Comparison of targeted next-generation sequencing and the Xpert MTB/RIF assay for detection of Mycobacterium tuberculosis in clinical isolates and sputum specimens.

Targeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of many genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and another molecular testing tool, Xpert MTB/rifampicin (RIF), have been empirical. Here, using a dilution series of a RIF-resistant clinical isolate of MTB, we found that tNGS had a slightly lower limit of bacterial detection (102 CFU/mL) compared with Xpert MTB/RIF (103 CFU/mL) in culture medium. However, the minimum detection limit of the rpoB S450L mutation in this isolate was significantly lower with tNGS (102 CFU/mL) than with Xpert MTB/RIF (106 CFU/mL). Sputum samples collected from 129 suspected pulmonary tuberculosis patients were also prospectively studied with the clinical diagnosis as a reference, revealing that the sensitivity of tNGS (48.6%) was higher than those of culture (46.8%), Xpert MTB/RIF (39.4%), and smear microscopy (34.9%) testing. Notably, AMR analysis of 56 MTB-positive samples as determined by tNGS revealed high mutation frequencies of 96.4%, 35.7%, 26.8%, and 19.6% in the following AMR-associated genes: rrs, rpoB, katG, and pncA, respectively. The findings of this study provide theoretical support for the differential clinical application of tNGS and Xpert MTB/RIF and suggest that tNGS has greater application value in tuberculosis drug resistance monitoring and prevention.IMPORTANCETargeted next-generation sequencing (tNGS) can be used to perform Mycobacterium tuberculosis (MTB) complex-specific amplification or target capture directly from sputum samples, yielding simultaneous coverage of genes and DNA regions associated with antimicrobial resistance (AMR). Performance comparisons of tNGS and Xpert MTB/rifampicin (RIF) have been empirical. The Xpert MTB/RIF assay is a commercial system that uses the nucleic acid amplification detection method for rapid (2 hours) diagnosis of tuberculosis (TB). The cost of the tNGS and Xpert MTB/RIF assays in this study was similar, at USD 98 and USD 70-104 per sample, respectively, but the time required for tNGS (3 days) was much longer than that required for the Xpert MTB/RIF assay. However, tNGS yielded more accurate results and a larger number of AMR-associated gene mutations, which compensated for the extra time and highlighted the greater application value of tNGS in TB drug resistance monitoring and prevention.

Pathways of soil organic carbon accumulation are related to microbial life history strategies in fertilized agroecosystems.

Although the formation, turnover, and accumulation of soil organic carbon (SOC) are driven by different fertilizer inputs and their subsequent microbial-mediated transformation, the relationship between changes in plant-derived and microbial-derived components and soil microbial life history strategies under different fertilization regimes has not been well explored. In this study, the changes in microbial necromass carbon (MNC), lignin phenols, and glomalin-related soil protein (GRSP), as well as soil microbial life history strategy were determined in a 16-year field experiment in response to different fertilization regimes, including a no-fertilizer control (C), conventional chemical NPK fertilization (NPK), and partial substitutions of the NPK in chemical fertilizers with a low (30 %) or high (60 %) level of straw (0.3S and 0.6S) or cattle manure (0.3M and 0.6M). The results showed that total lignin phenol content and its contribution to SOC were significantly increased by 88.7 % and 74.2 %, respectively, in high-level straw substitution treatment as compared to chemical fertilization. Both high-level straw and cattle manure substitution increased MNC and total GRSP contents, but did not alter their contributions to SOC compared to chemical fertilization. In fertilized treatments, the high-level cattle manure substitution had the lowest and highest bacterial and fungal K/r ratio, respectively. Bacterial K/r ratio was an important factor in predicting bacterial necromass carbon content and there was a significant negative correlation between them. The ratio of ectomycorrhizal to saprotrophic fungi and fungal diversity were important factors for predicting lignin phenol and GRSP contents, respectively. In addition, the SEMs modeling indicated that straw substitution directly affected lignin phenol and MNC accumulation, whereas cattle manure substitution indirectly affected MNC accumulation by affecting microbial life history strategies. In conclusions, agricultural residues inputs support the formation of a multiple carbon pool of SOC compared to chemical fertilization; and microbial life history strategy is an important driver of SOC formation and affects SOC accumulation and stability in agroecosystems.

