Genetic surveillance and outcomes of pyrazinamide and fluoroquinolones-resistant tuberculosis in Taiwan.

BACKGROUND: Pyrazinamide (PZA) and fluoroquinolone (FQ), particularly moxifloxacin (MXF), are essential drugs in the World Health Organization (WHO) recommended short-course regimen to treat drug-susceptible tuberculosis (TB). METHODS: To understand the extent of PZA and MXF susceptibility in general TB cases in Taiwan, we conducted retrospective analyses of 385 conservative Mycobacterium tuberculosis complex (MTBC) isolates identified from 4 TB laboratories in different regions of Taiwan. The case information was obtained from the TB registry. Genotypic drug susceptibility testing (DST) was performed by sequencing drug-resistance associated genes, PZA (pncA) and FQ (gyrA, and gyrB). Phenotypic DST was determined using the Bactec MGIT 960 system or the agar proportion method. Genotyping was carried out using spacer oligonucleotide typing. RESULTS: In this study, 4.7% (18/385) cases' isolates harbored pncA mutations and 7.0% (27/385) cases' isolates harbored gyrA or gyrB mutation. Notably, pncA mutation was associated with Beijing family genotypes (P = 0.028), East African-Indian (EAI) genotypes (P = 0.047) and MDR-TB (P < 0.001). Whereas, gyrA or gyrB mutation was associated with EAI genotypes (P = 0.020) and MDR-TB (P = 0.006). In addition, a statistically significant difference was found between the favorable outcomes using active and inactive PZA (P = 0.009) in 38 case isolates with any pncA, gyrA, or gyrB mutation. CONCLUSION: We concluded that routine PZA and FQ susceptibility tests are recommended for guiding the treatment of TB.

Whole-genome sequencing-based analyses of drug-resistant Mycobacterium tuberculosis from Taiwan.

Drug-resistant tuberculosis (DR-TB) posed challenges to global TB control. Whole-genome sequencing (WGS) is recommended for predicting drug resistance to guide DR-TB treatment and management. Nevertheless, data are lacking in Taiwan. Phenotypic drug susceptibility testing (DST) of 12 anti-TB drugs was performed for 200 Mycobacterium tuberculosis isolates. WGS was performed using the Illumina platform. Drug resistance profiles and lineages were predicted in silico using the Total Genotyping Solution for TB (TGS-TB). Using the phenotypic DST results as a reference, WGS-based prediction demonstrated high concordance rates of isoniazid (95.0%), rifampicin (RIF) (98.0%), pyrazinamide (98.5%) and fluoroquinolones (FQs) (99.5%) and 96.0% to 99.5% for second-line injectable drugs (SLIDs); whereas, lower concordance rates of ethambutol (87.5%), streptomycin (88.0%) and ethionamide (84.0%). Furthermore, minimum inhibitory concentrations confirmed that RIF rpoB S450L, FQs gyrA D94G and SLIDs rrs a1401g conferred high resistance levels. Besides, we identified lineage-associated mutations in lineage 1 (rpoB H445Y and fabG1 c-15t) and predominant lineage 2 (rpoB S450L and rpsL K43R). The WGS-based prediction of drug resistance is highly concordant with phenotypic DST results and can provide comprehensive genetic information to guide DR-TB precision therapies in Taiwan.

Surveillance of multidrug-resistant tuberculosis in Taiwan, 2008-2019.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) is a major contributor to global cases of antimicrobial resistance and remains a public health challenge. To understand the extent and trend of DR-TB under an enhanced multidrug-resistant TB (MDR-TB) management program, we conducted a population-based retrospective study of 1511 Taiwanese MDR-TB cases reported from 2008 to 2019. METHODS: We obtained patient demographics and clinical and bacteriological information from the National TB Registry and the Infectious Disease Notification System. RESULTS: Of the 1511 MDR-TB patients, 941 were new cases, 485 were previously treated, and 85 had an unknown history of treatment. The male to female ratio was 2.75, and the median age of the patients was 57 years (IQR: 45-72). We observed a significant decline in MDR-TB cases, with annual percentage change (APC) of -4.17%. However, new and previously treated MDR-TB cases had APCs of -1.41% and -9.18%, respectively. The rates of MDR-TB resistance to ethambutol, streptomycin and pyrazinamide were 47.2%, 42.4% and 28.9%, respectively, whereas the rates of resistance to fluoroquinolones and second-line injectable drugs (SLIDs) were 4.1-7.1%, 9.0-14.1%; and the rate of extensively drug-resistant TB was 1.9%, respectively. Furthermore, we observed a decreasing trend of resistance to SLIDs (APCs -7.0% to -8.2%) in new cases and a significant decreasing trend of resistance to moxifloxacin (-24.6%) and levofloxacin (-23.3%) in previously treated cases. CONCLUSION: The decreasing trend of MDR-TB and resistance to second-line drugs suggested that our programmatic management of TB was effective and that the impact on TB control was profound.

