Diagnostic efficacy of an optimized nucleotide MALDI-TOF-MS assay for anti-tuberculosis drug resistance detection.

PURPOSE: We aimed at evaluating the diagnostic efficacy of a nucleotide matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) assay to detect drug resistance of Mycobacterium tuberculosis. METHODS: Overall, 263 M. tuberculosis clinical isolates were selected to evaluate the performance of nucleic MALDI-TOF-MS for rifampin (RIF), isoniazid (INH), ethambutol (EMB), moxifloxacin (MXF), streptomycin (SM), and pyrazinamide (PZA) resistance detection. The results for RIF, INH, EMB, and MXF were compared with phenotypic microbroth dilution drug susceptibility testing (DST) and whole-genome sequencing (WGS), and the results for SM and PZA were compared with those obtained by WGS. RESULTS: Using DST as the gold standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 98.2%, 98.7%, and 0.97 for RIF; 92.8%, 99%, and 0.90 for INH; 82.4%, 98.0%, and 0.82 for EMB; and 92.6%, 99.5%, and 0.94 for MXF, respectively. Compared with WGS as the reference standard, the sensitivity, specificity, and kappa values of the MALDI-TOF-MS assay for the detection of resistance were 97.4%, 100.0%, and 0.98 for RIF; 98.7%, 92.9%, and 0.92 for INH; 96.3%, 100.0%, and 0.98 for EMB; 98.1%, 100.0%, and 0.99 for MXF; 98.0%, 100.0%, and 0.98 for SM; and 50.0%, 100.0%, and 0.65 for PZA. CONCLUSION: The nucleotide MALDI-TOF-MS assay yielded highly consistent results compared to DST and WGS, suggesting that it is a promising tool for the rapid detection of sensitivity to RIF, INH, EMB, and MXF.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

Tuberculosis screening characteristics amongst freshmen in Changping District, Beijing, China.

BACKGROUND: Screening for Tuberculosis (TB) is a critical tactic for minimizing the prevalence of illness within schools. Tuberculosis Preventive Therapy (TPT), in turn, effectively staves off the development of TB from latent tuberculosis infection (LTBI). Unfortunately, there is limited research on LTBI and TPT among students. This study aimed to assess LTBI among freshmen in Changping District and advocate for the implementation of TPT. METHODS: The prospective study collected data from 12 educational institutions within the Changping District of Beijing. The Kolmogorov - Smirnov test and other statistical methods were used for statistical analysis, [Formula: see text] was obtained using the formula [Formula: see text] nΣA2/nRnC-1, df = (C-1) (R-1). We analyzed potential factors impacting the LTBI rate, and scrutinized the possible causes behind the low application of TPT and its efficacy for LTBI treatment, China. RESULTS: Among 19,872 freshmen included in this study, 18 active TB cases (91 per 10,0000) and 2236 LTBI cases (11.6% of 19,223) were identified, respectively. Furthermore, of those with LTBI, 1045 (5.4% of 19,223) showed a strong positive for purified protein derivative (PPD), but only 312 opted for TB preventive treatment. There appeared to be no significant difference in the prevalence of LTBI and TPT rate between male and female students. Concurrently, 11 (71 per 100,000) and 7 (158 per 100,000) cases of active tuberculosis were identified in 6 universities and 6 higher vocational colleges, respectively. Interestingly, almost all freshmen who underwent TPT came from universities, suggesting a statistically significant disparity in TPT rate (χ2 = 139.829, P < 0.001) between these two types of educational institutions. Meanwhile, as for the age-wise distribution of latent infection among 17-20 years old freshmen, the LTBI rate exhibited 10.5%, 11.6%, 12.1% and 13.5%, respectively. Correlation between LTBI rate, the strong positive rate was statistically significant among different ages (χ2 = 34.559, P < 0.001). Over a follow-up period of 2 years, three students were diagnosed with active tuberculosis, one of which was resistant to rifampicin. All three students manifested a strong positive for PPD and declined preventive treatment during TB screening. CONCLUSIONS: The data indicates a high rate of LTBI amongst students in areas with a heavy TB burden, potentially leading to cross-regional TB transmission due to the migration of students. Education level might contribute to the limited uptake of TPT. Therefore, improving the implementation of TB preventive treatments is crucial in controlling and preventing TB across schools.

