Fengmin Huo

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.

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.

The reliability analysis of Xpert-positive result for smear-negative and culture-negative specimen collected from bone and joint tuberculosis suspects

BACKGROUND The Xpert MTB/RIF assay (Xpert; Cepheid, Sunnyvale, CA, USA) has been widely used for pulmonary and extra-pulmonary tuberculosis (TB) diagnosis. In clinical practice, specimen yielding smear-negative, culture-negative but Xpert-positive results is frequently confronted. Due to the notorious possibility of contamination that molecular tests always been thought of, Xpert-positive results without bacteriological supporting evidence arouse great confusions to clinicians. METHODS A retrospective study was performed. From April 2014 to February 2015, 852 clinical specimens were Xpert-positive. The results of Xpert assay, bacteriological and pathological examinations from either the same specimens or from the specimens collected during same clinical operations were investigated. RESULTS A total of 90 specimens with Xpert-positive but smear-negative and culture-negative results were recruited, and 81 of them were pus specimens collected from Bone and Joint Tuberculosis (BJTB) patients. According to the pathological examination results, 77 of the 81 pus specimens, 8 of 9 other types of specimens were confirmed as either TB or strongly suggestive of TB; three pus specimens and one biopsy tissue were also suggested TB but with less stronger evidence; only one pus specimen was not TB suggestive. CONCLUSIONS Our study demonstrated that Xpert could be trusted for BJTB diagnosis even when no supporting bacteriological evidence is available in high TB prevalence settings. Our results will alleviate the confusion among clinicians in such scenarios.

Transdermal delivery of isoniazid and rifampin in guinea pigs by electro-phonophoresis

Abstract Electro-phonophoresis (EP) has been used as a drug delivery approach in clinical fields. The objective of the present study is to evaluate the skin permeability of isoniazid and rifampin in guinea pigs by EP to provide reference basis for clinical applications of such transdermal delivery system in the treatment of patients with superficial tuberculosis. Isoniazid and rifampin solutions were delivered transdermally with or without EP in health guinea pigs for 0.5 h. Local skin and blood samples were collected serially at 0, 1/2, 1, 2, 4, 6 and 24 h after dosing. Drug concentrations in local skin and blood were evaluated by high-performance liquid chromatography. Isoniazid concentrations in local skin of guinea pigs receiving isoniazid through EP transdermal delivery were significantly higher than in animals receiving only isoniazid with transdermal patch. However, for rifampin, patches alone group presented almost uniform concentration versus time curve with that of EP group, and both groups had concentrations much higher than the therapeutic concentration of the drug over sustainable time. After EP transdermal delivery, the mean peak concentrations of isoniazid and rifampin in skin were 771.0 ± 163.4 μg/mL and 81.2 ± 17.3 μg/mL respectively. Neither isoniazid nor rifampin concentration in blood could be detected (below the lower detection limit of 1 μg/mL) at any time point. The present study showed that application of EP significantly enhanced INH penetration through skin in guinea pigs, while RIF patch alone obtained therapeutic concentration in local skin. Our work suggests several possible medication approaches for efficient treatment of superficial tuberculosis.

The Mono-Prep system increases the detection rate of sputum smear microscopy for diagnosing tuberculosis

Objective Direct sputum smear microscopy (DSSM) has a low detection rate. This study investigated whether an alternative method called Mono-Prep smear microscopy (MPSM) can enhance the diagnosis of tuberculosis in tuberculosis laboratories that perform direct smear microscopy in China. Methods A total of 117 sputum samples were collected from outpatients who attended Beijing Chest Hospital. DSSM, MPSM, solid culture, and Xpert MTB/RIF were performed on the samples. Results The positive rates of DSSM, MPSM, solid culture, and Xpert MTB/RIF were 27.4% (32/117), 40.2% (47/117), 35.9% (42/117), and 52.1% (61/117), respectively. MPSM could detect 15 more cases of tuberculosis compared with DSSM (47 vs 32) among 117 sputum samples. This represented a significantly higher positive rate and sensitivity of MPSM compared with DSSM. However, MPSM appeared to have a lower specificity (81.3%) compared with DSSM (90.7%) with the solid culture used as a standard. Conclusion Use of MPSM can increase the number of positive sputum samples, but it still needs improvement.

Relapse Versus Reinfection of Recurrent Tuberculosis Patients in a National Tuberculosis Specialized Hospital in Beijing, China

Tuberculosis (TB) recurrence can result from either relapse of an original infection or exogenous reinfection with a new strain of Mycobacterium tuberculosis (MTB). The aim of this study was to assess the roles of relapse and reinfection among recurrent TB cases characterized by a high prevalence rate of drug-resistant TB within a hospital setting. After 58 paired recurrent TB cases were genotyped to distinguish relapse from reinfection, 37 (63.8%) were demonstrated to be relapse cases, while the remaining 21 were classified as reinfection cases. Statistical analysis revealed that male gender was a risk factor for TB reinfection, odds ratios and 95% confidence interval (OR [95% CI]: 4.188[1.012–17.392], P = 0.049). Of MTB isolates obtained from the 37 relapse cases, 11 exhibited conversion from susceptible to resistance to at least one antibiotic, with the most frequent emergence of drug resistance observed to be levofloxacin. For reinfection cases, reemergence of rifampicin-resistant isolates harboring double gene mutations, of codon 531 of rpoB and codon 306 of embB, were observed. In conclusion, our data demonstrate that relapse is a major mechanism leading to TB recurrence in Beijing Chest Hospital, a national hospital specialized in TB treatment. Moreover, male patients are at higher risk for reinfection. The extremely high rate of multidrug-resistant tuberculosis (MDR-TB) among reinfection cases reflects more successful transmission of MDR-TB strains versus non-resistant strains overall.