Development and Assessment of a Novel Whole-Gene-Based Targeted Next-Generation Sequencing Assay for Detecting the Susceptibility of Mycobacterium tuberculosis to 14 Drugs.

Targeted next-generation sequencing (tNGS) has emerged as an alternative method for detecting drug-resistant tuberculosis (DR-TB). To provide comprehensive drug susceptibility information and to address mutations missed by available commercial molecular diagnostics, we developed and evaluated a tNGS panel with 22 whole-gene targets using the Ion Torrent platform to predict drug resistance to 14 drugs, namely, rifampicin (RIF), isoniazid (INH), ethambutol (EMB), pyrazinamide (PZA), moxifloxacin (MFX), levofloxacin (LFX), amikacin (AMK), capreomycin (CM), kanamycin (KM), streptomycin (SM), bedaquiline (BDQ), clofazimine (CFZ), linezolid (LZD), and delamanid (DLM). We selected 50 and 35 Mycobacterium tuberculosis isolates with various DR profiles as the training set and the challenge set, respectively. Comparative variant analyses of the DR genes were performed using Sanger sequencing and whole-genome sequencing (WGS). Phenotypic drug susceptibility testing (pDST) results were used as gold standards. Regarding the limit of detection, the tNGS assay detected 2.9 to 3.8% minority variants in 4% mutant mixtures. The sensitivity and specificity of tNGS were 97.0% (95% confidence interval [CI] = 93.1 to 98.7%) and 99.1% (95% CI = 97.7 to 99.7%), respectively. The concordance of tNGS with pDST was 98.5% (95% CI = 97.2 to 99.2%), which was comparable to that of WGS (98.7%, 95% CI = 97.4 to 99.3%) and better than that of Sanger sequencing (96.9%, 95% CI = 95.3 to 98.0%). The agreement between tNGS and pDST was almost perfect for RIF, INH, EMB, MFX, LFX, AMK, CM, KM, SM, BDQ, and LZD (kappa value = 0.807 to 1.000) and substantial for PZA (kappa value = 0.791). Our customized novel whole-gene-based tNGS panel is highly consistent with pDST and WGS for comprehensive and accurate prediction of drug resistance in a strengthened and streamlined DR-TB laboratory program. IMPORTANCE We developed and validated a tNGS assay that was the first to target 22 whole genes instead of regions of drug resistance genes and comprehensively detected susceptibility to 14 anti-TB drugs, with great flexibility to include new or repurposed drugs. Notably, we demonstrated that our custom-designed Ion AmpliSeq TB research panel platform had high concordance with pDST and could significantly reduce turnaround time (by approximately 70%) to meet a clinically actionable time frame. Our tNGS assay is a promising DST solution for providing needed clinical information for precision medicine-guided therapies for DR-TB and allows the rollout of active pharmacovigilance.

Molecular Epidemiological Characteristics of Mycobacterium abscessus Complex Derived from Non-Cystic Fibrosis Patients in Japan and Taiwan.

