Optimization of the Zhou Tian Formula extraction technology based on AHP-CRITIC method and analysis of transfer efficiency of key components based on HPLC fingerprinting.

INTRODUCTION: Zhou Tian Formula (ZTF) is an antidepressant traditional Chinese medicine utilized widely in clinical settings for the treatment of patients with depression. However, shortcomings persist in its extraction technology and quality control. OBJECTIVE: This study aimed to propose a methodology for ZTF extraction technology based on the analytic hierarchy process (AHP)-criteria importance through intercriteria correlation (CRITIC) method and to establish a quality control framework for the efficient transfer of index components. METHOD: Firstly, we analyzed the chemical components of ZTF and determined the optimal extraction technology. Secondly, we calculated the transfer efficiency of the index components during the conversion of water decoction to extract powder and subsequently to granules. Thirdly, we established HPLC fingerprints for 15 batches of ZTF water decoction, extract powder, and granules. We employed SIMCA software to analyze the chemicals responsible for variations in quality among different batches of ZTF granules. RESULTS: We determined the optimal extraction process. The average transfer efficiency of ferulic acid, puerarin, mirificin, isoferulic acid, and calycosin during the conversion of water decoction to extract powder and subsequently to granules exceeded 41%. The HPLC fingerprints of ZTF exhibited a similarity exceeding 0.890. Variable importance in projection values indicated that calycosin, ferulic acid, and puerarin were the primary contributors to quality variations. CONCLUSIONS: The AHP-CRITIC method, coupled with an orthogonal array design, could be used for exploring extraction technology. In addition, the rules governing the transfer of index components from water decoction to extract powder, and subsequently to granules, could be applied for the evaluation and quality assessment of ZTF.

StyleTalk++: A Unified Framework for Controlling the Speaking Styles of Talking Heads.

Individuals have unique facial expression and head pose styles that reflect their personalized speaking styles. Existing one-shot talking head methods cannot capture such personalized characteristics and therefore fail to produce diverse speaking styles in the final videos. To address this challenge, we propose a one-shot style-controllable talking face generation method that can obtain speaking styles from reference speaking videos and drive the one-shot portrait to speak with the reference speaking styles and another piece of audio. Our method aims to synthesize the style-controllable coefficients of a 3D Morphable Model (3DMM), including facial expressions and head movements, in a unified framework. Specifically, the proposed framework first leverages a style encoder to extract the desired speaking styles from the reference videos and transform them into style codes. Then, the framework uses a style-aware decoder to synthesize the coefficients of 3DMM from the audio input and style codes. During decoding, our framework adopts a two-branch architecture, which generates the stylized facial expression coefficients and stylized head movement coefficients, respectively. After obtaining the coefficients of 3DMM, an image renderer renders the expression coefficients into a specific person's talking-head video. Extensive experiments demonstrate that our method generates visually authentic talking head videos with diverse speaking styles from only one portrait image and an audio clip.

Effects of fertilization gradient on the production performance and nutritional quality of cultivated grasslands in karst areas.

Optimal fertilization is an important measure for managing cultivated grasslands, and a necessary means for maintaining the nutrient balance, yield, and quality of grassland ecosystems. This study aimed to explore the effects of organic fertilizers on the production performance and nutritional quality of cultivated grasslands in karst areas. Two types of monocultured cultivated grasslands (i.e., Medicago sativa and Dactylis glomerata) were employed as the research objects, and a randomized block design was adopted to investigate the effects of five fertilization gradients on the forage height, coverage, yield, and nutritional quality of the cultivated grasslands. According to the results, the plant height, coverage, and yield of M. sativa first presented an increasing trend, then decreased with increasing fertilization gradient, with a peak at 20-30 t/hm2 fertilization gradient. The height, coverage, and yield of D. glomerata increased gradually with increase in fertilization gradient, and peaked at 40 t/hm2. Meanwhile, the crude protein (CP) and ether extract (EE) contents of both grassland types displayed first presented an increasing trend, then decreased with increasing fertilization gradient, and peaked at 10-30 t/hm2 fertilization gradient. The neutral detergent fiber (NDF) and acidic detergent fiber (ADF) contents of M. sativa presented "N-shaped" and "M-shaped" change trends with increasing fertilization gradient, while those of D. glomerata showed "V-shaped" and "M-shaped" change trends, reaching minimum values under fertilization gradients of 30 and 20 t/hm2, respectively. Year, fertilization, and year × fertilization (Y×F) significantly affected the plant heights, coverages, dry/fresh weight ratios, and yields of M. sativa and D. glomerata. The contribution of coverage to the subordinate function of M. sativa was greatest at a fertilization gradient of 20 t/hm2. Meanwhile, the subordinate function values of the height and coverage of D. glomerata increased gradually with increasing fertilization gradient, but the difference in the subordinate function value of height was only 0.09%-0.18% under the fertilization gradient of 20-40 t/hm2. Evaluation of forage nutrition revealed 10-30 t/hm2 and 20-30 t/hm2 as the optimal organic fertilizer application rates for M. sativa and D. glomerata, respectively.

