Efflux pump effects on levofloxacin resistance in Mycobacterium abscessus.

Mycobacterium abscessus (M. abscessus) inherently displays resistance to most antibiotics, with the underlying drug resistance mechanisms remaining largely unexplored. Efflux pump is believed to play an important role in mediating drug resistance. The current study examined the potential of efflux pump inhibitors to reverse levofloxacin (LFX) resistance in M. abscessus. The reference strain of M. abscessus (ATCC19977) and 60 clinical isolates, including 41 M. abscessus subsp. abscessus and 19 M. abscessus subsp. massilense, were investigated. The drug sensitivity of M. abscessus against LFX alone or in conjunction with efflux pump inhibitors, including verapamil (VP), reserpine (RSP), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or dicyclohexylcarbodiimide (DCC), were determined by AlarmarBlue microplate assay. Drug-resistant regions of the gyrA and gyrB genes from the drug-resistant strains were sequenced. The transcription level of the efflux pump genes was monitored using qRT-PCR. All the tested strains were resistant to LFX. The drug-resistant regions from the gyrA and gyrB genes showed no mutation associated with LFX resistance. CCCP, DCC, VP, and RSP increased the susceptibility of 93.3% (56/60), 91.7% (55/60), 85% (51/60), and 83.3% (50/60) isolates to LFX by 2 to 32-fold, respectively. Elevated transcription of seven efflux pump genes was observed in isolates with a high reduction in LFX MIC values in the presence of efflux pump inhibitors. Efflux pump inhibitors can improve the antibacterial activity of LFX against M. abscessus in vitro. The overexpression of efflux-related genes in LFX-resistant isolates suggests that efflux pumps are associated with the development of LFX resistance in M. abscessus.

Mycobacterium vicinigordonae sp. nov., a slow-growing scotochromogenic species isolated from sputum.

A slow-growing, scotochromogenic mycobacterial strain (24T) was isolated from the sputum of a Chinese male human. Phylogenetic analysis using the 16S rRNA gene assigned strain 24T to the Mycobacterium gordonae complex, which includes Mycobacterium gordonae and Mycobacterium paragordonae. The phenotypic characteristics, unique mycolic acid profile and the results of phylogenetic analysis based on hsp65 and rpoB sequences strongly supported the taxonomic status of strain 24T as a representative of a species distinct from the other members of the M. gordonae complex. The genomic G+C content of strain 24T was 65.40mol%. Genomic comparisons showed that strain 24T and M. gordonae ATCC 14470T had an average nucleotide identity (ANI) value of 81.00 % and a DNA-DNA hybridization (DDH) value of 22.80 %, while the ANI and DDH values between strain 24Tand M. paragordonae 49 061T were 80.98 and 22.80 %, respectively. In terms of phylogenetic, phenotypic and chemotaxonomic features, strain 24T is distinguishable from its closest phylogenetic relatives and represents a novel species of the genus Mycobacterium, therefore the name Mycobacterium vicinigordonae sp. nov. is proposed. The type strain is 24T (=CMCC 93559T=DSM 105979T).

External quality control of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China.

The aim of our study was to evaluate the performance of conventional drug susceptibility testing (DST) among the tuberculosis (TB)-specialized hospitals in China. A total of 40 hospitals participated in the external quality assurance program for assessment of DST results from each hospital. The sensitivity, specificity, and accuracy of DST were analyzed. The mean accuracy was 96.5% for isoniazid (INH), 95.8% for rifampin (RIF), 97.0% for ethambutol (EMB), 96.8% for ofloxacin (OFX), 97.1% for kanamycin (KAN), 96.1% for amikacin (AMK), and 93.6% for capreomycin (CAP), respectively. Of the 40 participating laboratories, 4 (10.0%) and 6 (15%) failed to achieve 90% accuracy for INH and RIF, respectively. In addition, six hospitals (15%) were confirmed as certified to provide reliable DST results for both first-line and second-line drugs. The certified proportion for DST dropped from 73.9% in the non-western region to 59.2% in the western region. The significant difference was observed in the certified proportion for first-line drugs between the western and non-western region (P = 0.013). Our results demonstrate that the quality of phenotypical DST is frequently unsatisfactory, with approximately one-third of participated laboratories failing to produce certified phenotypical DST results. In addition, the uncertified laboratories majorly come from the western region in China.