Mycobacterium abscessus complex (MABC) is a group of emerging, highly antimicrobial-resistant non-tuberculous mycobacteria. Specific MABC clones are spreading globally in patients with cystic fibrosis (CF); however, associated genomic epidemiology is lacking in East Asia, with very few patients with CF. Here, we investigated MABC populations derived from non-CF patients in Japan and Taiwan. Analysis of whole-genome sequencing data of 220 MABC isolates revealed that 112, 105, and 3 were M. abscessus subsp. abscessus (ABS), M. abscessus subsp. massiliense (MAS), and M. abscessus subsp. bolletii (BOL), respectively. Moreover, >50% of ABS and >70% of MAS were related to four predominant clones in the region. Known mutations conferring macrolide resistance were rare (1.4%) and were not enriched in the predominant clones. Conversely, the macrolide-susceptible erm(41) T28C mutation was significantly enriched in one predominant ABS clone. The most predominant ABS clone was genetically related to the previously described dominant circulating clone (DCC)1 in patients with CF, whereas no isolates were related to DCC2; isolates related to DCC3 were not necessarily predominant in our sample set. We found that the erm(41) T28C mutants spread globally, and some of them reacquired the functional erm(41) gene through both point mutation and recombination. This study revealed predominant MABC clones in Japan and Taiwan and their relationship with the globally superadding clones in the patient community with CF. Our study provides insights into the genetic characteristics of globally dominant and area-specific strains isolated from patients with or without CF and differences between globally spread and regionally specific strains. IMPORTANCE Members of Mycobacterium abscessus complex (MABC) are frequently isolated from patients. Studies have reported that predominant clones of MABC (known as dominant circulating clones; DCCs) are distributed worldwide and transmitted from humans to humans in patients with cystic fibrosis (CF). However, associated genomic epidemiology has not yet been conducted in East Asia, including Japan and Taiwan, where there are only a few patients with CF. Using whole-genome sequencing data derived from non-CF patients in Japan and Taiwan, we revealed prevalent clones and the incidence of macrolide resistance-associated mutations in the MABC population in this region. We also clarified the associations between these predominant clones and DCCs in the global CF patient community. Our results would assist further studies in elucidating the genetic characteristics of strains isolated from patients with or without CF, the differences between globally spread and regionally specific strains, and the adaptive evolution of MABC within the host.

Bovine tuberculosis in Taiwan, 2008-2019.

Bovine tuberculosis (bTB) is a zoonosis caused by Mycobacterium bovis. The impact of bTB on global TB control has been underestimated. We adopted the One Health approach to human bTB surveillance in Taiwan. Of 20,972 human TB cases, 202 (1.0%) were bTB, 78.2% were in males, 85.1% were new cases, 83.2% were pulmonary TB, and most were in Central (52.5%) and Southern (24.8%) Taiwan. Only 18.8% of bTB patients had known animal contact. Of the 202 human M. bovis strains, 100% were resistant to pyrazinamide (PZA), 30.2% were concurrently resistant to isoniazid (INH) and 2.0% were multidrug resistant, defined as being resistant to at least INH and rifampin. Whereas, of the 22 animal M. bovis strains, 100% and 22.7% were resistant to PZA and INH, respectively. Seven spoligotypes and 25 mycobacterial interspersed repetitive unit genotypes were identified. The predominant genotype, SB0265, was also prevalent in livestock. Notably, six animal-specific M. bovis genotypes were identified. bTB differential diagnosis and drug resistance detection are crucial for TB control. Comprehensive surveillance and human-animal interface investigations are needed.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-Resistant Tuberculosis (MDR-TB): a 5-Year Prospective In Vitro Surveillance Study of Bedaquiline and Other Second-Line Drug Susceptibility Testing in MDR-TB Isolates.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant tuberculosis (MDR-TB) (DREAM) was a 5-year (2015 to 2019) phenotypic drug resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5,036 MDR-TB isolates of bedaquiline treatment-naive patients to bedaquiline and other antituberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) MIC methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015 to 0.12 µg/ml) was adjusted compared with ranges from a multilaboratory, multicountry reproducibility study conforming to Clinical and Laboratory Standards Institute Tier-2 criteria. Epidemiological cutoff values of 0.12 µg/ml by BMD and 0.25 µg/ml by AD were consistent with previous bedaquiline breakpoints. An area of technical uncertainty or intermediate category was set at 0.25 µg/ml and 0.5 µg/ml for BMD and AD, respectively. When applied to the 5,036 MDR-TB isolates, bedaquiline-susceptible, -intermediate, and -resistant rates were 97.9%, 1.5%, and 0.6%, respectively, for BMD and 98.8%, 0.8%, and 0.4% for AD. Resistance rates were the following: 35.1% ofloxacin, 34.2% levofloxacin, 33.3% moxifloxacin, 1.5% linezolid, and 2% clofazimine. Phenotypic cross-resistance between bedaquiline and clofazimine was 0.4% in MDR-TB and 1% in pre-extensively drug-resistant (pre-XDR-TB)/XDR-TB populations. Coresistance to bedaquiline and linezolid and clofazimine and linezolid were 0.1% and 0.3%, respectively, in MDR-TB and 0.2% and 0.4%, respectively, in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naive population. No treatment-limiting patterns for cross-resistance and coresistance have been identified with key TB drugs to date.

Primary Bedaquiline Resistance Among Cases of Drug-Resistant Tuberculosis in Taiwan.