Strengthen interactions among fungal and protistan taxa by increasing root biomass and soil nutrient in the topsoil than in the soil-rock mixing layer.

Soil depth plays a crucial role in shaping the interactions between soil microbes and nutrient availability. However, there is limited understanding of how bacterial, fungal, and protistan communities respond to different soil depths, particularly in the unique geological context and soil properties of karst regions. Organic matter, total nitrogen, and phosphorus, ammonium, nitrate, and plant root biomass, as well as bacterial and fungal abundances, bacterial and protistan diversity were higher in the 0-20 cm soil layer than those in the 20-40 cm and soil-rock mixing layers. In contrast, soil pH was higher in the 20-40 cm and soil-rock mixing layers than that in the 0-20 cm soil layer. The soil exchange of calcium, nitrate, and root biomass were identified as the primary factors regulating microbial assemblages across the depth transect. Moreover, co-occurrence network analysis revealed a greater degree of connectivity between protistan taxa and fungal taxa in the 0-20 cm soil layer than those in the 20-40 cm and soil-rock mixing layers. In contrast, the number of association links between protist-bacteria and bacteria-bacteria was higher in the soil-rock mixing layers compared to the 0-20 cm soil layer. Actinobacteria, Ascomycota, and unclassified protistan taxa were identified as keystones, displaying the highest number of connections with other microbial taxa. Collectively, these results suggested that the increased plant root biomass, coupled with sufficient available nutrient inputs in the upper 0-20 cm soil layer, facilitates strong interactions among fungal and protistan taxa, which play crucial roles in the topsoil. However, as nutrients become less available with increasing depth, competition among bacterial taxa and the predation between bacterial and protistan taxa intensify. Therefore, these findings indicate the interactions among keystone taxa at different soil depths has the potential to generate ecological implications during vegetation restoration in fragile ecosystems.

Lithology and niche habitat have significant effect on arbuscular mycorrhizal fungal abundance and their interspecific interactions.

The chemical properties of bedrock play a crucial role in shaping the communities of soil and root-associated arbuscular mycorrhizal fungi (AMF). We investigate AMF community composition and diversity in bulk soil, rhizosphere soil, and roots in karst and non-karst forests. Chemical properties of bedrock of the calcium oxide (CaO) and ratio of calcium oxide and magnesium oxide (Ca/Mg), soil pH, and exchangeable Ca2+ were higher in karst carbonate rocks compared to non-karst clastic rocks. Conversely, bedrock phosphorus content (P-rock), silicon dioxide (SiO2) content, and tree diversity exhibited an opposing trend. AMF abundance was higher in non-karst clastic rocks than in karst carbonate rocks. Stronger interspecific interactions among AMF taxa occurred in the bulk soil and rhizosphere soil of non-karst clastic rocks compared to karst carbonate rocks. AMF abundance and diversity were higher in rhizosphere soil and roots, attributed to increasing nutrient availability when compared to the bulk soil. A more complex network within AMF taxa was observed in rhizosphere soil and roots compared to bulk soil due to an increase in AMF abundance and diversity in rhizosphere soil and roots. Comparing non-karst clastic rocks, karst carbonate rocks increased soil nitrogen (N) and P levels, which can be attributed to the elevated content of soil Ca2+ and Mg2+ content, facilitated by the high CaO content and Ca/Mg ratio in the bedrock of karst forests. However, the thicker soil layer exhibited higher soil nutrient storage, resulting in greater tree diversity in non-karst forests. These findings suggest that high tree richness may increase root biomass and secretion of root exudates in non-karst regions, thereby enhancing the abundance of AMF and their interspecies interactions. Consequently, the diverse bedrock properties that drive variations in soil properties, nutrients, and plant diversity can impact AMF communities, ultimately promoting plant growth and contributing to vegetation recovery.

Prevalence, Transmission and Genetic Diversity of Pyrazinamide Resistance Among Multidrug-Resistant Mycobacterium tuberculosis Isolates in Hunan, China.