The Recent Transmission and Associated Risk Factor of Mycobacterium tuberculosis in Golmud City, China.

Background: Tuberculosis (TB) remains a severe public health problem globally, and it is essential to comprehend the transmission pattern to control tuberculosis. Herein, we evaluated the drug-resistant characteristics, recent transmission, and associated risk factors of TB in Golmud, Qinghai, China. Methods: In this study, we performed a population-based study of patients diagnosed with TB in Golmud from 2013 to 2018. Drug-susceptibility testing and whole-genome sequencing were performed on 133 Mycobacterium tuberculosis strains. The genomic clustering rate was calculated to evaluate the level of recent transmission. Risk factors were identified by logistic regression analysis. Results: Our results showed that 46.97% (62/132) of strains were phylogenetically clustered and formed into 23 transmission clusters, suggesting a high recent transmission of TB in the area. 12.78% (17/133) strains were multidrug-resistant/rifampicin tuberculosis (MDR/RR-TB), with a high drug-resistant burden. Based on drug resistance gene analysis, we found 23 strains belonging to genotype MDR/RR-TB, where some strains may have borderline mutations. Among these strains, 65.2% (15/23) were found within putative transmission clusters. Additionally, risk factor analysis showed that recent transmission of TB happened more in patients with Tibetan nationality or older age. Conclusion: Overall our study indicates that the recent transmissions of MTB strains, especially genotypic MDR/RR strains, drive the tuberculosis epidemic in Golmud, which could contribute to developing effective TB prevention and control strategies.

Impact of MSMEG5257 Deletion on Mycolicibacterium smegmatis Growth.

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.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Whole Genomic Analysis Revealed High Genetic Diversity and Drug-Resistant Characteristics of Mycobacterium tuberculosis in Guangxi, China.

Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a major public health issue in China. Nevertheless, the prevalence and drug resistance characteristics of isolates vary in different regions and provinces. In this study, we investigated the population structure, transmission dynamics and drug-resistant profiles of Mtb in Guangxi, located on the border of China. Methods: From February 2016 to April 2017, 462 clinical M. tuberculosis isolates were selected from 5 locations in Guangxi. Drug-susceptibility testing was performed using 6 common anti-tuberculosis drugs. The genotypic drug resistance and transmission dynamics were analyzed by the whole genome sequence. Results: Our data showed that the Mtb in Guangxi has high genetic diversity including Lineage 1 to Lineage 4, and mostly belong to Lineage 2 and Lineage 4. Novelty, 9.6% of Lineage 2 isolates were proto-Beijing genotype (L2.1), which is rare in China. About 12.6% of isolates were phylogenetically clustered and formed into 28 transmission clusters. We observed that the isolates with the high resistant rate of isoniazid (INH, 21.2%), followed by rifampicin (RIF, 13.2%), and 6.7%, 12.1%, 6.7% and 1.9% isolates were resistant to ethambutol (EMB), streptomycin (SM), ofloxacin (OFL) and kanamycin (KAN), respectively. Among these, 6.5% and 3.3% of isolates belong to MDR-TB and Pre-XDR, respectively, with a high drug-resistant burden. Genetic analysis identified the most frequently encountered mutations of INH, RIF, EMB, SM, OFL and KAN were katG_Ser315Thr (62.2%), rpoB_Ser450Leu (42.6%), embB_Met306Vol (45.2%), rpsL_Lys43Arg (53.6%), gyrA_Asp94Gly (29.0%) and rrs_A1401G (66.7%), respectively. Additionally, we discovered that isolates from border cities are more likely to be drug-resistant than isolates from non-border cities. Conclusion: Our findings provide a deep analysis of the genomic population characteristics and drug-resistant of M. tuberculosis in Guangxi, which could contribute to developing effective TB prevention and control strategies.

Endogenous relapse and exogenous reinfection in recurrent pulmonary tuberculosis: A retrospective study revealed by whole genome sequencing.