The Effects of Auditory Working Memory Task on Situation Awareness in Complex Dynamic Environments: An Eye-movement Study.

OBJECTIVE: This study investigated the effect of auditory working memory task on situation awareness (SA) and eye-movement patterns in complex dynamic environments. BACKGROUND: Many human errors in aviation are caused by a lack of SA, and distraction from auditory secondary tasks is a serious threat to SA. However, it remains unclear how auditory working memory tasks affect SA and eye-movement patterns. METHOD: Participants (n = 28) were randomly allocated to two groups and received different periods of visual search training (short versus long). They subsequently completed a situation awareness measurement task in three auditory secondary task conditions (without secondary task, auditory calculation task, and auditory 2-back task). Eye-movement data were collected during the situation awareness measurement task. RESULTS: The auditory 2-back task significantly reduced overall SA, Level 1 SA, dwell times, and total percentage of fixation time on task-related areas of interests in the SA measurement task. Overall SA and Level 3 SA were not reduced by the auditory 2-back task in individuals in the longer visual search training time condition. CONCLUSION: Auditory working memory load impairs SA in the perception and projection stage; however, greater experience can overcome impairment of SA in the projection stage. APPLICATION: This study provided possible approaches to preventing loss of SA: (1) improving crew members' communication skills to ensure the accurate and clear transmission of information, reducing the difficulty of processing information, and (2) providing targeted cognitive training tailored to each pilot's level of experience.

In Vitro and Ex Planta Gold-Bonded and Gold-Mineralized Tobacco Mosaic Virus.

Biotemplated mineralization is a promising and ecofriendly approach to manufacture metal nanoparticles and composites with precise size control. Plant viruses are suitable templates for biomineralization because they are chemically robust and highly scalable through molecular farming. Here, we report a gold-nanoparticle-coated tobacco mosaic virus (TMV) synthesized in a test tube or in plant extracts making use of a TMV displaying a gold-binding peptide (GBP). The methods developed are a step toward engineered living materials, where gold nanowires could be formed in plant tissues for sensing or energy harvest applications.

Potato Virus X Inactivation and Characterization.

The nucleoprotein components of plant viruses self-assemble into monodisperse, nanoscale structures with a high degree of symmetry and polyvalency. Of particular interest are the filamentous plant viruses which provide uniform high aspect ratio nanostructures-such structures remain challenging to obtain using purely synthetic approaches. Potato virus X (PVX) has drawn interest by the materials science community because of its filamentous structure measuring 515 × 13 nm; and both genetic engineering and chemical conjugation methods have been reported to impart new functionalities and develop PVX-based nanomaterials for applications in the health and materials sector. Toward environmentally safe materials-i.e., materials that are not infectious toward crops, such as potato, we reported methods to inactivate PVX. In this chapter, we describe the three methods to inactivate PVX and render it non-infectious toward plants, while maintaining structure and function.

Biological interpretation of the sporadic sputum smear-positive-culture-negative outcome for patients with tuberculosis undertaking treatments.