Bedaquiline (BDQ), which is recommended for the treatment of drug-resistant tuberculosis (DR-TB), was introduced in Taiwan in 2014. Due to the alarming emergence of BDQ resistance, we conducted BDQ resistance analyses to strengthen our DR-TB management program. This retrospective population-based study included initial Mycobacterium tuberculosis isolates from 898 rifampicin-resistant (RR) or multidrug-resistant (MDR) TB cases never exposed to BDQ during 2008-2019. We randomly selected 65 isolates and identified 28 isolates with BDQ MIC<0.25µg/ml and MIC≥0.25µg/ml as the control and study groups, respectively. BDQ drug susceptibility testing (DST) using the MGIT960 system and Sanger sequencing of the atpE, Rv0678, and pepQ genes was conducted. Notably, 18 isolates with BDQ MIC=0.25µg/ml, 38.9% (7/18), and 61.1% (11/18) isolates were MGIT-BDQ resistant and susceptible, respectively. Consequently, we recommended redefining MIC=0.25µg/ml as an intermediate-susceptible category to resolve discordance between different DST methods. Of the 93 isolates, 22 isolates were MGIT-BDQ-resistant and 77.3% (17/22) of MGIT-BDQ-resistant isolates harbored Rv0678 mutations. After excluding 2 MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day), 100% (15/15) harbored Rv0678 gene mutations, including seven novel mutations [g-14a, Ile80Ser (N=2), Phe100Tyr, Ala102Val, Ins g 181-182 frameshift mutation (N=2), Del 11-63 frameshift mutation, and whole gene deletion (N=2)]. Since the other 22.7% (5/22) MGIT-BDQ-resistant isolates with borderline resistance (GU400growth control-GU100BDQ≤1day) had no mutation in three analyzed genes. For isolates with phenotypic MGIT-BDQ borderline resistance, checking for GU differences or conducting genotypic analyses are suggested for ruling out BDQ resistance. In addition, we observed favorable outcomes among patients with BDQ-resistant isolates who received BDQ-containing regimens regardless of Rv0678 mutations. We concluded that based on MIC≥0.25µg/ml, 3.1% (28/898) of drug-resistant TB cases without BDQ exposure showed BDQ resistance, Rv0678 was not a robust marker of BDQ resistance, and its mutations were not associated with treatment outcomes.

Acquired Resistance to Isoniazid During Isoniazid Monotherapy in a Subject with Latent Infection Following Household Rifampicin-Resistant Tuberculosis Contact: A Case Report.

Appropriate treatment is the key element in eliminating tuberculosis (TB), and requires prompt diagnosis. We presented a case of a household contact of rifampicin-resistant TB revealing reactive IFN-gamma release assay with unsuspicious clinical and radiologic examinations. She was diagnosed with latent tuberculosis infection (LTBI) and treated with isoniazid monotherapy. On the ninth month, she developed a progressive cough and was found to harbor active TB disease with added resistance to isoniazid. An individualized anti-TB regimen consisting of moxifloxacin, kanamycin, prothionamide, ethambutol, and pyrazinamide was prescribed for 20 months, leading to sputum culture conversion and improvement of the reported symptom. No recurrence was observed on one-year follow-up. Assuming high compliance to therapy, we propose that the patient may have been underdiagnosed and received sub-optimal treatment leading to acquired-drug resistance. Conventional diagnosis methods based on immunological assay and radiographical findings may be insufficient to distinguish the incipient and subclinical states of TB from LTBI.

Disputed rpoB Mutations in Mycobacterium tuberculosis and Tuberculosis Treatment Outcomes.

