithromycin showed excellent activity against M. abscessus in vitro. Imipenem, linezolid, cefoxitin, and moxifloxacin also showed good activity. Levofloxacin was inactive against M. abscessus. Although clarithromycin showed excellent activity against M. abscessus on day 3, inducible resistance occurred, and after 14 days clarithromycin showed little activity against M. abscessus subsp. abscessus, but still had good activity against M. abscessus subsp. bolletii.

Performance of the MTBDRsl Line probe assay for rapid detection of resistance to second-line anti-tuberculosis drugs and ethambutol in China

This study aimed to assess the performance of the GenoType MTBDRsl Line Probe Assay (LPA) for the detection of resistance to levofloxacin, amikacin, capreomycin and ethambutol on sputum specimens. Sputum samples from patients with smear positive multidrug-resistant tuberculosis, identified with GenoType MTBDRplus LPA, were further evaluated using the MTBDRsl LPA, while phenotypic drug susceptibility testing was used as control. Sputa with discordant outcomes were assessed by gene sequencing. 189 cases were included. The interpretability of the MTBDRsl LPA was 96.8%. The sensitivity and specificity of the MTBDRsl test were 82.5% and 91.5% for levofloxacin, 52.6% and 99.2% for amikacin, 58.1% and 97.7% for capreomycin, and 70.8% and 93.3% for ethambutol respectively. For the diagnosis of extensively drug-resistant tuberculosis, the sensitivity and specificity were 56.1% and 100%. The MTBDRsl LPA presents a specific screening tool to detect resistance to several key second-line anti-tuberculosis drugs and ethambutol in smear positive specimens.

Clinical Significance of Nontuberculous Mycobacteria Isolated From Respiratory Specimens in a Chinese Tuberculosis Tertiary Care Center

The clinical relevance of non-tuberculous mycobacteria (NTM) has been reported to be different dramatically by species or by regions, however, no such evaluation has been performed in China.A retrospective study was performed in Beijing Chest Hospital. All the NTM strains isolated from respiratory specimens in the past 5 years, and patients’ clinical records (symptoms and radiographic information etc.) were investigated. The clinical relevance was evaluated according to the criteria recommended by the American Thoracic society. Totally 232 NTM strains were recruited, among them, M. intracellulare was the dominant species (40.5%), followed by M. abscessus (28.4%). 109 patients, with 185 total isolates, had full clinical records available for review. 84.4% (38/45), 85.7% (24/28%) and 63.6% (7/11) of patients with isolation of M. intracellulare, M. abscessus and M. kansasii, respectively, were categorized as definite NTM disease. Whereas all the 10 patients with isolation of M. gordonae were defined as unlikely NTM disease. The majority of NTMs isolates yielded from respiratory specimens in Beijing Chest Hospital were clinically significant, and M. intracellulare and M. abscessus was the dominated species of NTM lung disease. NTM lung infections demonstrated some specific chest radiograph characteristics.

Transregional movement of multidrug-resistant tuberculosis in north China: an underlying threat to tuberculosis control.

Due to unbalanced distribution of health care resource in China, tuberculosis patients, especially multidrug-resistant tuberculosis (MDR-TB), prefer to suffer transregional movement for seeking better health care service in the first-tier cities. Here, we performed a study on the current status of transregional movement of tuberculosis in northern China by reviewing the medical record of TB patients hospitalized in Beijing Chest Hospital from 2011 to 2015. From 2011 to 2015, the proportion of non-Beijing in-patients had increased from 55.0% (996/1810) to 67.2% (1860/2769). In addition, we found that the rate of re-treated among non-Beijing group was significantly higher than that among Beijing-group. Compared with the proportion of pulmonary TB patients from non-Beijing, there were more extra-pulmonary TB patients from non-Beijing. In addition, 10.5% (164/1568) of Beijing in-patients and 26.5% (491/1858) of non-Beijing in-patients had MDR tuberculosis, and statistical analysis revealed that there was significantly higher proportion of MDR cases among non-Beijing group than Beijing group. In conclusion, our data demonstrate that an increasing proportion of TB patients from northern China seek health care in Beijing. In view of higher prevalence of MDR-TB cases among these patients, the transregional movement of MDR-TB will lead to ongoing MDR TB transmission in the community.

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.