Background: China is a country with a burden of high rates of both TB and multidrug-resistant TB (MDR-TB). However, published data on pyrazinamide (PZA) resistance are still limited in Hunan province, China. This study investigated the prevalence, transmission, and genetic diversity of PZA resistance among multidrug-resistant Mycobacterium tuberculosis isolates in Hunan province. Methods: Drug susceptibility testing (DST) with the Bactec MGIT 960 PZA kit and pyrazinamidase (PZase) testing were conducted on all 298 MDR clinical isolates. Moreover, 24-locus MIRU-VNTR and DNA sequencing of pncA, rpsA, and panD genes were conducted on 180 PZA-resistant (PZA-R) isolates. Results: The prevalence of PZA resistance among MDR-TB strains reached 60.4%. Newly diagnosed PZA-R TB patients and clustered isolates with identical pncA, rpsA, and panD mutations showed that transmission of PZA-R isolates played a significant role in the formation of PZA-R TB. Ninety-eight mutation patterns were observed in the pncA among 180 PZA-R isolates, and seventy-one (72.4%) were point mutations. Twenty-four of these mutations are new, including 2 base substitutions (V93G and T153S) and 22 nucleotide deletions or insertions. The W119C was found in PZA-S isolates, on the other hand, F94L and V155A mutations were found in both PZA resistant and susceptible isolates with positive PZase activity, indicating that they were not associated with PZA resistance. This is not entirely in line with the WHO catalogue. Ten novel rpsA mutations were found in 10 PZA-R isolates, which all combined with mutations in pncA. Thus, it is unpredictable whether these mutations in rpsA can impact PZA resistance. No panD mutation was found in all PZA-R isolates. Conclusion: DNA sequencing of pncA and PZase activity testing have great potential in predicting PZA resistance.

Omics analysis of Mycobacterium tuberculosis isolates uncovers Rv3094c, an ethionamide metabolism-associated gene.

Global control of the tuberculosis epidemic is threatened by increasing prevalence of drug resistant M. tuberculosis isolates. Many genome-wide studies focus on SNP-associated drug resistance mechanisms, but drug resistance in 5-30% of M. tuberculosis isolates (varying with antibiotic) appears unrelated to reported SNPs, and alternative drug resistance mechanisms involving variation in gene/protein expression are not well-studied. Here, using an omics approach, we identify 388 genes with lineage-related differential expression and 68 candidate drug resistance-associated gene pairs/clusters in 11 M. tuberculosis isolates (variable lineage/drug resistance profiles). Structural, mutagenesis, biochemical and bioinformatic studies on Rv3094c from the Rv3093c-Rv3095 gene cluster, a gene cluster selected for further investigation as it contains a putative monooxygenase/repressor pair and is associated with ethionamide resistance, provide insights on its involvement in ethionamide sulfoxidation, the initial step in its activation. Analysis of the structure of Rv3094c and its complex with ethionamide and flavin mononucleotide, to the best of our knowledge the first structures of an enzyme involved in ethionamide activation, identify key residues in the flavin mononucleotide and ethionamide binding pockets of Rv3094c, and F221, a gate between flavin mononucleotide and ethionamide allowing their interaction to complete the sulfoxidation reaction. Our work broadens understanding of both lineage- and drug resistance-associated gene/protein expression perturbations and identifies another player in mycobacterial ethionamide metabolism.

Bedrock outcrops weakly promote rather than inhibit soil carbon sequestration after vegetation restoration.

Vegetation restoration can increase soil carbon (C) content in karst regions characterized by highly exposed carbonate rocks; however, it remains unclear whether and how bedrock outcrops contribute to soil C-accumulation after vegetation restoration. We aimed to investigate the magnitude and mechanisms of bedrock outcrops on soil C-accumulation after vegetation restoration. Here, we selected 362 fixed locations to investigate changes in soil organic carbon (SOC) content and density before and after cropland restoration in a karst catchment with varying bedrock exposure ratios and initial soil C pools prior to restoration. Active vegetation restoration (i.e., cropland converted to forage grass, plantation forest, and a combination of grass and forest) and natural regeneration (cropland abandoned) were compared, with croplands maintained with no change as the control. Compared to croplands maintained with no change, SOC density significantly increased in the four vegetation restoration types. The SOC accumulation rate was higher for natural regeneration (39 g C m-2 yr-1) than for the three active restoration strategies (18-27 g C m-2 yr-1). SOC accumulation decreased with a higher initial pool size of soil C but increased with nitrogen accumulation and soil exchangeable calcium (Ca2+) concentration. Higher bedrock outcrops reduced soil volume but increased SOC content through their indirect effects on the initial pool size of soil C, external nitrogen inputs, and soil Ca2+ concentration. This weakly promoted rather than inhibited SOC sequestration. Our findings highlight the effectiveness of various restoration strategies in promoting SOC accumulation in karst areas, as well as the need to take bedrock outcrops and initial soil C pools into consideration when modeling SOC dynamics and maximizing C sinks for vegetation restoration.