Background: Tuberculosis may reoccur due to reinfection or relapse after initially successful treatment. Distinguishing the cause of TB recurrence is crucial to guide TB control and treatment. This study aimed to investigate the source of TB recurrence and risk factors related to relapse in Hunan province, a high TB burden region in southern China. Methods: A population-based retrospective study was conducted on all culture-positive TB cases in Hunan province, China from 2013 to 2020. Phenotypic drug susceptibility testing and whole-genome sequencing were used to detect drug resistance and distinguish between relapse and reinfection. Pearson chi-square test and Fisher exact test were applied to compare differences in categorical variables between relapse and reinfection. The Kaplan-Meier curve was generated in R studio (4.0.4) to describe and compare the time to recurrence between different groups. p < 0.05 was considered statistically significant. Results: Of 36 recurrent events, 27 (75.0%, 27/36) paired isolates were caused by relapse, and reinfection accounted for 25.0% (9/36) of recurrent cases. No significant difference in characteristics was observed between relapse and reinfection (all p > 0.05). In addition, TB relapse occurs earlier in patients of Tu ethnicity compared to patients of Han ethnicity (p < 0.0001), whereas no significant differences in the time interval to relapse were noted in other groups. Moreover, 83.3% (30/36) of TB recurrence occurred within 3 years. Overall, these recurrent TB isolates were predominantly pan-susceptible strains (71.0%, 49/69), followed by DR-TB (17.4%, 12/69) and MDR-TB (11.6%, 8/69), with mutations mainly in codon 450 of the rpoB gene and codon 315 of the katG gene. 11.1% (3/27) of relapse cases had acquired new resistance during treatment, with fluoroquinolone resistance occurring most frequently (7.4%, 2/27), both with mutations in codon 94 of gyrA. Conclusion: Endogenous relapse is the main mechanism leading to TB recurrences in Hunan province. Given that TB recurrences can occur more than 4 years after treatment completion, it is necessary to extend the post-treatment follow-up period to achieve better management of TB patients. Moreover, the relatively high frequency of fluoroquinolone resistance in the second episode of relapse suggests that fluoroquinolones should be used with caution when treating TB cases with relapse, preferably guided by DST results.

Insight into the drug-resistant characteristics and genetic diversity of multidrug-resistant Mycobacterium tuberculosis in China.

Multidrug-resistant tuberculosis (MDR-TB) has a severe impact on public health. To investigate the drug-resistant profile, compensatory mutations and genetic variations among MDR-TB isolates, a total of 546 MDR-TB isolates from China underwent drug-susceptibility testing and whole genome sequencing for further analysis. The results showed that our isolates have a high rate of fluoroquinolone resistance (45.60%, 249/546) and a low proportion of conferring resistance to bedaquiline, clofazimine, linezolid, and delamanid. The majority of MDR-TB isolates (77.66%, 424/546) belong to Lineage 2.2.1, followed by Lineage 4.5 (6.41%, 35/546), and the Lineage 2 isolates have a strong association with pre-XDR/XDR-TB (P < 0.05) in our study. Epidemic success analysis using time-scaled haplotypic density (THD) showed that clustered isolates outperformed non-clustered isolates. Compensatory mutations happened in rpoA, rpoC, and non-RRDR of rpoB genes, which were found more frequently in clusters and were associated with the increase of THD index, suggesting that increased bacterial fitness was associated with MDR-TB transmission. In addition, the variants in resistance associated genes in MDR isolates are mainly focused on single nucleotide polymorphism mutations, and only a few genes have indel variants, such as katG, ethA. We also found some genes underwent indel variation correlated with the lineage and sub-lineage of isolates, suggesting the selective evolution of different lineage isolates. Thus, this analysis of the characterization and genetic diversity of MDR isolates would be helpful in developing effective strategies for treatment regimens and tailoring public interventions. IMPORTANCE Multidrug-resistant tuberculosis (MDR-TB) is a serious obstacle to tuberculosis prevention and control in China. This study provides insight into the drug-resistant characteristics of MDR combined with phenotypic drug-susceptibility testing and whole genome sequencing. The compensatory mutations and epidemic success analysis were analyzed by time-scaled haplotypic density (THD) method, suggesting clustered isolates and compensatory mutations are associated with MDR-TB transmission. In addition, the insertion and deletion variants happened in some genes, which are associated with the lineage and sub-lineage of isolates, such as the mpt64 gene. This study offered a valuable reference and increased understanding of MDR-TB in China, which could be crucial for achieving the objective of precision medicine in the prevention and treatment of MDR-TB.