Cross-sectional Whole-genome Sequencing and Epidemiological Study of Multidrug-resistant Mycobacterium tuberculosis in China.

BACKGROUND: The increase in multidrug-resistant tuberculosis (MDR-TB) severely hampers tuberculosis prevention and control in China, a country with the second highest MDR-TB burden globally. The first nationwide drug-resistant tuberculosis surveillance program provides an opportunity to comprehensively investigate the epidemiological/drug-resistance characteristics, potential drug-resistance mutations, and effective population changes of Chinese MDR-TB. METHODS: We sequenced 357 MDR strains from 4600 representative tuberculosis-positive sputum samples collected during the survey (70 counties in 31 provinces). Drug-susceptibility testing was performed using 18 anti-tuberculosis drugs, representing the most comprehensive drug-resistance profile to date. We used 3 statistical and 1 machine-learning methods to identify drug-resistance genes/single-nucleotide polymorphisms (SNPs). We used Bayesian skyline analysis to investigate changes in effective population size. RESULTS: Epidemiological/drug-resistance characteristics showed different MDR profiles, co-resistance patterns, preferred drug combination/use, and recommended regimens among 7 Chinese administrative regions. These factors not only reflected the serious multidrug co-resistance and drug misuse but they were also potentially significant in facilitating the development of appropriate regimens for MDR-TB treatment in China. Further investigation identified 86 drug-resistance genes/intergenic regions/SNPs (58 new), providing potential targets for MDR-TB diagnosis and treatment. In addition, the effective population of Chinese MDR-TB displayed a strong expansion during 1993-2000, reflecting socioeconomic transition within the country. The phenomenon of expansion was restrained after 2000, likely attributable to the advances in diagnosis/treatment technologies and government support. CONCLUSIONS: Our findings provide an important reference and improved understanding of MDR-TB in China, which are potentially significant in achieving the goal of precision medicine with respect to MDR-TB prevention and treatment.

In Vitro Activity of Clofazimine against Nontuberculous Mycobacteria Isolated in Beijing, China.

Due to the natural resistance of nontuberculous mycobacteria (NTM) to many antibiotics, the treatment of diseases caused by NTM is often long-term but unsuccessful. The main goal of this study was to evaluate the in vitro susceptibilities to clofazimine of 209 isolates consisting of different NTM species isolated in Beijing, China. Furthermore, 47 reference strains were also tested, including 30 rapidly growing mycobacterium (RGM) species and 17 slowly growing mycobacterium (SGM) species. The potential molecular mechanism contributing to clofazimine resistance of NTM was investigated as well. Clofazimine exhibited excellent activity against both reference strains and clinical isolates of different SGM species, and most of the strains had MICs far below 1 µg/ml. Although the majority of the clinical isolates of Mycobacterium abscessus and Mycobacterium fortuitum had MICs higher than 2 µg/ml, 17 out of the 30 reference strains of different RGM species had MICs below 1 µg/ml in vitro According to the MIC distributions, the tentative epidemiological cutoff (ECOFF) values for Mycobacterium kansasii, Mycobacterium avium, and Mycobacterium intracellulare were defined at 0.5 µg/ml, 1 µg/ml, and 2 µg/ml, respectively. Intriguingly, single-direction cross-resistance between bedaquiline- and clofazimine (Cfz)-resistant isolates was observed among the tested NTM species. This study demonstrates that clofazimine had strong activity against most SGM species in vitro, as well as some RGM species. The data provide important insights into the possible clinical application of Cfz to treat NTM infections.

National survey of drug-resistant tuberculosis in China.