Whole-Genome Sequencing and Epidemiological Investigation of Tuberculosis Outbreaks in High Schools in Hunan, China.

Background: Tuberculosis (TB) seriously threatens individual and public health. Recently, TB outbreaks in schools have been reported more frequently in China and have attracted widespread attention. We reported three TB outbreaks in high schools in Hunan Province, China. Methods: When a tuberculosis patient was reported in a school, we carried out field epidemiological investigations, including tuberculin skin testing (TST), chest X-ray (CXR) and laboratory test for all close contacts, and whole-genome sequencing (WGS) analyses to understand the transmission patterns, the causes and the risk factors for the outbreaks, thereby providing a foundation for the control of TB epidemics in schools. Results: A total of 49 students with TB patients were identified in the three schools where TB outbreaks occurred, including nine patients in School A, 14 patients in School B, and 26 patients in School C. In Schools A, B and C, the putative attack rates in the classes of the index case were 13.8% (8/58), 7.6% (5/66), and 40.4% (21/52), while the putative attack rates of expanding screening in the school were 0.3% (1/361), 0.2% (9/3955), and 0.2% (5/2080), respectively. Thirteen patients had patient delay, with a median delay interval of 69 days (IQR 30.5-113 days). Twelve patients had a healthcare diagnostic delay with a median delay interval of 32 days (IQR 24-82 days). Phylogenetic analysis of culture-positive patients revealed that most of them shared a small genetic distance (≤12 SNPs), with three separate genetic clusters (including one MDR-TB genomic cluster), indicating the recent transmission of Mycobacterium tuberculosis strains. Conclusion: This combination of field investigation and WGS analysis revealed the transmission of three TB outbreaks in schools. Reinforced implementation is needed to improve timely case finding and reduce diagnosis delay in routine TB control in the school population.

CD84 is a Suppressor of T and B Cell Activation during Mycobacterium tuberculosis Pathogenesis.

Interest in host-directed therapies as alternatives/adjuncts to antibiotic treatment has resurged with the increasing prevalence of antibiotic-resistant tuberculosis (TB). Immunotherapies that reinvigorate immune responses by targeting immune checkpoints like PD-1/PD-L1 have proved successful in cancer therapy. Immune cell inhibitory receptors that trigger Mycobacterium tuberculosis-specific immunosuppression, however, are unknown. Here, we show that the levels of CD84, a SLAM family receptor, increase in T and B cells in lung tissues from M. tuberculosis-infected C57BL/6 mice and in peripheral blood mononuclear cells (PBMCs) from pulmonary TB patients. M. tuberculosis challenge experiments using CD84-deficient C57BL/6 mice suggest that CD84 expression likely leads to T and B cell immunosuppression during M. tuberculosis pathogenesis and also plays an inhibitory role in B cell activation. Importantly, CD84-deficient mice showed improved M. tuberculosis clearance and longer survival than M. tuberculosis-infected wild-type (WT) mice. That CD84 is a putative M. tuberculosis infection-specific inhibitory receptor suggests it may be a suitable target for the development of TB-specific checkpoint immunotherapies. IMPORTANCE Immune checkpoint therapies, such as targeting checkpoints like PD-1/PD-L1, have proved successful in cancer therapy and can reinvigorate immune responses. The potential of this approach for treating chronic infectious diseases like TB has been recognized, but a lack of suitable immunotherapeutic targets, i.e., immune cell inhibitory receptors that trigger immunosuppression specifically during Mycobacterium tuberculosis pathogenesis, has limited the application of this strategy in the development of new TB therapies. Our focus in this study was to address this gap and search for an M. tuberculosis-specific checkpoint target. Our results suggest that CD84 is a putative inhibitory receptor that may be a suitable target for the development of TB-specific checkpoint immunotherapies.

Comparison of bacterial and fungal diversity and network connectivity in karst and non-karst forests in southwest China.