Association of lineage 4.2.2 of Mycobacterium tuberculosis with the 63-bp deletion variant of the mpt64 gene.

IMPORTANCE: To date, rapid diagnostic methods based on the MPT64 antigen assay are increasingly utilized to differentiate between non-tuberculous mycobacteria and TB disease in clinical settings. Furthermore, numerous novel techniques based on the MPT64 release assay are continuously being developed and applied for the identification of both pulmonary and extrapulmonary TB. However, the diagnostic accuracy of the MPT64 antigen assay is influenced by the presence of 63 bp deletion variants within the mpt64 gene. To our knowledge, this is the first report on the association between the 63 bp deletion variant in mpt64 and Mycobacterium tuberculosis L4.2.2 globally, which highlights the need for the cautious utilization of MPT64-based testing in regions where L4.2.2 isolates are prevalent, such as China and Vietnam, and MPT64 negative results should be confirmed with another assay. In addition, further studies on vaccine development and immunology based on MPT64 should consider these isolates with 63 bp deletion variant.

Clinical symptoms associated with fatality of severe fever with thrombocytopenia syndrome: A systematic review and meta-analysis.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infection disease that is deadly if left untreated. Identification of potential risk factors with prognosis value may help to focus clinical management and reduce case fatality. However, information about these factors is conflicting and scattered. In this study, documents on SFTS were collected by searching PubMed, Web of Science, Chinese National Knowledge Infrastructure (CNKI) and Chinese Wan Fang Database through 12 January 2021. Meta-analysis was performed by using Stata and Review Manage software. 29 studies were included, involving 3011 participants with 38 clinical symptoms. The following factors were strongly (OR>5) and significantly (P-value<0.05) associated with mortality: hematemesis (OR=13.73), slurred speech (OR=5.05), disturbance of consciousness (OR=9.20), coma (OR=47.84), disseminated intravascular coagulation (OR=11.79), multiple organ dysfunction (OR=21.30), shock (OR=8.20) and acute kidney injury (OR=6.22). Non-specific symptoms, underlying diseases, respiratory symptoms and gastrointestinal symptoms were not associated with mortality of SFTS patients. Neurological symptoms and severe complications significantly increasing the death risk of SFTS cases can be identified by medical staff in resource-constrained settings and should be considered as core factors in future researchers that aim at improving the prognosis of SFTS patients.

Clinical laboratory parameters and fatality of Severe fever with thrombocytopenia syndrome patients: A systematic review and meta-analysis.

BACKGROUND: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne infectious disease with high case fatality rate. Unfortunately, no vaccine or antiviral specifically targeting SFTS virus (SFTSV) are available for the time being. Our objective was to investigate the association between clinical laboratory parameters and fatality of SFTS patients. METHODS: The systematic review was conducted in accordance with The Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines. We searched (from inception to 24th February 2022) Web of Science, PubMed, National Knowledge Infrastructure databases and Wan Fang Data for relevant researchers on SFTS. Studies were eligible if they reported on laboratory parameters of SFTS patients and were stratified by clinical outcomes. A modified version of Newcastle-Ottawa scale was used to evaluate the quality of included studies. Standardized mean difference (SMD) was used to evaluate the association between laboratory parameters and outcomes. The between-study heterogeneity was evaluated quantitatively by standard Chi-square and the index of heterogeneity (I2). Heterogeneity was explored by subgroup and sensitivity analyses, and univariable meta-regression. Publication bias was determined using funnel plots and Egger's test. RESULTS: We identified 34 relevant studies, with over 3300 participants across three countries. The following factors were strongly (SMD>1 or SMD<-0.5) and significantly (P<0.05) associated mortality: thrombin time (TT) (SMD = 1.53), viral load (SMD = 1.47), activated partial-thromboplastin time (APTT) (SMD = 1.37), aspartate aminotransferase (AST) (SMD = 1.19), lactate dehydrogenase (LDH) (SMD = 1.13), platelet count (PLT) (SMD = -0.47), monocyte percentage (MON%) (SMD = -0.47), lymphocyte percentage (LYM%) (SMD = -0.46) and albumin (ALB) (SMD = -0.43). Alanine aminotransferase, AST, creatin phosphokinase, LDH, PLT, partial-thromboplastin time and viral load contributed to the risk of dying of SFTS patients in each subgroup analyses. Sensitivity analysis demonstrated that the results above were robust. CONCLUSIONS/SIGNIFICANCE: The abnormal levels of viral load, PLT, coagulation function and liver function, significantly increase the risk of SFTS mortality, suggesting that SFTS patients with above symptoms call for special concern.