BACKGROUND: The available information on the epidemic of drug-resistant tuberculosis in China is based on local or regional surveys. In 2007, we carried out a national survey of drug-resistant tuberculosis in China. METHODS: We estimated the proportion of tuberculosis cases in China that were resistant to drugs by means of cluster-randomized sampling of tuberculosis cases in the public health system and testing for resistance to the first-line antituberculosis drugs isoniazid, rifampin, ethambutol, and streptomycin and the second-line drugs ofloxacin and kanamycin. We used the results from this survey and published estimates of the incidence of tuberculosis to estimate the incidence of drug-resistant tuberculosis. Information from patient interviews was used to identify factors linked to drug resistance. RESULTS: Among 3037 patients with new cases of tuberculosis and 892 with previously treated cases, 5.7% (95% confidence interval [CI], 4.5 to 7.0) and 25.6% (95% CI, 21.5 to 29.8), respectively, had multidrug-resistant (MDR) tuberculosis (defined as disease that was resistant to at least isoniazid and rifampin). Among all patients with tuberculosis, approximately 1 of 4 had disease that was resistant to isoniazid, rifampin, or both, and 1 of 10 had MDR tuberculosis. Approximately 8% of the patients with MDR tuberculosis had extensively drug-resistant (XDR) tuberculosis (defined as disease that was resistant to at least isoniazid, rifampin, ofloxacin, and kanamycin). In 2007, there were 110,000 incident cases (95% CI, 97,000 to 130,000) of MDR tuberculosis and 8200 incident cases (95% CI, 7200 to 9700) of XDR tuberculosis. Most cases of MDR and XDR tuberculosis resulted from primary transmission. Patients with multiple previous treatments who had received their last treatment in a tuberculosis hospital had the highest risk of MDR tuberculosis (adjusted odds ratio, 13.3; 95% CI, 3.9 to 46.0). Among 226 previously treated patients with MDR tuberculosis, 43.8% had not completed their last treatment; most had been treated in the hospital system. Among those who had completed treatment, tuberculosis developed again in most of the patients after their treatment in the public health system. CONCLUSIONS: China has a serious epidemic of drug-resistant tuberculosis. MDR tuberculosis is linked to inadequate treatment in both the public health system and the hospital system, especially tuberculosis hospitals; however, primary transmission accounts for most cases. (Funded by the Chinese Ministry of Health.).

Apramycin has high in vitro activity against Mycobacterium tuberculosis.

Introduction. Aminoglycoside antibiotics such as amikacin and kanamycin are important components in the treatment of Mycobacterium tuberculosis (Mtb) infection. However, more and more clinical strains are found to be aminoglycoside antibiotic-resistant. Apramycin is another kind of aminoglycoside antibiotic that is commonly used to treat infections in animals.Hypothesis. Apramycin may have in vitro activity against Mtb.Aim. This study aims to evaluate the efficacy of apramycin against Mtb in vitro and determine its epidemiological cut-off (ECOFF) value.Methodology. One hundred Mtb isolates, including 17 pansusceptible and 83 drug-resistant tuberculosis (DR-TB) strains, were analysed for apramycin resistance using the MIC assay.Results. Apramycin exhibited significant inhibitory activity against Mtb clinical isolates, with an MIC50 of 0.5 µg ml-1 and an MIC90 of 1 µg ml-1. We determined the tentative ECOFF value as 1 µg ml-1 for apramycin. The resistant rates of multidrug-resistant tuberculosis (MDR-TB), pre-extensively drug-resistant (pre-XDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) strains were 12.12 % (4/33), 20.69 % (6/29) and 66.67 % (14/21), respectively. The rrs gene A1401G is associated with apramycin resistance, as well as the cross-resistance between apramycin and other aminoglycosides.Conclusion. Apramycin shows high in vitro activity against the Mtb clinical isolates, especially the MDR-TB clinical isolates. This encouraging discovery calls for more research on the functions of apramycin in vivo and as a possible antibiotic for the treatment of drug-resistant TB.

Discordant results between Xpert MTB/RIF assay and Bactec MGIT 960 culture system regarding the detection of rifampin-resistant Mycobacterium tuberculosis isolates in Wenzhou, China.