Microbial communities contribute to sustaining the function of terrestrial ecosystems and are influenced by soil type and climate gradients. The effects of karst and non-karst soils on bacterial and fungal profiles for seven climate gradients were assessed to better understand bacterial and fungal diversity and community composition in response to soil type with changes in soil physicochemical properties under different temperatures and precipitations. Bacterial and fungal abundance, diversity, and community composition differed between karst and non-karst forests. Bacterial and fungal richness, Shannon index, and bacterial abundance in karst forests were higher than non-karst forests, but the fungal abundance was lower. Mean annual temperature was negatively correlated with bacterial diversity in the karst forest and fungal abundance in karst and non-karst forests. The community composition of bacteria and fungi differed among these two soil types. The karst forest had greater connectivity among bacterial and fungal communities than non-karst forests. The bacterial members of Acidobacteria, Proteobacteria, Actinobacteria, and fungal groups of Ascomycota and Basidiomycota were mainly connected with other taxa in the network, implying that taxa were associated with highly functional potential. The relative abundance of Actinobacteria and Ascomycota was higher in karst than in non-karst forests. Proteobacteria and Basidiomycota showed the opposite results. A random forest and multiple regression tree analyses revealed that soil properties, specifically pH, calcium, and total nitrogen, were the main factors influencing the variation in bacterial and fungal profiles between karst and non-karst forests. This study provides novel evidence that the abundant microbial taxa were kinless hubs in co-occurrence patterns. Controlling complex networks of species interactions may contribute to improving soil nutrient processes rather than microbial diversity, enhancing our understanding of developing sustainable recovery strategies in fragile ecosystems.

The formation of large macroaggregates induces soil organic carbon sequestration in short-term cropland restoration in a typical karst area.

Cropland restoration induces litter and root inputs and promotes the development of biological soil crusts (BSCs), which may promote aggregate formation and soil organic carbon (SOC) sequestration. However, litter, roots and BSCs have not been simultaneously considered when assessing soil aggregate and aggregate-associated SOC fraction responses to cropland restoration in subtropical areas. Here, we measured particulate organic carbon (POC) and mineral-associated organic carbon (MOC) in bulk soils and soil aggregates after 15 years of cropland restoration. Soil samples of cropland (CL) and four cropland restoration types (plantation forest [PF], forage grassland [FG], mixed plantation of forest and forage grassland [FF], and abandoned natural grassland [NG]) from depths of 0-30 cm were collected. Cropland restoration significantly increased SOC and POC in bulk soil at the 0-5 cm depth. However, only in FG did SOC significantly increase at depths of 5-15 cm, and POC significantly increased at depths of 5-30 cm. The large macroaggregate (5-10 mm and 2-5 mm) proportions increased significantly at the 0-15 cm depth after cropland restoration, and FG, FF and NG also increased the 5-10 mm aggregate proportions at the 15-30 cm depth. The SOC sequestration in bulk soil with cropland restoration was attributed to increases in the aggregate-associated organic carbon (AAOC) pool in large macroaggregates, which was mainly attributed to the increased aggregate amount rather than the increased AAOC concentration in large macroaggregates. Our results also indicated that an increase in aggregate-associated particulate organic carbon (AAPOC) led to an increase in AAOC. Variation partitioning indicated that the formation of large macroaggregates was controlled by the litter-moss-root interactive effect in this karst area. FG could be a better short-term cropland restoration strategy, increasing large macroaggregates in deeper soil layers better than the other vegetation types and promoting soil carbon sequestration in deeper soil layers.

Soil carbon accumulation with increasing temperature under both managed and natural vegetation restoration in calcareous soils.

Vegetation restoration has been proposed as an effective strategy for increasing soil organic carbon (SOC) sequestration. However, the responses of SOC to managed and natural vegetation restoration strategies at a large scale are poorly understood due to the varying SOC components and changing climatic conditions. Here, we measured bulk SOC, particulate organic carbon (POC), and mineral-associated organic carbon (MOC) after 15 years of vegetation restoration along an elevation gradient with a corresponding temperature gradient in the calcareous soils of karst region, Southwest China. We compared managed plantation forest and naturally recovered shrubland vegetation restoration strategies, using cropland and mature forest as references. Overall, we found that the SOC and POC densities in both plantation forest and shrubland were significantly higher than in the cropland but lower than in the mature forest. There were no significant differences in the SOC pool between the plantation forest and shrubland. Furthermore, the relative changes in the SOC and POC densities increased with increasing mean annual temperature in the plantation forest and shrubland. Our results showed that both vegetation restoration strategies, characterized by higher soil microbial abundance and exchangeable Ca concentration, were beneficial to POC but not MOC accumulation, and sufficiently compensated SOC decomposition at lower elevation with higher MAT. Our results highlight the potential of both vegetation restoration strategies for promoting SOC accumulation in warmer karst regions and emphasize the necessity to understand soil carbon stabilization mechanisms in calcareous soils.

