Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly. In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1–26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis. The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Background: Bedaquiline (BDQ) has been designated as a Group A drug by the World Health Organization (WHO) for the management of multi-drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). This systematic review and meta-analysis aim to evaluate the efficacy and safety of BDQ-containing regimens for the treatment of patients with pulmonary TB. Methods: PubMed (MEDLINE), Elton B. Stephens Company (EBSCO) database, the Cochrane Register of Controlled Trials, and the China National Knowledge Infrastructure (CNKI) database were initially searched on 15 June 2022 and again on 20 March 2023. We included randomized controlled trials (RCTs) and non-randomized studies (NRSs) that administered BDQ to TB patients. The outcomes of interest were as follows: (1) efficacy, including the rate of sputum culture conversion at 8 weeks, 24 weeks, and during follow-up, as well as the rates of completion cure, death, treatment failure, and loss at follow-up and at the end of the treatment; and (2) safety, which encompassed the incidences of cardiotoxicity, hepatotoxicity, and grade 3–5 adverse events during the treatment period. Results: A total of 29 articles were included in this meta-analysis, representing 23,358 individuals. Patients who were treated with BDQ were compared with patients who were not exposed to BDQ. The use of BDQ-containing regimens demonstrated improved rates of sputum conversion in RCTs at 24 weeks (RR = 1.27, 95% CI: 1.10 to 1.46) and during follow-up (RR = 1.33, 95% CI: 1.06 to 1.66). Additionally, BDQ-containing regimens showed increased cure rates (RR = 1.60, 95% CI: 1.13 to 2.26) and decreased failure rates (RR = 0.56, 95% CI: 0.56 to 0.88). In NRSs, BDQ-containing regimens improved the sputum culture conversion rate during follow-up (RR = 1.53, 95% CI: 1.07 to 2.20), increased the rate of cure (RR = 1.86, 95% CI: 1.23 to 2.83), reduced deaths from all causes (RR = 0.68, 95% CI: 0.48 to 0.97), and reduced failure rates (RR = 0.57, 95% CI: 0.46 to 0.71). However, the use of BDQ-containing regimens was associated with increased incidences of cardiotoxicity (RR = 4.54, 95% CI: 1.74 to 11.87) and grade 3–5 adverse events (RR = 1.42, 95% CI: 1.17 to 1.73) in RCTs. NRSs also showed an association between BDQ-containing regimens and cardiotoxicity (RR = 6.00, 95% CI: 1.32 to 27.19). No significant differences were observed between intervention groups and control groups with respect to other outcomes. Conclusions: Data from both RCTs and NRSs support the efficacy of BDQ for the treatment of pulmonary tuberculosis. However, the use of BDQ is associated with a higher incidence of cardiotoxicity and serious adverse events. Comparative data on efficacy and safety are limited, and further confirmation is required, due to potential bias and discrepancies in the available studies.

Pulmonary tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB). Whole-genome sequencing (WGS) holds great promise as an advanced technology for accurately predicting anti-TB drug resistance. The development of a reliable method for detecting drug resistance is crucial in order to standardize anti-TB treatments, enhance patient prognosis, and effectively reduce the risk of transmission. In this study, our primary objective was to explore and determine the potential of WGS for assessing drug resistance based on genetic variants recommended by the World Health Organization (WHO). A total of 1105 MTB strains were selected from samples collected from 2014–2018 in Zhejiang Province, China. Phenotypic drug sensitivity tests (DST) of the anti-TB drugs were conducted for isoniazid (INH), rifampicin (RFP), streptomycin, ethambutol, fluoroquinolones (levofloxacin and moxifloxacin), amikacin, kanamycin, and capreomycin, and the drug-resistance rates were calculated. The clean WGS data of the 1105 strains were acquired and analyzed. The predictive performance of WGS was evaluated by the comparison between genotypic and phenotypic DST results. For all anti-TB drugs, WGS achieved good specificity values (>90%). The sensitivity values for INH and RFP were 91.78% and 82.26%, respectively; however, they were ≤60% for other drugs. The positive predictive values for anti-TB drugs were >80%, except for ethambutol and moxifloxacin, and the negative predictive values were >90% for all drugs. In light of the findings from our study, we draw the conclusion that WGS is a valuable tool for identifying genome-wide variants. Leveraging the genetic variants recommended by the WHO, WGS proves to be effective in detecting resistance to RFP and INH, enabling the identification of multi-drug resistant TB patients. However, it is evident that the genetic variants recommended for predicting resistance to other anti-TB drugs require further optimization and improvement.