This study aimed to assess the possible causes of discordant results between Xpert MTB/RIF (Xpert) and Bactec MGIT 960 Culture System (MGIT960) regarding rifampicin (RIF) susceptibility in Mycobacterium tuberculosis. Patients with previous RIF-resistant tuberculosis who were admitted to Wenzhou Central Hospital from January 2020 to December 2022 were enrolled. The isolates obtained from these patients were subjected to RIF susceptibility tests using Xpert and MGIT960, and the minimum inhibitory concentration (MIC) of RIF was determined by the MYCOTB MIC plate test. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to evaluate the binding efficacy of rpoB and RIF based on rpoB mutations detected in the isolates with discordant RIF susceptibility results. A total of 28 isolates with discordant RIF susceptibility test results were detected, 15 of them were RIF susceptible with MICs ≤ 0.5 µg/mL. Twelve out of 15 isolates contained borderline RIF resistance-associated mutations [L430P (n = 6), H445N (n = 6)], 1 isolate had D435Y and Q429H double mutation, and the remaining 2 isolates had a silent (Q432Q) mutation. Compared with the affinity of RIF toward the wild type (WT) (-45.83 kcal/mol) by MD, its affinity toward L452P (-55.52 kcal/mol), D435Y (-47.39 kcal/mol), L430P (approximately -69.72 kcal/mol), H445N (-49.53 kcal/mol), and Q429H (-55.67 kcal/mol) increased. Borderline RIF resistance-associated mutations were the main cause for the discordant RIF susceptibility results between Xpert and MGIT960, and the mechanisms of the resistance need further investigated.IMPORTANCEThis study is aimed at assessing discordant results between Xpert MTB/RIF (Xpert) assay and Bactec MGIT 960 Culture System (MGIT960) regarding the detection of rifampicin (RIF)-resistant Mycobacterium tuberculosis isolates in Wenzhou, China. The discordant results of RIF between these two assays were mainly caused by borderline RIF resistance-associated mutations, subsequently by silent mutations of rpoB. Borderline RIF resistance- associated mutations detected in our study were demonstrated to not be affected by the affinity of rpoB and RIF by molecular dynamics, and the mechanism of resistance was needed to be clarified. For the discordant results of RIF by Xpert and MGIT960 that occurred, rpoB DNA sequencing was recommended to investigate its association with resistance to RIF.

An early and trustable indicator suggestive of non-tuberculosis mycobacteria isolation in a high tuberculosis burden setting.

BACKGROUND: Distinguishing between nontuberculous mycobacterial (NTM) lung infections and pulmonary tuberculosis becomes challenging due to their similar clinical manifestations and radiological images. Consequently, instances of delayed diagnosis or misdiagnosis are highly frequent. A feasible and reliable indicator of the existence of NTM in the early stages of the disease would help to solve this dilemma. METHODS: In this study, we evaluated the potential of smear-positive and Xpert assay (Cepheid, USA) negative outcomes as an early indicator of possible NTM infection in a high TB-burden setting retrospectively and prospectively. RESULTS: During the study period, 12·77% (138/1081) of the smear-positive cases yielded negative outcomes with the simultaneous Xpert assay. From the 110 patients who yielded smear-positive/Xpert-negative outcomes and cultivated strain as well, 105 (95·45%) were proved to have NTM isolated. By incorporating an additional criterion of a negative result from the Interferon-gamma release assay, the accuracy of the screening method reached 100%. Regarding the NTM presence prediction value, smear-positive/Xpert-negative has a sensitivity of 24·86% (45/181) in all NTM isolated cases but 93·75-96·55% accuracy in retrospective study or 93·75% accuracy in prospective study in smear-positive NTM isolated cases. In addition, the specificity was ∼99·47% (943/948) in smear-positive tuberculosis cases. CONCLUSION: The clue of the presence of NTM could be obtained on the first day of the hospital visit due to the point of care (POC) feature of smear testing and Xpert assay. About one-fourth of the NTM-isolated patients would benefit from this rapid, convenient, and reliable screening strategy in the given circumstance. Smear-positive/Xpert-negative outcome is an early, trustable indicator that is indicative of NTM isolation.

New generation fluoroquinolone sitafloxacin could potentially overcome the majority levofloxacin and moxifloxacin resistance in multidrug-resistant Mycobacterium tuberculosis.