Evaluation of the Ribosomal Protein S1 Gene (rpsA) as a Novel Biomarker for Mycobacterium Species Identification

Objectives. To evaluate the resolution and reliability of the rpsA gene, encoding ribosomal protein S1, as a novel biomarker for mycobacteria species identification. Methods. A segment of the rpsA gene (565 bp) was amplified by PCR from 42 mycobacterial reference strains, 172 nontuberculosis mycobacteria clinical isolates, and 16 M. tuberculosis complex clinical isolates. The PCR products were sequenced and aligned by using the multiple alignment algorithm in the MegAlign package (DNASTAR) and the MEGA program. A phylogenetic tree was constructed by the neighbor-joining method. Results. Comparative sequence analysis of the rpsA gene provided the basis for species differentiation within the genus Mycobacterium. Slow- and rapid-growing groups of mycobacteria were clearly separated, and each mycobacterial species was differentiated as a distinct entity in the phylogenetic tree. The sequences discrepancy was obvious between M. kansasii and M. gastri, M. chelonae and M. abscessus, M. avium and M. intracellulare, and M. szulgai and M. malmoense, which cannot be achieved by 16S ribosomal DNA (rDNA) homologue genes comparison. 183 of the 188 (97.3%) clinical isolates, consisting of 8 mycobacterial species, were identified correctly by rpsA gene blast. Conclusions. Our study indicates that rpsA sequencing can be used effectively for mycobacteria species identification as a supplement to 16S rDNA sequence analysis.

The Difference in Pharmacokinetics and Pharmacodynamics between Extended-Release Fluvastatin and Immediate-Release Fluvastatin in Healthy Chinese Subjects

The aim of this study was to evaluate the difference in pharmacokinetics and pharmacodynamics between extended-release (ER) fluvastatin tablet and its immediate-release (IR) capsule in Chinese healthy subjects. This was an open-label, single/multiple-dose, two-period, two-treatment, crossover, randomized trial with a minimum washout period of 7 days. Twenty healthy male adult subjects were given fluvastatin ER tablet 80 mg QD by oral administration or fluvastatin IR capsule 40 mg BID for seven days. Blood samples were collected up to 24 hours after dosing on day 1 and day 7. Serum concentrations of fluvastatin were determined by LC-MS/MS. For fluvastatin ER tablet 80 mg QD, Cmax was 61.0 ± 39.0 and 63.9 ± 29.7 ng/mL, and AUC0−24 h was 242 ± 156 and 253 ± 91.1 ng·h/mL on day 1 and 7, respectively. For fluvastatin IR capsule 40 mg BID, Cmax was 283 ± 271 and 382 ± 255 ng/mL, and AUC0−24 h was 720 ± 776 and 917 ± 994 ng·h/mL on day 1 and day 7, respectively. The relative bioavailability of fluvastatin ER tablet 80 mg QD to fluvastatin IR capsule 40 mg BID is (45.3 ± 23.9)% and (43.3 ± 24.1)% on day 1 and day 7, respectively. Tmax for fluvastatin ER tablet was 2.50 and 2.60 h and for capsule was 0.78 and 0.88 h on day 1 and day 7, respectively. In the first period, compared to baseline, cholesterol decreased 15.3% in fluvastatin ER tablet 80 mg QD and 16.9% in fluvastatin IR capsule 40 mg BID. Triglyceride decreased 3.7% in fluvastatin ER tablet 80 mg QD and 19.1% in fluvastatin IR capsule 40 mg BID. The difference has no statistical significance at P > 0.05 in reduction percent of cholesterol and triglyceride between the two groups. No adverse events were recorded. The results indicated that Cmax of fluvastatin ER tablet is reduced and Tmax is prolonged compared with IR capsule. There is no accumulation for ER formulation after multiple doses.