Introduction: The objective of the study was to identify the causes of smear-positive-culture-negative (S+/C-) outcomes of patients with tuberculosis during the treatment course. Methods: A laboratory-based retrospective study was performed at the Beijing Chest Hospital in China. Within the study period, all patients with pulmonary tuberculosis (PTB) who undertook anti-TB treatments and yielded smear positive outcomes with simultaneous culture outcomes on sputa were considered. Patients were classified into three groups: (I) performed LJ medium culture only; (II) performed BACTEC MGIT960 liquid culture only; and (III) performed both LJ culture and MGIT960 culture. The S+/C- rates of each group were analyzed. The clinical medical records regarding patient category, follow-up bacteriologic examination data, and treatment response were investigated. Results: In total, 1,200 eligible patients were enrolled, and the overall S+/C- rate was 17.5% (210/1,200). Group I had obviously higher S+/C- rate (37%) than group II (18.5%) and group III (9.5%). When solid and liquid cultures were considered independently, the S+/C- outcome was observed more frequently in the solid culture group than in the liquid culture group (30.4%, 345/1,135 vs. 11.5%, 100/873; p < 0.001, χ2 = 102.64). Among the 102 S+/C- patients who had follow-up cultures performed, 35 (34.3%) had positive culture outcomes. Whereas among the 67 patients with follow-up information for more than 3 months but without supportive bacteriological evidence, 45 (67.2%, 45/67) had unfavorable prognosis (including relapse and unimproved conditions), and only 22 (32.8%, 22/67) patients had improved conditions. Compared with new cases, retreated cases produced S+/C- outcomes more frequently and had more chances to be cultivated bacilli successfully afterward. Conclusions: Among our patients, the sporadic smear positive and culture negative outcomes for sputa are more likely associated with the technical failures of culture than with dead bacilli, and this is especially noteworthy for LJ medium culture.

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA.

Methods for the real-time monitoring of the substrate acceptance of modified nucleotides by DNA polymerases are in high demand. In a step towards this aim, we have incorporated ferrocene-based abasic nucleotides into DNA templates and evaluated their compatibility with enzymatic synthesis of unmodified and modified DNA. All canonical nucleotides can be incorporated opposite ferrocene sites with a strong preference for purines. DNA polymerases with lesion-bypass capacity such as Dpo4 allow DNA synthesis to be resumed beyond the site of incorporation. Modified purine nucleotides can readily be incorporated opposite ferrocene basic site analogs, while pyrimidine nucleotides decorated with simple side-chains are also readily tolerated. These findings open up directions for the design of electrochemical sensing devices for the monitoring of enzymatic synthesis of natural or modified DNA.

Toward Plant Cyborgs: Hydrogels Incorporated onto Plant Tissues Enable Programmable Shape Control.

Engineered living materials (ELMs) that incorporate living organisms and synthetic materials enable advanced functional properties. Here, we seek to create plant cyborgs by combining plants or plant tissues with stimuli-responsive polymeric materials. Plant tissues with integrated shape control may find applications in regenerative medicine, and the shape control of living plants enables another dimension of adaptability and response to environmental threats, which can be applied to next-generation precision farming. In this work, we develop chemistry to integrate stimuli-responsive poly(N-isopropylacrylamide) (PNIPAM) hydrogels with decellularized plant tissues assisted by 3D printing. We demonstrate programmable shape morphing in response to thermal cues and ultraviolet (UV) light. Specifically, by taking advantage of the extrusion-based 3D printing method, we deposit nanocomposite PNIPAM precursors onto silane-treated decellularized leaf surface with prescribed shapes and spatial control. When subjected to external stimuli, the strain mismatch generated between the swellable nanocomposite PNIPAM and nonswellable decellularized leaf enables folding and bending to occur. This strategy to integrate the plant tissues with stimuli-responsive hydrogels allows the control of leaf morphology, opening avenues for plant-based biosensors and soft actuators to enhance food security; such materials also may find applications in biomedicine as tissue-engineering scaffolds.

Overexpression of Peroxisome-Localized GmABCA7 Promotes Seed Germination in Arabidopsis thaliana.