Introduction. Pre-existing fluoroquinolones (FQs) resistance is a major threat in treating multidrug-resistant (MDR) tuberculosis. Sitafloxacin (Sfx) is a new broad-spectrum FQ.Hypothesis. Sfx is more active against drug-resistant Mycobacterium tuberculosis (Mtb) isolates.Aim. To determine whether there is cross-resistance between Sfx and ofloxacin (Ofx), levofloxacin (Lfx) and moxifloxacin (Mfx) in MDR Mtb.Methods. A total of 106 clinical Mtb isolates, including 23 pan-susceptible and 83 MDR strains, were analysed for Sfx, Lfx and Mfx resistance using MIC assay. The isolates were also subjected to whole-genome sequencing to analyse drug-resistant genes.Results. Sfx exhibited the most robust inhibition activity against Mtb clinical isolates, with a MIC50 of 0.0313 µg ml-1 and MIC90 of 0.125 µg ml-1, which was lower than that of Mfx (MIC50 = 0.0625 µg ml-1, MIC90 = 1 µg ml-1) and Lfx (MIC50 = 0.125 µg ml-1, MIC90 = 2 µg ml-1). We determined the tentative epidemiological cut-off values as 0.5 µg ml-1 for Sfx. Also, 8.43% (7/83), 43.37% (36/83), 42.17% (35/83) and 51.81% (43/83) MDR strains were resistant to Sfx, Mfx, Lfx and Ofx, respectively. Cross-resistance between Ofx, Lfx and Mfx was 80.43% (37/46). Only 15.22% (7/46) of the pre-existing FQs resistance isolates were resistant to Sfx. Among the 30 isolates with mutations in gyrA or gyrB, 5 (16.67%) were Sfx resistant. The combination of Sfx and rifampicin could exert partial synergistic effects, and no antagonism between Sfx and six clinically important anti-Mtb antibiotics was evident.Conclusion. Sfx exhibited superior activity against MDR isolates comparing to Lfx and Mfx, and could potentially overcome the majority pre-existing FQs resistance in Mtb strains.

Co-occurrence of amikacin-resistant and -susceptible Mycobacterium tuberculosis isolates in clinical samples from Beijing, China.

OBJECTIVES: This study examined the phenomenon of heteroresistance in Mycobacterium tuberculosis clinical isolates obtained from retreated patients in Beijing, China between 2006 and 2011. METHODS: The iPLEX Gold assay platform was used to determine the prevalence of heteroresistance to injectable second-line drugs (amikacin, kanamycin and capreomycin) in resistant isolates. RESULTS: Heteroresistance was identified in 10.9% of 220 phenotypic amikacin-resistant isolates. CONCLUSIONS: Heteroresistance was related mainly to the short duration and repeated use of amikacin and capreomycin during retreatment. These findings further our understanding of the evolution of resistance to injectable drugs used for tuberculosis treatment and help guide the rational use of injectable drugs during therapy.

[Evaluation of the reliability of thiophen-2-carboxylic acid hydrazine result for the identification of Mycobacterium bovis].

OBJECTIVE: To evaluate the reliability of thiophen-2-carboxylic acid hydrazine (TCH) test for identification of Mycobacterium bovis (M. bovis). METHODS: A total of 4069 clinically isolated strains were identified by P-Nitrobenzoic acid medium (500 mg/L) and TCH medium (5 mg/L). Mycobacterium tuberculosis (M. tuberculosis) complex strains susceptible to 5 mg/L TCH were further tested for susceptibility to 2 mg/L TCH. Spacer oligonucleotide typing (spoligotyping) and multi-loci PCR were also performed to identify TCH susceptible strains. RESULTS: Among the 4069 isolated strains there were 3929 strains belonging to M. tuberculosis complex (MTBC) of which 245 were susceptible to 5 mg/L TCH. Of these 245 strains, 20 were also susceptible to 2 mg/L TCH, while only 1 strain was identified to be M. bovis by both spoligotyping and multi-loci PCR. CONCLUSION: TCH susceptibility test (either 5 mg/L or 2 mg/L) is not a reliable method for identification of M. bovis.

In vitro activity of tubercidin against Mycobacterium tuberculosis and nontuberculosis Mycobacteria.

Tubercidin is an adenosine analogue that has been shown to exhibit good activity against some tumours and parasites. In this study, the in vitro activity of tubercidin was evaluated against Mycobacterium tuberculosis (Mtb) and nontuberculosis Mycobacteria (NTM). For determining the MICs of tubercidin, 23 fully drug-sensitive (DS) Mtb strains, 33 multi-drug resistance tuberculosis (MDR-TB) strains, 29 pre-extensively drug-resistant tuberculosis (pre-XDR-TB) strains, 21 extensively drug-resistant tuberculosis (XDR-TB) strains, 17 rapidly growing mycobacteria (RGM) and nine slowly growing mycobacteria (SGM) references strains were tested by microplate-based Alamar Blue assay (MABA) method. The results indicate that tubercidin has high in vitro activity against some drug-resistance Mtb strains and NTM reference strains, which warrants further investigation on the actions of tubercidin and its derivatives as potential drugs for mycobacterial infections.