Anti-tuberculosis drug prescribing for inpatients in a National Tuberculosis Hospital of China, 2011-2015

OBJECTIVES This study aimed to describe trends in antituberculosis drug prescribing for inpatients from 2011-2015 in a Chinese national tuberculosis (TB) hospital. METHODS This retrospective study, performed in March 2016, reviewed the medical records of all inpatients from Beijing Chest Hospital diagnosed with TB between 2011-2015. Medication used for TB treatment during the inpatient period was recorded. RESULTS A total of 11465 inpatients were enrolled in the study. The most frequently prescribed drug for inpatients was isoniazid (71.2%; 8164/11465), followed by ethambutol (67.5%; 7738/11465), pyrazinamide (59.7%; 6839/11465) and rifampicin (40.0%; 4589/11465). In addition, amikacin (16.5%; 1889/11465), levofloxacin (33.0%; 3789/11465), para-aminosalicylic acid (12.4%; 1422/11465) and clarithromycin (3.5%; 406/11465) were the most common drugs used in the treatment of inpatients for Group II, III, IV and V drugs, respectively. A significant increasing trend in prescribing was found for rifampicin, pyrazinamide, capreomycin, moxifloxacin, prothionamide, para-aminosalicylic acid, cycloserine, clofazimine and linezolid, respectively, whilst there was a significant decreasing trend in the rate of prescribing of ethambutol, amikacin, levofloxacin, amoxicillin/clavulanic acid and clarithromycin during the 5-year study period (Ptrend<0.01). CONCLUSIONS These data demonstrate that prescription of anti-TB drugs varied greatly across clinical diagnostic categories, treatment history and drug susceptibility profiles of TB patients. The World Health Organization (WHO)-endorsed standard regimen should be more extensively employed under conditions where drug susceptibility testing is unavailable in order to guide clinicians to formulate a suitable treatment regimen for TB patients.

Clofazimine improves clinical outcomes in multidrug-resistant tuberculosis: a randomized controlled trial

OBJECTIVES We carried out a randomized multicentre study in China to investigate whether the clofazimine would improve the efficacy of the standardized regimen in patients with multidrug-resistant tuberculosis (MDR-TB). METHODS Patients with MDR-TB managed in 17 TB specialist hospitals in China between September 2009 and September 2011 were randomly assigned to the treatment groups at enrolment. In the intervention group, 100 mg clofazimine per day was added to the standardized regimen. The primary outcome was the proportion of patients with successful outcomes. RESULTS From the 156 patients that were screened, 74 were assigned to the control group and 66 to the clofazimine group. Of the 66 cases analysed for clinical outcome in the clofazimine group, 36 patients were cured, and seven completed treatment, yielding a favourable outcome rate of 65.1%. The proportion of patients with favourable outcomes receiving the control regimen was 47.3% (35/74), which was significantly lower than that in the clofazimine group (p 0.034, relative risk 0.661, 95% CI 0.243-0.949). CONCLUSIONS The addition of clofazimine to the standard regimen improved the treatment of MDR-TB.

Validation of Cycloserine Efficacy in Treatment of Multidrug-Resistant and Extensively Drug-Resistant Tuberculosis in Beijing, China

ABSTRACT Cycloserine (Cs) is recommended by the World Health Organization as a second-line drug to treat multidrug-resistant tuberculosis (MDR-TB); however, its efficacy has never been sufficiently evaluated. To gain some insights into the value of cycloserine for MDR-TB treatment, in vitro bacteriostatic effect was determined and patient validations were performed prospectively. The in vitro activity of Cs against 104 wild-type Mycobacterium tuberculosis strains was determined, and serum Cs concentrations were measured for 73 MDR TB patients 2 h after administration. The treatment outcomes for 27 MDR-TB patients who had baseline isolates and were treated with Cs-containing regimens were followed up. The MICs for 90% of the recruited 104 wild-type strains were below 32 μg/ml. Eighteen out of 52 patients had peak serum concentrations (Cmax) below 20 μg/ml at the dosage of 500 mg daily, while 13 out of 21 patients had peak serum concentrations higher than 35 μg/ml at the dosage of 750 mg daily. The percentage of favorable treatment outcomes among patients with a Cmax/MIC ratio of ≥1 was statistically significantly higher than that among the group with a Cmax/MIC ratio of <1 (P = 0.022). The epidemiological cutoff value for Cs susceptibility testing was 32 μg/ml. A high percentage of patients receiving the recommended dosage of 10 mg/kg for Cs administration could not acquire desirable blood concentrations; therefore, adjusting the dosage according to drug concentration monitoring is necessary. The Cmax/MIC ratio might be a good indicator for predicting the treatment outcome for patients with MDR-TB or extensively drug-resistant TB (XDR-TB) who are being administered Cs-containing regimens.