Peroxisome is one of the important organelles for intracellular lipid metabolism in plant cells and ß-oxidation of fatty acids in peroxisomes provides the energy for oil-containing seed germination. In this study, we identified an ATP-binding cassette (ABC) transporter gene, GmABCA7 from soybean, which is highly expressed in the different developmental stages of seeds. Transient expression of GmABCA7 in tobacco epidermal cells showed that GmABCA7 was specifically localized at the peroxisomes. Overexpression of GmABCA7 in Arabidopsis does not change seed phenotypes, or the overall levels of lipid, protein and sugar stored in the seeds; however, the transgenic seeds produced more gluconeogenic pathway precursors such as succinate and malate and germinated earlier compared to the wild type seeds. These results suggest that GmABCA7 may affect the ß-oxidation of fatty acids and play an important role in seed germination.

Intra-Articular Drug Delivery for Osteoarthritis Treatment.

Osteoarthritis (OA) is the most prevalent degenerative joint disease affecting millions of people worldwide. Currently, clinical nonsurgical treatments of OA are only limited to pain relief, anti-inflammation, and viscosupplementation. Developing disease-modifying OA drugs (DMOADs) is highly demanded for the efficient treatment of OA. As OA is a local disease, intra-articular (IA) injection directly delivers drugs to synovial joints, resulting in high-concentration drugs in the joint and reduced side effects, accompanied with traditional oral or topical administrations. However, the injected drugs are rapidly cleaved. By properly designing the drug delivery systems, prolonged retention time and targeting could be obtained. In this review, we summarize the drugs investigated for OA treatment and recent advances in the IA drug delivery systems, including micro- and nano-particles, liposomes, and hydrogels, hoping to provide some information for designing the IA injected formulations.

In situ vaccine application of inactivated CPMV nanoparticles for cancer immunotherapy.

Cowpea mosaic virus (CPMV) is currently in the development pipeline for multiple biomedical applications, including cancer immunotherapy. In particular the application of CPMV as in situ vaccine has shown promise; here the plant viral nanoparticle is used as an adjuvant and is injected directly into a tumor to reverse immunosuppression and prime systemic anti-tumor immunity. Efficacy of this CPMV-based cancer immunotherapy has been demonstrated in multiple tumor mouse models and canine cancer patients. However, while CPMV is non-infectious to mammals, it is infectious to legumes and therefore, from a safety perspective, it is desired to develop non-infectious CPMV formulations. Non-infectious virus-like particles of CPMV devoid of nucleic acids have been produced; nevertheless, efficacy of such empty CPMV nanoparticles does not match efficacy of nucleic acid-laden CPMV. The multivalent capsid activates the innate immune system through pathogen pattern recognition receptors (PRRs) such as toll-like receptors (TLRs); the RNA cargo provides additional signaling through TLR-7/8, which boosts the efficacy of this adjuvant. Therefore, in this study, we set out to develop RNA-laden, but non-infectious CPMV. We report inactivation of CPMV using UV light and chemical inactivation using ß-propiolactone (ßPL) or formalin. 7.5 J cm-2 UV, 50 mM ßPL or 1 mM formalin was determined to be sufficient to inactivate CPMV and prevented plant infection. We compared the immunogenicity of native CPMV and inactivated CPMV formulations in vitro and in vivo using RAW-Blue™ reporter cells and a murine syngeneic, orthotropic melanoma model (using B16F10 cells and C57BL6 mice). While the in vitro assay indicated activation of the RAW-Blue™ reporter cells by formaldehyde and UV-inactivated CPMV at levels comparable to native CPMV; ßPL-inactivated CPMV appeared to have diminished activity. Tumor mouse model experiments indicate potent efficacy of the chemically-inactivated CPMV (UV-treated CPMV was not tested) leading to tumor regression and increased survival; efficacy was somewhat reduced when compared to CPMV, however these samples outperformed the empty CPMV nanoparticles. These results will facilitate the translational development of safe and potent CPMV-based cancer immunotherapies.

Diagnostic Yield of Oral Swab Testing by TB-LAMP for Diagnosis of Pulmonary Tuberculosis.