Discordant results between genotypic drug susceptibility testing (gDST) and phenotypic DST (pDST) for Mycobacterium tuberculosis isolates with disputed (discordance between gDST and pDST results) mutations affect rifampin (RIF)-resistant (RR) and multidrug-resistant (MDR) tuberculosis (TB) treatments due to a lack of practical clinical guidelines. To investigate the role of disputed rpoB mutations in M. tuberculosis and TB treatment outcomes, initial isolates of 837 clinical RR- or MDR-TB cases confirmed during 2014 to 2018 were retested using agar-based RIF pDST and rpoB gene sequencing. MICs were determined for isolates with disputed rpoB mutations. Disputed rpoB mutations were identified in 77 (9.2%) M. tuberculosis isolates, including 50 (64.9%) and 14 (18.2%) phenotypically RIF- and rifabutin (RFB)-resistant isolates, respectively. The predominant single mutations were those encoding L533P (a change of L to P at position 533) (44.2%) and L511P (20.8%). Most of the isolates harboring mutations encoding L511P (87.5%), H526N (100%), D516Y (70.0%), and L533P (63.6%) had MICs of ≤1 mg/liter, whereas isolates harboring the mutation encoding H526L (75%) had a MIC of >1 mg/liter. Of the 63 cases with treatment outcomes available, 11 (17.5%) cases died, 1 (1.6%) case transferred out, and 51 (81%) cases had favorable outcomes, including 8 and 20 cases treated with standard-dose RIF- and RFB-containing regimens, respectively. Excluding cases that transferred out or received no or 1-day treatment, we observed statistically significant differences between the outcomes using active and inactive fluoroquinolones (FQs) (P = 0.008, odds ratio = 0.05 [95% confidence interval, 0.01 to 0.38]) in 57 cases (where active means a case susceptible to the drug and inactive means a case resistant to the drug or drug not used). We concluded that disputed rpoB mutations are not rare. Depending on the resources available, sequencing and/or MIC testing is recommended for better management of RR- and MDR-TB cases.

Sensible Functional Linear Discriminant Analysis Effectively Discriminates Enhanced Raman Spectra of Mycobacterium Species.

Tuberculosis caused by Mycobacterium tuberculosis complex (MTBC) is one of the major infectious diseases in the world. Identification of MTBC and differential diagnosis of nontuberculous mycobacteria (NTM) species impose challenges because of their taxonomic similarity. This study describes a differential diagnosis method using the surface-enhanced Raman scattering (SERS) measurement of molecules released by Mycobacterium species. Conventional principal component analysis and linear discriminant analysis methods successfully separated the acquired spectrum of MTBC from those of NTM species but failed to distinguish between the spectra of different NTM species. A novel sensible functional linear discriminant analysis (SLDA), projecting the averaged spectrum of a bacterial specie to the subspace orthogonal to the within-species random variation, thereby eliminating its influence in applying linear discriminant analysis, was employed to effectively discriminate not only MTBC but also species of NTM. The successful demonstration of this SERS-SLDA method opens up new opportunities for the rapid differentiation of Mycobacterium species.

A Glutamine Insertion at Codon 432 of RpoB Confers Rifampicin Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G158R, RpoB V168A, RpoB S188P, and RpoB Q432insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H37Rv reference strain. The plasmid complementation of RpoB indicated that G158R, V168A, and S188P did not affect the MIC of RIF. However, the MIC of RIF was increased in H37Rv carrying RpoB Q432insQ. To confirm the correlation between RIF resistance and Q432insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q432insQ) into H37Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q432insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q432insQ causes RIF resistance in M. tuberculosis.

Two Pandemics, One Challenge-Leveraging Molecular Test Capacity of Tuberculosis Laboratories for Rapid COVID-19 Case-Finding.

In many settings, the ongoing coronavirus disease (COVID-19) pandemic coincides with other major public health threats, in particular tuberculosis. Using tuberculosis (TB) molecular diagnostic infrastructure, which has substantially expanded worldwide in recent years, for COVID-19 case-finding might be warranted. We analyze the potential of using TB diagnostic and research infrastructures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We focused on quality control by adapting the 12 Quality System Essentials framework to the COVID-19 and TB context. We conclude that diagnostic infrastructures for TB can in principle be leveraged to scale-up SARS-CoV-2 testing, in particular in resource-poor settings. TB research infrastructures also can support sequencing of SARS-CoV-2 to study virus evolution and diversity globally. However, fundamental principles of quality management must be followed for both TB and SARS-CoV-2 testing to ensure valid results and to minimize biosafety hazards, and the continuity of TB diagnostic services must be guaranteed at all times.

Two Novel katG Mutations Conferring Isoniazid Resistance in Mycobacterium tuberculosis.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, is among the top 10 leading causes of death worldwide. The treatment course for TB is challenging; it requires antibiotic administration for at least 6 months, and bacterial drug resistance makes treatment even more difficult. Understanding the mechanisms of resistance is important for improving treatment. To investigate new mechanisms of isoniazid (INH) resistance, we obtained three INH-resistant (INH-R) M. tuberculosis clinical isolates collected by the Taiwan Centers for Disease Control (TCDC) and sequenced genes known to harbor INH resistance-conferring mutations. Then, the relationship between the mutations and INH resistance of these three INH-R isolates was investigated. