Hui Xia

To evaluate the performance of the iFIND INH/FQ, a low-complexity molecular assay, for the rapid and simultaneous detection of resistance to isoniazid (INH) and fluoroquinolones (FQs) in Mycobacterium tuberculosis . Frozen sputum specimens stored at the Chengde Center for Disease Control and Prevention laboratory were used. Phenotypic drug susceptibility testing (pDST) and DNA sequencing served as reference standards. The limit of detection (LOD) was determined using probit regression with spiked samples. The assay’s ability to detect resistance-conferring mutations in katG , inhA , and gyrA genes was assessed using genotypically characterized strains. Diagnostic accuracy was evaluated against pDST. The LOD was 20.79 CFU/mL for INH and 9.34 CFU/mL for FQs. The assay detected all targeted mutations except ahpC c.-6 associated with INH resistance. Compared to pDST, the iFIND INH/FQ assay demonstrated a sensitivity of 97.59% (95% confidence interval [CI]: 91.63–99.34%) and specificity of 98.10% (95% CI: 94.57–99.35%) for INH resistance. For FQ resistance, sensitivity was 92.16% (95% CI: 81.50–96.91%) for levofloxacin and 92.00% (95% CI: 81.16–96.85%) for moxifloxacin, with specificities of 97.33% (95% CI: 93.89–98.85%) and 96.81% (95% CI: 93.21–98.53%), respectively. Sequencing confirmed iFIND results in the majority of discrepant cases (100% for INH and 55.65% for FQs). The iFIND INH/FQ LC-aNAAT is a highly accurate and rapid molecular assay for simultaneous detection of INH and FQ resistance. It is a promising tool for scaling up rapid drug susceptibility testing in clinical and peripheral laboratory settings. IMPORTANCE: As a low-complexity automated nucleic acid amplification test, the iFIND assay achieves the goal of simultaneously detecting isoniazid and fluoroquinolone resistance in approximately 90 min, perfectly meeting the TPP's core requirements for “rapid” and “simple operation.” Its fully integrated system minimizes manual steps and contamination risk, making it highly suitable for use in resource-limited, lower-biosafety-level primary laboratories.

Objective: Given the increase of treatment failure, relapse and acquired resistance observed in isoniazid (INH) resistance, there is an urgent to improve rifampin (RIF) -priority based diagnostic strategies. Therefore, we evaluated the performance of Innovo GenMax MTB-RIF/INH (GenMax), a moderate- complexity automated nucleic acid amplification test (NAAT), for detecting Mycobacterium tuberculosis complex (MTBC) and resistance to RIF and INH.Methods: Analytical sensitivity (limit of detection, LOD) was determined using serial dilutions of Mycobacterium tuberculosis H37Rv (ATCC 27249) strains. Diagnostic accuracy was assessed in clinical sputum specimens against microbiological reference standards (MRS: positive by smear microscopy, culture or Xpert MTB/RIF for diagnosis of TB) and phenotypic drug susceptibility testing (DST). Discordant results were resolved by sequencing resistance genes (IS6110, rpoB, katG, inhA, ahpC) and follow-up diagnosis results.Results: GenMax demonstrated a calculated LOD of 8.8 CFU/mL (95% CI: 7.4-11.4) for MTBC, 674.1 CFU/mL (95% CI: 578.8-923.5) for RIF resistance, and 747.3 CFU/mL (95% CI: 613.7-1081.3) for INH resistance. In clinical evaluation, the sensitivity and specificity for MTBC detection were 97.52% (95% CI: 92.38–99.36) and 93.65% (95% CI: 88.91–96.53), respectively. For RIF and INH resistance, sensitivities were 88.46% (95% CI: 68.72–96.97) and 85.19% (95% CI: 65.39–95.14), with specificity of 92.42% (95% CI: 82.50-97.18) and 94.12% (95% CI: 84.86-98.10).Conclusion: Innovo GenMax MTB-RIF/INH is a rapid and automated assay with high sensitivity for MTBC detection, suitable for decentralized settings. While its performance for RIF/INH resistance detection is competitive with existing assays, its sensitivity remains gaps relative to WHO targets. Further optimization, particularly through expanded probe coverage, is needed to bridge this gap and ensure reliable detection in clinical settings.