OBJECTIVE: A prospective study was conducted to ascertain the accuracy of oral swab specimens collected in the early morning, spot and at night for detecting pulmonary tuberculosis (TB). METHODS: We prospectively enrolled patients with symptoms suggestive of pulmonary TB in Beijing Chest Hospital. An early morning sputum specimen was collected from each patient for GeneXpert MTB/RIF (Xpert) and mycobacterial culture. In addition, three oral swabs were collected for TB-LAMP testing. RESULTS: With the combined results of three oral swab specimens, the proportion of Mycobacterium tuberculosis (MTB)-positive cases achieved 40.6%, which was comparable to results for Xpert and MGIT (P=0.603). Using Xpert plus MGIT as reference, the sensitivity of OS-LAMP on a single specimen ranged from 32.6% on the night oral swab to 50.0% on the morning swab. The combination of three oral swab specimens correctly identified 38 MTB-positive cases, indicating an overall sensitivity of 82.6%, which was significantly higher than that of a single oral swab specimen (P<0.001, P=0.001). CONCLUSION: Oral swab can be used as an alternative specimen for diagnosis of pulmonary TB using TB-LAMP. Morning oral swab exhibits the highest sensitivity, and the inclusion of more specimens at different time points provides compensation in diagnostic sensitivity with single oral swab.

Three Alternative Treatment Protocols for the Efficient Inactivation of Potato Virus X.

Filamentous nanomaterials are flexible with a high aspect ratio, conferring unique mechanical, electromagnetic, and optical properties; promoting tissue penetration; and allowing the formation of hierarchical superstructures. The fabrication of synthetic nanofilaments with uniform properties is challenging, but this can be addressed by the use of filamentous plant viruses such as potato virus X (PVX), which are produced as monodisperse structures from a genetic template. To take advantage of PVX without risks to agriculture and the environment, it is necessary to inactivate the virus efficiently without disrupting its chemical and material properties. Herein, we report experiments showing that PVX can be completely inactivated by exposure to UV irradiation (0.5 J cm-2) or chemical treatment (1 mM ß-propiolactone or 10 mM formalin) without interfering with the chemical addressability of lysine or cysteine residues, which are typically used as conjugation handles for virus nanoparticle functionalization.

Interpretation of Discordant Rifampicin Susceptibility Test Results Obtained Using GeneXpert vs Phenotypic Drug Susceptibility Testing.

BACKGROUND: The 3-month difference in turnaround time between Xpert and conventional phenotypic drug susceptibility testing (pDST) causes patient treatment challenges when pDST rifampin (RIF) susceptibility results and earlier Xpert results disagree, resulting in unnecessary tuberculosis (TB) patient exposure to toxic second-line drugs. Here, the prevalence of discordant RIF susceptibility test results, specifically Xpert (resistant) vs pDST (susceptible) results, was determined. METHODS: Tuberculosis patients enrolled between January 2015 and June 2018 at Beijing Chest Hospital who consecutively tested positive for RIF resistance using Xpert then negative using pDST were studied. DNA sequences and minimal inhibitory concentration (MIC) results provided insights for understanding discordant results. RESULTS: Of 26 826 patients with suggestive TB symptoms undergoing Xpert MTB/RIF testing, 728 diagnosed as RIF-resistant were evaluated. Of these, 118 (16.2%) exhibiting Xpert RIF resistance and phenotypic RIF susceptibility yielded 104 successfully subcultured isolates; of these, 86 (82.7%) harbored rpoB gene RIF resistance-determining region mutations and 18 (17.3%) did not. The Leu511Pro (25.0%) and Leu533Pro (17.3%) mutations were most frequently associated with discordant RIF susceptibility test results. Of the 86 isolates with rpoB mutations, 42 (48.8%) with MICs ≤1.0 mg/L were assigned to the RIF-susceptible group, with Leu511Pro being the most common mutation observed. Isolates with a very low bacterial load were most frequently misdiagnosed as RIF-resistant by Xpert. CONCLUSIONS: Approximately one-sixth of RIF-resistant TB isolates identified via Xpert yielded discordant pDST results due to questionable interpretation of specific "disputed" mutations. Thus, a diagnostic flowchart should be used to correctly interpret Xpert RIF resistance results to best guide patient treatment.