Systematic Evaluation of Mycobacterium tuberculosis Proteins for Antigenic Properties Identifies Rv1485 and Rv1705c as Potential Protective Subunit Vaccine Candidates.

The lack of efficacious vaccines against Mycobacterium tuberculosis (MTB) infection is a limiting factor in the prevention and control of tuberculosis (TB), the leading cause of death from an infectious agent. Improvement or replacement of the BCG vaccine with one that reliably protects all age groups is urgent. Concerns exist that antigens currently being evaluated are too homogeneous. To identify new protective antigens, we screened 1,781 proteins from a high-throughput proteome-wide protein purification study for antigenic activity. Forty-nine antigens (34 previously unreported) induced antigen-specific gamma interferon (IFN-γ) release from peripheral blood mononuclear cells (PBMCs) derived from 4,452 TB and suspected TB patients and 167 healthy donors. Three (Rv1485, Rv1705c, and Rv1802) of the 20 antigens evaluated in a BALB/c mouse challenge model showed protective efficacy, reducing lung CFU counts by 66.2%, 75.8%, and 60%, respectively. Evaluation of IgG2a/IgG1 ratios and cytokine release indicated that Rv1485 and Rv1705c induce a protective Th1 immune response. Epitope analysis of PE/PPE protein Rv1705c, the strongest candidate, identified a dominant epitope in its extreme N-terminal domain accounting for 90% of its immune response. Systematic preclinical assessment of antigens Rv1485 and Rv1705c is warranted.

Natural attenuation mechanism and health risk assessment of 1,1,2-trichloroethane in contaminated groundwater.

1,1,2-trichloroethane (TCA) is a contaminant in many pesticide and chemical fields. This study mainly described the potential effectiveness of the natural attenuation of 1,1,2-TCA in groundwater in a typical pesticide-contaminated field in east China. In this study, six typical 1,1,2-TCA-contaminated sites (MW14, MW21, MW25, MW31, MW36 and MW41) were selected, and the field investigation results indicated that there was an obvious decline in the concentrations of 1,1,2-TCA over time in all selected sites. Furthermore, the attenuation rate of 1,1,2-TCA concentration in sites MW14, MW21, MW25, MW31 and MW41 followed the first-order kinetic equation, and the first-order attenuation rate constants were calculated. The health risks of 1,1,2-TCA and its degradation product dichloroethane (DCA) were assessed and compared in site MW14. The result showed that the health risks of 1,1,2-TCA were much higher than those of DCA, and 1,1,2-TCA was the contamination source in this site. 16S rRNA sequencing was also conducted to investigate the diversity of the bacterial community in 1,1,2-trichloroethane (TCA)-contaminated groundwater, and Geobacter, Thauera, Pseudomonas, Diaphorobacter were the main species in the bacterial community.

Retrospective Analysis of False-Positive and Disputed Rifampin Resistance Xpert MTB/RIF Assay Results in Clinical Samples from a Referral Hospital in Hunan, China.

Concerns about the specificity of the Xpert MTB/RIF (Xpert) assay have arisen, as false-positive errors in the determination of Mycobacterium tuberculosis complex (MTBC) infection and rifampin (RIF) resistance in clinical practice have been reported. Here, we investigated 33 cases where patients were determined to be RIF susceptible using the Bactec MGIT 960 (MGIT) culture system but RIF resistant using the Xpert assay. Isolates from two of these patients were found not to have any mutations in the rifampin resistance determining region (RRDR) region of rpoB and had good treatment outcomes with first-line antituberculosis (anti-TB) drugs. The remaining 31 patients included 5 new cases and 26 previously treated patients. A large number of well-documented disputed mutations, including Leu511Pro, Asp516Tyr, His526Asn, His526Leu, His526Cys, and Leu533Pro, were detected, and mutations, including a 508 to 509 deletion and His526Gly, were described here as disputed mutations for the first time. Twenty-one (81%) of the 26 previously treated patients had poor treatment outcomes, and isolates from 19 (90%) of these 21 patients were resistant to isoniazid (INH) as determined using the MGIT culture system. Twenty-seven of the 31 isolates with disputed rpoB mutations were phenotypically resistant to INH, 21 (78%) being predicted by GenoType MTBDRplus to have a high level of INH resistance. Most (77.4%) of the isolates with disputed mutations were of the Beijing lineage. These findings have implications for the interpretation of false-positive and disputed rifampin resistance Xpert MTB/RIF results in clinical samples and provide guidance on how clinicians should manage patients carrying isolates with disputed rpoB mutations.