Background: The accurate identification of the Mycobacterium tuberculosis complex (MTBC) and different nontuberculous mycobacteria (NTM) species is crucial for the timely diagnosis of NTM infections and for reducing poor prognoses. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been extensively used for microbial identification with high accuracy and throughput. However, its efficacy for Mycobacterium species identification has been less studied. The objective of this study was to evaluate the performance of nucleotide MALDI-TOF-MS for Mycobacterium species identification.Methods: A total of 933 clinical Mycobacterium isolates were preliminarily identified as NTM by the MPB64 test. These isolates were identified by nucleotide MALDI-TOF-MS and Sanger sequencing. The performance of nucleotide MALDI-TOF MS for identifying various Mycobacterium species was analyzed based on Sanger sequencing as the gold standard.Results: The total correct detection rate of all 933 clinical Mycobacterium isolates using nucleotide MALDI-TOF-MS was 91.64% (855/933), and mixed infections were detected in 18.65% (174/933) of the samples. The correct detection rates for Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium avium, MTBC, Mycobacterium gordonae, and Mycobacterium massiliense were 99.32% (585/589), 100% (86/86), 98.46% (64/65), 94.59% (35/37), 100.00% (34/34), 95.65% (22/23), and 100% (19/19), respectively. For the identification of the MTBC, M. intracellulare, M. abscessus, M. kansasii, M. avium, M. gordonae, and M. massiliense, nucleotide MALDI-TOF-MS and Sanger sequencing results were in good agreement (k > 0.7).Conclusion: In conclusion, nucleotide MALDI-TOF-MS is a promising approach for identifying MTBC and the most common clinical NTM species.

Background: Pulmonary tuberculosis (PTB) is a severe infection, and its factors that influence survival have not been explored in some developed regions. Therefore, this study aimed to identify the factors that influence TB survival in China. Methods: We acquired data of PTB deaths through matching two manage systems of PTB and mortality surveillance. The Kaplan–Meier method and Cox regression model were used to identify the significant factors under various survival times. Results: A total of 283 deaths were caused by PTB, in which 53.4% occurred during the first year. In the Cox regression model, regular sputum smear test could reduce the risk of PTB death from the result of one-, three-, and five-year survival; and history of PTB treatment could decline the risk of PTB death in the first year. Additionally, age of “ 75– 90 years” and positive result of sputum smear test significantly increased the risk of PTB death for the findings of three- and five-year survival, while age of “ 60– 75 years” could increase the probability of PTB death for five-year survival. Conclusion: To reduce PTB deaths, more attention should be paid to the older group especially in the first year. Regular sputum smear test could effectively reduce the mortality of PTB cases. More evidence-based interventions should be implemented to enhance the survival of patients with PTB.

Purpose: This study aimed to determine the prevalence and molecular characterization of bedaquiline (BDQ) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates collected from Zhejiang, China. Patients and Methods: A total of 245 RR-TB isolates were collected from 19 municipal TB hospitals in Zhejiang province, China between January and December 2021. Microplate assays were used to determine the minimum inhibitory concentrations (MIC) of BDQ. Whole-genome sequencing (WGS) was performed on isolates with MIC values for BDQ ≥ 0.25 μg/mL. Results: Five (2.04%) BDQ-resistant strains were isolated from 245 tuberculosis patients. The resistance rate of BDQ was not correlated to the sex, age, treatment history, or occupation of patients. Four BDQ-resistant isolates and three BDQ-sensitive isolates were found to carry Rv0678 mutations, and one BDQ-resistant strain carried both Rv0678 and pepQ mutations. No mutations within the atpE and Rv1979c genes were observed. Conclusion: BDQ demonstrated strong in vitro antibacterial activity against RR-TB isolates, and the Rv0678 gene was identified as the primary mechanism contributing to BDQ resistance among RR-TB isolates from Zhejiang, China. Furthermore, in addition to the four currently known resistance-associated genes (atpE, Rv0678, Rv1979c, and pepQ), other mechanisms of resistance to BDQ may exist that need further study. Plain Language Summary: This study looked at a bacterial species called Mycobacterium tuberculosis, which causes the highly problematic disease, tuberculosis. Certain strains of this bacterium have developed resistance to conventional antibiotics used in tuberculosis treatment, necessitating an investigation into the efficacy of the newer antibiotic, bedaquiline. We collected 245 rifampicin-resistant tuberculosis samples from patients in Zhejiang, China, subjecting them to bedaquiline susceptibility testing. Concurrently, we conducted a genetic analysis of the bacteria to pinpoint mutations linked to bedaquiline resistance. Out of the 245 samples, 5 were found to be resistant to bedaquiline. We found that mutations in a gene called Rv0678 were the main reason for this resistance. This gene had mutations in four of the bedaquiline-resistant samples and three of the bedaquiline-susceptible samples. One of the bedaquiline-resistant samples had mutations in both Rv0678 and another gene called pepQ. We also found that bedaquiline was effective at killing drug-resistant tuberculosis bacteria in the lab. However, there may be other genes or mechanisms that make other bacteria resistant to the drug, which will need further study. Overall, this study helps us understand how bedaquiline works against drug-resistant tuberculosis bacteria and identifies a genetic mechanism that can cause resistance to the drug.