Specific gyrA Gene Mutations Correlate with High Prevalence of Discordant Levofloxacin Resistance in Mycobacterium tuberculosis Isolates from Beijing, China.

Although rapid, highly sensitive molecular diagnostics tests are useful for diagnosing fluoroquinolone-resistant tuberculosis (TB), results of molecular testing versus conventional sequential phenotypic drug susceptibility testing (pDST) are frequently discordant. This article determined the discordance rate of levofloxacin (LFX) resistance results, obtained via MeltPro TB molecular testing, versus pDST of clinical TB isolates collected in Beijing, China, between January and December 2018. Isolates with discordant results were further subjected to LFX minimal inhibitory concentration (MIC) determinations and DNA sequence analysis to explore causes of discordance. Of 571 total TB cases, 126 (22.1%) were identified as LFX resistant using the MeltPro TB assay. However, 34 of these 126 LFX-resistant isolates yielded LFX-susceptible test results using pDST, for an overall discordance rate of 27.0%. LFX MICs mainly clustered around the critical LFX concentration, with 7 (21.2%) and 13 (39.4%) of isolates exhibiting MICs of 2.0 and 4.0 mg/L, respectively. The most prevalent LFX resistance mutations associated with discordant results were involved in DNA gyrase subunit A amino acid substitutions Ala90Val (13, 39.4%) and Asp94Ala (11, 33.3%). Notably, more than one-quarter of isolates deemed LFX resistant via the MeltPro assay were scored as LFX susceptible on the basis of pDST results. Ultimately, highly discordant LFX-resistance test results were associated with specific gyrA mutations in isolates with MICs approaching the critical LFX concentration.

Comparison of Lowenstein-Jensen medium and MGIT culture system for recovery of Mycobacterium tuberculosis from abscess samples.

PURPOSE: In this study, our aim was to assess Lowenstein-Jensen (L-J) medium and MGIT culture system for recovery of Mycobacterium tuberculosis (MTB) from abscess samples in skeletal tuberculosis (TB) cases. METHODS: Abscess samples were collected from patients suggestive of skeletal TB in Beijing Chest Hospital for laboratory examination, including smear microscopy, L-J culture and MGIT culture. RESULTS: Of the 232 abscess samples, 72 (31.0%) were culture-positive for mycobacteria. Of 72 isolates recovered, 94.4% were detected in MGIT 960 and 75.0% on L-J medium. MGIT could recover significantly higher rate of MTB isolates from smear-positive specimens than L-J medium. The mean time to detection of MTB in MGIT 960 was significantly lower than that on L-J medium. CONCLUSION: The BACTEC MGIT 960 outperforms the conventional L-J medium in recovering MTB from abscess samples. The combination of MGIT and L-J method also increases the overall recovery rate of MTB in culture.

Increased prevalence of levofloxacin-resistant Mycobacterium tuberculosis in China is associated with specific mutations within the gyrA gene.

OBJECTIVES: To compare the prevalence of levofloxacin (LFX) resistance and the population structure of Mycobacterium tuberculosis (MTB) with different mutations conferring LFX resistance between 2005 and 2015. METHODS: A total 542 MTB isolates were randomly selected from pulmonary tuberculosis (TB) patients in 2005 and 2015 and analyzed regarding minimum inhibitory concentrations (MICs) and quinolone resistance-determining regions (QRDR). RESULTS: One hundred and eleven of the 542 MTB isolates analyzed (20.5%) were resistant to LFX. There were 42 and 69 LFX-resistant isolates from 2005 and 2015, respectively, and MIC high-level LFX resistance was significantly higher in 2015 (40.6%, 28/69) than in 2005 (16.7%, 7/42) (p = 0.02). There were 87 (78.4%) mutations of these 111 LFX-resistant isolates. In addition, a significant difference in proportion was observed in the isolates with mutations in codon 90 of the gyrA gene between 2005 and 2015 (11.9% in 2005 versus 29.0% in 2015, p = 0.04). CONCLUSIONS: There was an alarming increase in prevalence of LFX-resistant TB in China between 2005 and 2015. This dynamic change is mostly attributed to the increase in high-level LFX resistance. Moreover, a significant difference was noted in the proportion of LFX-resistant isolates harboring specific mutations within the gyrA gene between 2005 and 2015.