In vitro and Intracellular Antibacterial Activity of Sudapyridine (WX-081) Against Tuberculosis.

Background: Sudapyridine (WX-081) has exhibited equivalent efficacy than its counterpart parent drug bedaquiline (BDQ) but better safety profile against Mycobacterium tuberculosis (Mtb). Our study was aimed to evaluate in vitro activity of WX-081 against the clinical isolates of Mtb with different drug-resistance profiles and the intracellular bactericidal activity against the reference strain. Methods: The minimum inhibitory concentrations (MICs) of WX-081 and BDQ were tested against 114 Mtb clinical isolates. The intracellular activity of WX-081 and BDQ against the Mtb reference strain H37Rv in THP-1 cells was also evaluated in parallel. Results: The MICs for WX-081 of the enrolled isolates ranged from 0.0156 µg/mL to 1 µg/mL. The MIC50 and MIC90 of WX-081 were, respectively, 0.25 µg/mL and 0.5 µg/mL, with 95.6% of the enrolled strains having MICs ≤0.25 µg/mL. For a given strain, the MIC value of WX-081 was generally equivalent to or 2-fold than MIC of BDQ. The intracellular bacterial killing was acquired with the tested drug concentrations that were presumed attainable during clinical usage. Conclusion: WX-081 exhibited potent efficacy against the clinical isolates in vitro. The intracellular killing effect of sudapyridine against the reference strain supports its potential efficacy in treating TB patients.

Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex.

Since the onset of the COVID-19 pandemic in 2020, global efforts towards tuberculosis (TB) control have encountered unprecedented challenges. There is an urgent demand for efficient and cost-effective diagnostic technologies for TB. Recent advancements in CRISPR-Cas technologies have improved our capacity to detect pathogens. The present study established a CRISPR-Cas12a-based multiplex detection (designated as MCMD) that simultaneously targets two conserved insertion sequences (IS6110 and IS1081) to detect Mycobacterium tuberculosis complex (MTBC). The MCMD integrated a graphene oxide-assisted multiplex recombinase polymerase amplification (RPA) assay with a Cas12a-based trans-cleavage assay identified with fluorescent or lateral flow biosensor (LFB). The process can be performed at a constant temperature of around 37°C and completed within 1 h. The limit of detection (LoD) was 4 copies µL-1, and no cross-reaction was observed with non-MTBC bacteria strains. This MCMD showed 74.8% sensitivity and 100% specificity in clinical samples from 107 patients with pulmonary TB and 40 non-TB patients compared to Xpert MTB/RIF assay (63.6%, 100%). In this study, we have developed a straightforward, rapid, highly sensitive, specific, and cost-effective assay for the multiplex detection of MTBC. Our assay showed superior diagnostic performance when compared to the widely used Xpert assay. The novel approach employed in this study makes a substantial contribution to the detection of strains with low or no copies of IS6110 and facilitates point-of-care (POC) testing for MTBC in resource-limited countries.

Bedaquiline resistance pattern in clofazimine-resistant clinical isolates of tuberculosis patients.

OBJECTIVES: Bedaquiline (BDQ) is a potent drug for treating drug-resistant tuberculosis (TB). Here, we analysed the resistance profiles of BDQ in CFZ-resistant clinical isolates and investigated the clinical risk factors of BDQ and CFZ cross/co-resistance. METHODS: The AlarmarBlue microplate assay was performed to determine the minimum inhibitory concentration (MIC) of the CFZ-resistant Mycobacterium tuberculosis (MTB) clinical isolates to CFZ and BDQ. The clinical characteristics of the respective patients were analysed to explore the possible risk factors of BDQ resistance. The drug-resistance-associated genes including Rv0678, Rv1979c, atpE, pepQ and Rv1453 were sequenced and analysed. RESULTS: A total of 72 clinical CFZ-resistant MTB isolates were collected; among these, half were identified as BDQ-resistant. The MIC value of BDQ closely correlated with CFZ (Spearman's q = 0.766, P < 0.005). Among the isolates with a MIC of CFZ ≥4 mg/L, 92.31% (12/13) were resistant to BDQ. Pre-XDR and exposure to BDQ or CFZ are the major risk factors for concurrent BDQ resistance. Among the 36 cross/co-resistant isolates, 50% (18/36) had mutations in Rv0678, 8.3% (3/36) had mutations in Rv0678+Rv1453, 5.6% (2/36) had mutations in Rv0678+Rv1979c, 2.8% (1/36) had mutations in Rv0678+Rv1979c+Rv1453, 2.8% (1/36) had mutations in atpE+Rv0678+Rv1453, 2.8% (1/36) had mutations in Rv1979c, and 27.7% (10/36) had no variations in the target genes. CONCLUSION: Nearly half of the CFZ-resistant isolates were still sensitive to BDQ, whereas this rate dramatically decreased among patients with pre-XDR TB or those who had been exposed to BDQ or CFZ.