Whole-genome sequencing reveals genomic characterization of Listeria monocytogenes from food in China.

Introduction: Listeria monocytogenes is a foodborne bacterium that could persist in food and food processing environments for a long time. Understanding the population structure and genomic characterization of foodborne L. monocytogenes is essential for the prevention and control of listeriosis. Methods: A total of 322 foodborne L. monocytogenes isolates from 13 geographical locations and four food sources in China between 2000 and 2018 were selected for whole-genome sequencing. Results: In silico subtyping divided the 322 isolates into five serogroups, 35 sequence types (STs), 26 clonal complexes (CCs) and four lineages. Serogroup IIa was the most prevalent serogroup and ST9 was the most prevalent ST of foodborne L. monocytogenes strains isolated in China. The in-depth phylogenetic analysis on CC9 revealed that ST122 clone might be original from ST9 clone. Furthermore, 23 potentially relevant clusters were identified by pair-wised whole-genome single nucleotide polymorphism analysis, indicating that persistent- and/or cross-contamination had occurred in markets in China. ST8 and ST121 were the second and third top STs of L. monocytogenes in China, which had heterogeneity with that of L. monocytogenes isolates from other countries. The antibiotic resistance genes aacA4, tetM, tetS, dfrG carried by different mobile elements were found in L. monocytogenes strains. One lineage II strain carrying Listeria Pathogenicity Island 3 was first reported. In addition, a novel type of premature stop codon in inlA gene was identified in this study. Discussion: These findings revealed the genomic characteristics and evolutionary relationship of foodborne L. monocytogenes in China on a scale larger than previous studies, which further confirmed that whole-genome sequencing analysis would be a helpful tool for routine surveillance and source-tracing investigation.

The population structure and genetic diversity of Listeria monocytogenes ST9 strains based on genomic analysis.

Listeria monocytogenes is a ubiquitous foodborne pathogen causing both invasive and non-invasive listeriosis. Sequence type (ST) 9 strains is common in food and food processing environments. In this study, the whole-genome sequences (WGS) of 207 ST9 isolates from different sources, geographical locations (14 countries), and isolated years were analyzed. The ST9 isolates were divided into three clusters after phylogenetic analysis; 67.63% of ST9 isolates contained putative plasmids with different sizes and genomic structure, the putative prophages inserted in the chromosome at ten hotspots, and seven types of premature stop codon (PMSC) mutations in inlA were found in 81.86% of the ST9 isolates. In addition, 78.26% of ST9 isolates harbored Tn554-like elements carrying arsenic resistance genes. All the ST9 isolates conservatively contained environment-resistance genes on the chromosome. This analysis of population structures and features of ST9 isolates was aimed to help develop effective strategies to control this prevalent pathogen in the food chain.

Evaluation Study of xMAP TIER Assay on a Microsphere-Based Platform for Detecting First-Line Anti-Tuberculosis Drug Resistance.

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.

Drug-Resistant Characteristics, Genetic Diversity, and Transmission Dynamics of Rifampicin-Resistant Mycobacterium tuberculosis in Hunan, China, Revealed by Whole-Genome Sequencing.

