Lingling Dong

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.

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.

Wild-Type and Non-Wild-Type Mycobacterium tuberculosis MIC Distributions for the Novel Fluoroquinolone Antofloxacin Compared with Those for Ofloxacin, Levofloxacin, and Moxifloxacin

ABSTRACT Antofloxacin (AFX) is a novel fluoroquinolone that has been approved in China for the treatment of infections caused by a variety of bacterial species. We investigated whether it could be repurposed for the treatment of tuberculosis by studying its in vitro activity. We determined the wild-type and non-wild-type MIC ranges for AFX as well as ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), using the microplate alamarBlue assay, of 126 clinical Mycobacterium tuberculosis strains from Beijing, China, of which 48 were OFX resistant on the basis of drug susceptibility testing on Löwenstein-Jensen medium. The MIC distributions were correlated with mutations in the quinolone resistance-determining regions of gyrA (Rv0006) and gyrB (Rv0005). Pharmacokinetic/pharmacodynamic (PK/PD) data for AFX were retrieved from the literature. AFX showed lower MIC levels than OFX but higher MIC levels than LFX and MFX on the basis of the tentative epidemiological cutoff values (ECOFFs) determined in this study. All strains with non-wild-type MICs for AFX harbored known resistance mutations that also resulted in non-wild-type MICs for LFX and MFX. Moreover, our data suggested that the current critical concentration of OFX for Löwenstein-Jensen medium that was recently revised by the World Health Organization might be too high, resulting in the misclassification of phenotypically non-wild-type strains with known resistance mutations as wild type. On the basis of our exploratory PK/PD calculations, the current dose of AFX is unlikely to be optimal for the treatment of tuberculosis, but higher doses could be effective.

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.

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.

Bacteriological and virulence study of a Mycobacterium chimaera isolate from a patient in China.

Abstract A clinical isolate from a patient was identified as Mycobacterium chimaera, a recently identified species of nontuberculous Mycobacteria. The biochemical and molecular identity, drug sensitivity and virulence of this isolated strain were investigated. 16S rRNA, the 16S-23S ITS, hsp65 and rpoB were amplified, and their sequence similarities with other mycobacteria were analyzed. The minimum inhibitory concentrations of 22 anti-microbial agents against this isolate were established, and the virulence of the isolate was evaluated by intravenous injection into C57BL/6 mice using Mycobacterium tuberculosis H37Rv as a control strain. Growth and morphological characteristics and mycolic acid profile analysis revealed that this isolated strain was a member of the Mycobacterium avium complex. BLAST analysis of the amplified sequences showed that the isolated strain was closely related to M. chimaera. Susceptibility testing showed that the isolate was sensitive to rifabutin, rifapentine, clarithromycin, azithromycin, imipenem and cefoxitin. Bacterial load determination and tissue histopathology of the infected mice indicated that the isolate was highly virulent. The first case of M. chimaera infection in China was evaluated. The information derived from this case may offer valuable guidance for clinical diagnosis and treatment.

GeneXpert MTB/RIF assay in the diagnosis of urinary tuberculosis from urine specimens

Conventional bacteriological methods are not generally helpful in diagnosing urinary tuberculosis (UTB). GeneXpert is endorsed for the detection of pulmonary tuberculosis, whereas the data on its utility for urine specimens is limited. In this study, we aimed to evaluate its performance on urine specimens in a country with high TB incidence. A total of 163 suspected UTB patients were consecutively enrolled in the analysis, including 37 (22.7%) culture-positive and 44 (27.0%) clinically diagnosed UTB cases. Compared with conventional culture, the sensitivity of GeneXpert (94.6%) was significantly higher than that of smear microscopy (40.5%, P < 0.001). When setting clinical diagnosis as gold standard, 51 out of 81 clinically diagnosed UTB cases were detected by GeneXpert, demonstrating a sensitivity of 63.0%, which was significantly higher than that of smear microscopy (18.5%, P < 0.001) and culture (45.7%, P = 0.027), respectively. In addition, the proportion of UTB cases in the migrant population was significantly higher than that in the resident population (P = 0.019). To conclude, our data demonstrate that GeneXpert outperforms AFB smear and culture for the detection of MTB in urine samples, which provides an alternative for the diagnosis of UTB. The migrant population and previously diagnosed TB cases are high risk factors for developing UTB cases.

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.