Accuracy of Xpert MTB/RIF Ultra for the Diagnosis of Pleural TB in a Multicenter Cohort Study.

BACKGROUND: The Xpert MTB/RIF (Xpert) assay has greatly improved the diagnosis of TB and identification of resistance to rifampicin (RIF). However, sensitivity of Xpert remains poor for pleural fluid detection. This study evaluated the performance of the novel next-generation Xpert MTB/RIF Ultra (Xpert Ultra) in comparison with Xpert for pleural TB diagnosis. METHODS: Patients with suspected pleural TB were enrolled consecutively in four hospitals, and pleural fluids were subjected to smear, culture, and Xpert. Defrosted pleural fluid (-80°C) was examined using Xpert Ultra. Drug susceptibility testing (DST) was conducted for all of the recovered isolates. RESULTS: In total, 317 individuals with suspected pleural TB were recruited; 208 of them were diagnosed with pleural TB according to the composite reference standard, which was composed of clinical, laboratory, histopathologic, and radiologic examination features and ≥ 12 months of follow-up data. The direct head-to-head comparison for Mycobacterium tuberculosis detection showed that Xpert Ultra (44.23%, 92 of 208) produced a higher sensitivity than culture (26.44%, 55 of 208, P < .001), Xpert (19.23%, 40 of 208, P < .001), and smear (1.44%, three of 208, P < .001). When Xpert Ultra outcomes were integrated, the percentage of definite pleural TB cases increased from 56.25% (117 of 208) to 64.90% (135 of 208). The specificities of smear, culture, Xpert, and Xpert Ultra were 100% (84 of 84), 100% (84 of 84), 98.67% (83 of 84), and 98.67% (83 of 84), respectively. Xpert Ultra was 100% concordant with phenotype DST for the detection of RIF resistance. CONCLUSIONS: Xpert Ultra has great potential in diagnosis of pleural TB and its RIF resistance, which could speed up the initiation of appropriate treatment.

In Vitro Susceptibility Testing of GSK656 against Mycobacterium Species.

In this study, we aimed to assess the in vitro susceptibility to GSK656 among multiple mycobacterial species and to investigate the correlation between leucyl-tRNA synthetase (LeuRS) sequence variations and in vitro susceptibility to GSK656 among mycobacterial species. A total of 187 mycobacterial isolates, comprising 105 Mycobacterium tuberculosis isolates and 82 nontuberculous mycobacteria (NTM) isolates, were randomly selected for the determination of in vitro susceptibility. For M. tuberculosis, 102 of 105 isolates had MICs of ≤0.5 mg/liter, demonstrating a MIC50 of 0.063 mg/liter and a MIC90 of 0.25 mg/liter. An epidemiological cutoff value of 0.5 mg/liter was proposed for identification of GSK656-resistant M. tuberculosis strains. For NTM, the MIC50 and MIC90 values were >8.0 mg/liter for both Mycobacterium intracellulare and Mycobacterium avium In contrast, all Mycobacterium abscessus isolates had MICs of ≤0.25 mg/liter, yielding a MIC90 of 0.063 mg/liter. LeuRS from M. abscessus showed greater sequence similarity to M. tuberculosis LeuRS than to LeuRSs from M. avium and M. intracellulare Sequence alignment revealed 28 residues differing between LeuRSs from M. avium and M. intracellulare and LeuRSs from M. tuberculosis and M. abscessus; among them, 15 residues were in the drug binding domain. Structure modeling revealed that several different residues were close to the tRNA-LeuRS interface or the entrance of the drug-tRNA binding pocket. In conclusion, our data demonstrate significant species diversity in in vitro susceptibility to GSK656 among various mycobacterial species. GSK656 has potent efficacy against M. tuberculosis and M. abscessus, whereas inherent resistance was noted for M. intracellulare and M. avium.