To gain a deep insight into the additional drug-resistant profiles, genetic diversity, and transmission dynamics of rifampicin-resistant tuberculosis (RR-TB) circulating in Hunan province, drug susceptibility testing and whole-genome-sequencing were performed among RR-TB strains collected from Jan. 2013 to Jun. 2018 in Hunan province. A total of 124 RR-TB strains were recovered successfully and included into the final analysis. Lineage 2.2.1 was the dominant sublineage, accounting for 72.6% (90/124), followed by lineage 4.5 (11.3%, 14/124), lineage 4.4 (8.1%, 10/124), lineage 4.2 (6.5%, 8/124) and lineage 2.2.2 (1.6%, 2/124). Overall, 83.1% (103/124) and 3.2% (4/124) of RR-TB were MDR-TB and XDR-TB, respectively. Nearly 30% of RR-TB isolates were resistant to fluoroquinolones, and 26.6% (33/124) were pre-XDR-TB. Moreover, 30.6% (38/124) of RR-TB strains were identified as phenotypically resistance to pyrazinamide. Totally, 17 clusters containing 48 (38.7%, 48/124) RR-TB strains were identified, ranging in size from 2 to 10 isolates. No significant difference was detected in clustering rate between lineage 2 and lineage 4 (χ2 = 0.027, P = 0.870). Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions. IMPORTANCE Comprehensive information such as genetic background and drug-resistant profile of MTB strains could help to tailor public interventions. However, these data are limited in Hunan province, one of the provinces with high-TB burden in China. So, this study aimed to provide us with deep insight into the molecular epidemiology of RR-TB isolates circulating in Hunan province by combining phenotypic drug susceptibility testing and whole-genome sequencing. To our knowledge, this is the first study to use whole-genome sequencing data of RR-TB strains spanning more than 5 years for molecular epidemiology analysis in Hunan province, which allows us to identify genetic background information and clustered strains more accurately. Our study revealed the complexity of RR-TB strains circulating in Hunan province with complex additional drug-resistant profile and relatively higher clustering rates. Comprehensive information based on WGS should be used to guide the design of treatment regimens and tailor public interventions.

High diversity of clinical Mycobacterium intracellulare in China revealed by whole genome sequencing.

Mycobacterium intracellulare is the most common cause of nontuberculous mycobacterial lung disease, with a rapidly growing prevalence worldwide. In this study, we performed comparative genomic analysis and antimicrobial susceptibility characteristics analysis of 117 clinical M. intracellulare strains in China. Phylogenetic analysis showed that clinical M. intracellulare strains had high genetic diversity and were not related to the geographical area. Notably, most strains (76.07%, 89/117) belonged to Mycobacterium paraintracellulare (MP) and Mycobacterium indicus pranii (MIP) in the genome, and we named them MP-MIP strains. These MP-MIP strains may be regarded as a causative agent of chronic lung disease. Furthermore, our data demonstrated that clarithromycin, amikacin, and rifabutin showed strong antimicrobial activity against both M. intracellulare and MP-MIP strains in vitro. Our findings also showed that there was no clear correlation between the rrs, rrl, and DNA gyrase genes (gyrA and gyrB) and the aminoglycosides, macrolides, and moxifloxacin resistance, respectively. In conclusion, this study highlights the high diversity of M. intracellulare in the clinical setting and suggests paying great attention to the lung disease caused by MP-MIP.

Function and distribution of the conjugative plasmid pLM1686 in foodborne Listeria monocytogenes in China.

Listeria monocytogenes, a fatal foodborne pathogen has the extraordinary capacity to survive in harsh conditions and is a potential threat to public health. A novel 91 kb plasmid pLM1686 was found in the prevalent L. monocytogenes sequence type (ST) 87 strain in China. In this study, the function and distribution of pLM1686 were firstly investigated in L. monocytogenes. The results showed plasmid pLM1686 had self-transmissible ability and existed in various types of L. monocytogenes isolates belonging to two lineages (lineage I and II), four serotypes (1/2b, 3b, 1/2c and 1/2a) and four STs (ST87, ST59, ST9 and ST120). The wild strain LM1686 and transconjugant strain 10403SP1686 exhibited significantly higher growth rate and biofilm formation in Modification of Welshimer's medium (MWB), greater salinity tolerance, stronger cell invasion and higher cytotoxicity than plasmid-cured strain and reference strain 10403S. Moreover, plasmid curing caused the loss of cadmium resistance of strain, and the recipient strain acquired cadmium resistance after conjugation. Thus, pLM1686 would provide L. monocytogenes advantages of surviving in adverse environments.