Comparison of the in vitro antibacterial activity of ofloxacin, levofloxacin, moxifloxacin, sitafloxacin, finafloxacin, and delafloxacin against Mycobacterium tuberculosis strains isolated in China.

Objective: The resistance of Mycobacterium tuberculosis (Mtb) to currently available fluoroquinolones (FQs), namely ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MFX), renders the treatment of TB infections less successful. In this study, we aimed to evaluate the susceptibility and intracellular killing assay of Mtb to next-generation FQs in vitro and determine the correlation of FQs resistance and newly detected mutations in gyrB by molecular docking. Methods: Antimicrobial susceptibility test was performed to determine the minimum inhibitory concentrations (MICs) of six FQs, including currently available FQs (OFX, LFX, and MFX) and next-generation FQs, i.e., sitafloxacin (SFX), finafloxacin (FIN) and delafloxacin (DFX) against Mtb clinical isolates obtained in 2015 and 2022, respectively. Quinolone-resistance-determining regions of gyrA and gyrB were subjected to DNA sequencing and the correlation of FQs resistance and new mutations in gyrB were determined by molecular docking. Furthermore, the intracellular antibacterial activity of the six FQs against Mtb H37Rv in THP-1 cells was evaluated. Results: SFX exhibited the highest antibacterial activity against Mtb isolates (MIC90 = 0.25 µg/mL), whereas DFX and OFX exhibited comparable activity (MIC90 = 8 µg/mL). A statistically significant difference was observed among the MICs of the new generation FQs (SFX, P = 0.002; DFX, P = 0.008). Additionally, a marked increase in MICs was found in strains isolated in 2022 compared with those isolated in 2015. There might be correlation between FQs resistance and mutations in gyrB G520T and G520A. Cross-resistance rate between SFX and MFX was 40.6 % (26/64). At a concentration of 1 µg/mL, SFX exhibited high intracellular antibacterial activity (96.6 % ± 1.5 %) against the Mtb H37Rv, comparable with that of MFX at a concentration of 2 µg/mL. Conclusion: SFX exhibits the highest inhibitory activity against Mtb in vitro and THP-1 cell lines, which exhibits partial-cross resistance with MFX. There might be correlation between FQs resistance and mutations in gyrB G520T and G520A.Our findings provide crucial insights into the potential clinical application of SFX and DFX in the treatment of Mtb infections.

Evaluation of the diagnostic value of loop-mediated isothermal amplification assays targeting three different Mycobacterium tuberculosis genes.

BACKGROUND: The aim of the study was to evaluate a loop-mediated isothermal amplification (LAMP) assay for the ability to diagnose tuberculosis directly from clinical samples rapidly. METHODS: LAMP assays were performed using previously reported primer sets to amplify three specific Mycobacterium tuberculosis (MTB) gene targets, hspX, gyrB, and IS6110. Quantitated DNA from strain H37Rv were detected for assessment of analytical sensitivity; specificity was evaluated by testing eight species of non-tuberculosis Mycobacterium (NTM) and four unrelated bacterial species. Sputum samples from 68 pulmonary tuberculosis patients and a control group consisting of 45 lung cancer patients and 20 healthy controls were analyzed using LAMP assays, and then compared with smear, culture and quantitative real-time PCR (qRT-PCR) methods. RESULTS: All three LAMP assays showed 100% specificity for MTB when tested against NTM and other bacterial species. The gyrB-LAMP assay was able to detect 60 cfu/ml of H37Rv suspension within 1 h, similar to qRT-PCR, but 10 times more sensitive than the hspX-LAMP and IS6110-LAMP assays. In clinical samples, when qRT-PCR was used as the reference method, the sensitivity of the three LAMP assays targeting hspX, gyrB, and IS6110 genes was 94.6, 98.2 and 92.9%, respectively. CONCLUSIONS: LAMP is more sensitive than smear microscopy and close to qRT-PCR in sensitivity for the detection of MTB. LAMP has comparable specificity to qRT-PCR but was more rapid and convenient.