Objectives: To investigate epidemiological/drug-resistance characteristics and identify potential factors related to drug-resistant and clustered tuberculosis in Sichuan. Methods: A total of 295 Mycobacterium tuberculosis (MTB) isolates were collected from surveillance sites in Sichuan from 2019 to 2021. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole-genome sequencing (WGS) analysis. Results: Of 268 MTB isolates with both WGS and drug-susceptibility testing (DST) results, 159 (59.3%, 159/268) strains belonged to the Beijing lineage (L2). Isoniazid had the highest resistance rate (15.3%, 41/268), followed by rifampicin (9.3%, 25/268). The sensitivity of WGS to predict drug resistance varied from 75% to 97.6%, and the specificity was above 96.0% for all. rpoB Ser450Leu (41.7%, 10/24) and katG Ser315Thr (70%, 28/40) were the most frequent mutations in rifampicin and isoniazid resistance isolates, respectively. The clustering rate in Sichuan was 9.3% (25/268), and patients ≤ 24 years old (aOR = 11.697; 95% CI: 0.817–167.463) had a greater risk of clustering. Conclusions: Our findings prove that WGS is a promising tool for predicting drug resistance to isoniazid, rifampicin, ethambutol, moxifloxacin and levofloxacin in Sichuan. The higher resistance rate to isoniazid emphasizes the urgent need for susceptibility testing surveillance and application management. Improving the diagnosis, treatment and management of patients ≤ 24 years old may reduce the transmission of MTB in Sichuan.

To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.

Mycobacterial membrane proteins play a pivotal role in the bacterial invasion of host cells; however, the precise mechanisms underlying certain membrane proteins remain elusive. Mycolicibacterium smegmatis (Ms) msmeg5257 is a hemolysin III family protein that is homologous to Mycobacterium tuberculosis (Mtb) Rv1085c, but it has an unclear function in growth. To address this issue, we utilized the CRISPR/Cas9 gene editor to construct Δmsmeg5257 strains and combined RNA transcription and LC-MS/MS protein profiling to determine the functional role of msmeg5257 in Ms growth. The correlative analysis showed that the deletion of msmeg5257 inhibits ABC transporters in the cytomembrane and inhibits the biosynthesis of amino acids in the cell wall. Corresponding to these results, we confirmed that MSMEG5257 localizes in the cytomembrane via subcellular fractionation and also plays a role in facilitating the transport of iron ions in environments with low iron levels. Our data provide insights that msmeg5257 plays a role in maintaining Ms metabolic homeostasis, and the deletion of msmeg5257 significantly impacts the growth rate of Ms. Furthermore, msmeg5257, a promising drug target, offers a direction for the development of novel therapeutic strategies against mycobacterial diseases.

Early diagnosis of drug susceptibility for tuberculosis (TB) patients could guide the timely initiation of effective treatment. We evaluated a novel multiplex xMAP TIER (Tuberculosis-Isoniazid-Ethambutol-Rifampicin) assay based on the Luminex xMAP system to detect first-line anti-tuberculous drug resistance. Deoxyribonucleic acid samples from 353 Mycobacterium tuberculosis clinical isolates were amplified by multiplex polymerase chain reaction, followed by hybridization and analysis through the xMAP system. Compared with the broth microdilution method, the sensitivity and specificity of the xMAP TIER assay for detecting resistance was 94.9% (95%CI, 90.0–99.8%) and 98.9% (95%CI, 97.7–100.0%) for rifampicin; 89.1% (95%CI, 83.9–94.3%) and 100.0% (95%CI, 100.0–100.0%) for isoniazid; 82.1% (95% CI, 68.0–96.3%) and 99.7% (95% CI, 99.0–100.0%) for ethambutol. With DNA sequencing as the reference standard, the sensitivity and specificity of xMAP TIER for detecting resistance were 95.0% (95% CI, 90.2–99.8%) and 99.6% (95% CI, 98.9–100.0%) for rifampicin; 96.9% (95% CI, 93.8–99.9%) and 100.0% (95% CI, 100.0–100.0%) for isoniazid; 86.1% (95% CI, 74.8–97.4%) and 100.0% (95% CI, 100.0–100.0%) for ethambutol. The results achieved showed that the xMAP TIER assay had good performance for detecting first-line anti-tuberculosis drug resistance, and it has the potential to diagnose drug-resistant tuberculosis more accurately due to the addition of more optimal design primers and probes on open architecture xMAP system.

Mycobacterium abscessus (M. abscessus) and Mycobacterium massiliense (M. massiliense) are major pathogens that cause post-surgical wound infection and chronic pulmonary disease. Although they are closely related subspecies of M. abscessus complex, their infections are associated with different drug-resistance and cure rate. In the present study, a loop-mediated isothermal amplification (LAMP) coupled with lateral flow dipstick (LFD) method was developed to simultaneous detect M. abscessus and M. massiliense, via specific erm(41) gene. The amplification was carried out at 65 °C for only 60 min, and the results could be visualized on a lateral flow strip. Positive results only occurred in M. abscessus and M. massiliense, no cross-reaction with other mycobacterial species was observed. Therefore, the cost-effective MABC (M. abscessus complex)–LAMP–LFD method developed here was able to correct the diagnose of M. abscessus and M. massiliense infection in a short time. Thus, this method could be used to guide clinicians in treatment of M. abscessus group infections.