Evaluation of the Xpert MTB/RIF assay for diagnosis of tuberculosis and rifampin resistance in county-level laboratories in Hunan province, China.

BACKGROUND: The Xpert MTB/RIF showed high sensitivity and specificity in previous studies carried out in different epidemiological and geographical settings and patient populations in high-burden tuberculosis (TB) countries. However, there were little data obtained by validation or demonstration study of the assay in China. In this study, the performance of Xpert MTB/RIF was investigated in two county-level laboratories in Hunan Province, China. METHODS: Consecutive patients with suspected pulmonary tuberculosis (PTB) and suspicion for multidrug-resistant tuberculosis (MDR-TB) were enrolled. For each patient suspected to have PTB, three sputum specimens (one spot sputum, one night sputum, and one morning sputum) were collected and each sputum was tested with smear microscopy, Löwenstein-Jensen (LJ) culture, and Xpert MTB/RIF test. For comparison across subgroups and testing methods, 95% confidence intervals were calculated. All analyses were done with SPSS 16.0, and P < 0.05 was regarded as significant. RESULTS: For case detection, the sensitivity of Xpert MTB/RIF was 100% for smear- and culture-positive TB and 88.6% for smear-negative and culture-positive TB; the overall sensitivity was 94.5% for all culture-positive patients. The specificity was 99.8%. The sensitivity of Xpert MTB/RIF assay was 22.0% in clinical TB patients and the specificity reached 100.0% in the group of patients who are infected with nontuberculous mycobacteria. For the detection of rifampin resistance, the sensitivity of MTB/RIF RIF-resistance detection was 92.9%, and the specificity was 98.7%. Of the 26 Xpert MTB/RIF-positive and RIF-resistant patients confirmed by LJ proportion tests, 20 (76.9%) patients were infected by MDR-TB. CONCLUSIONS: The Xpert MTB/RIF assay is a highly sensitive and specific method for diagnosis of TB and RIF resistance, which will enable it to have the potential to be used in county-level laboratories and lead to the reduction of the infectious pool and improvements in TB control in China. Further evaluations in county-level laboratories for implementing the assay are still required.

Prevalence and molecular characteristics of drug-resistant Mycobacterium tuberculosis in Hunan, China.

To determine the prevalence and molecular characteristics of drug-resistant tuberculosis in Hunan province, drug susceptibility testing and spoligotyping methods were performed among 171 M. tuberculosis isolates. In addition, the mutated characteristics of 12 loci, including katG, inhA, rpoB, rpsL, nucleotides 388 to 1084 of the rrs gene [rrs(388-1084)], embB, pncA, tlyA, eis, nucleotides 1158 to 1674 of the rrs gene [rrs(1158-1674)], gyrA, and gyrB, among drug-resistant isolates were also analyzed by DNA sequencing. Our results indicated that the prevalences of isoniazid (INH), rifampin (RIF), streptomycin (SM), ethambutol (EMB), pyrazinamide (PZA), capreomycin (CAP), kanamycin (KAN), amikacin (AKM), and ofloxacin (OFX) resistance in Hunan province were 35.7%, 26.9%, 20.5%, 9.9% 15.2%, 2.3%, 1.8%, 1.2%, and 10.5%, respectively. The previously treated patients presented significantly increased risks for developing drug resistance. The majority of M. tuberculosis isolates belonged to the Beijing family. Almost all the drug resistance results demonstrated no association with genotype. The most frequent mutations of drug-resistant isolates were katG codon 315 (katG315), inhA15, rpoB531, rpoB526, rpoB516, rpsL43, rrs514, embB306, pncA96, rrs1401, gyrA94, and gyrA90. These results contribute to the knowledge of the prevalence of drug resistance in Hunan province and also expand the molecular characteristics of drug resistance in China.