Doubled Nontuberculous Mycobacteria Isolation as a Consequence of Changes in the Diagnosis Algorithm.

Background: Nontuberculous mycobacteria (NTM) and their associated diseases remain neglected. Through minor modifications in our diagnostic algorithm, we observed an unexpected higher number of cultivable NTM isolates. Therefore, a retrospective study was performed thoroughly to investigate the effect of changed laboratory procedures on NTM isolation in a specialized tuberculosis hospital. Methods: NTM isolation rates and composition of NTM species were compared for the two diagnostic algorithms: (1) by using traditional p-nitrobenzoic acid (PNB) selective medium as a preliminary test to identify NTM isolates among the positive cultures (procedure I) and (2) by using the MPT64 antigen detection method to distinguish between Mycobacterium tuberculosis complex (MTBC) isolates and possible NTM isolates after a positive MGIT960 liquid culture (procedure II). Results: The NTM isolation rate in procedure II was significantly higher than the procedure I (18.08% vs 9.71%; P<0.001). A noticeable increase in the ratio of NTM isolates among the identified mycobacteria was observed over the studied years (ie, from 58.18% in 2019 to 72.93% in 2021), which indicated a more precise prescription of species identification test after prompt information was provided in procedure II. In addition, the consistency of the identified species using multiple specimens from the same patient did not present a significant difference between the procedures. Conclusion: According to our study, NTM infection might be far more underestimated than it is. A diagnostic procedure combining MGIT960 culture and MPT64 antigen detection could timely and easily identify clues of NTM isolates and improve the diagnosis of NTM infections.

The Practical Value of Xpert MTB/RIF Ultra for Diagnosis of Pulmonary Tuberculosis in a High Tuberculosis Burden Setting: a Prospective Multicenter Diagnostic Accuracy Study.

Due to the probability of decreased specificity, the practical value of performing the Xpert MTB/RIF Ultra (Xpert Ultra) assay over the Xpert assay for diagnosing pulmonary tuberculosis (TB) and rifampicin (RIF) resistance in a high TB burden setting was evaluated. Participants were recruited consecutively in three tertiary hospitals in China and allocated to the TB case detection and/or rifampicin (RIF) resistance detection group. Each sputum specimen was subjected to smear, MGIT960 liquid culture, and Xpert, and Xpert Ultra assay in parallel. Drug susceptibility testing was conducted for all recovered isolates in the RIF resistance detection group. In total, 1,079 patients were recruited to the case detection group and 450 to the RIF resistance detection group. Xpert Ultra had higher sensitivity than Xpert (92.26%, 322/349 versus 89.40%, 312/349; P = 0.006), whereas the most prominent increase was identified in the smear-negative patients (83.70% versus 78.52%; P = 0.039). The specificity of Xpert Ultra was slightly lower than that of Xpert (96.30%, 495/514 versus 98.25%, 505/514; P = 0.055). Reclassifying trace results as negative resulted in a 4.01% loss of sensitivity (from 92.26% to 88.25%) accompanied by a 1.37% gain in specificity (from 96.30% to 97.67%). Both the sensitivity (97.64% versus 99.21%, P = 0.313) and specificity (96.90% versus 97.21%, P = 0.816) of Xpert Ultra and Xpert for detection RIF resistance were comparable. In conclusion, Xpert Ultra could improve the diagnosis of smear-negative pulmonary TB in contrast to the Xpert assay. A high percentage of TB history did not significantly decrease the specificity of the test, which supports the potential role of Xpert Ultra as an initial diagnostic tool for TB. IMPORTANCE Xpert Ultra is more sensitive than Xpert, especially in smear-negative TB. A high percentage of TB history in the non-TB population did not significantly affect the reliability of the assay, which supports the potential role of Xpert Ultra as an initial diagnostic tool for TB.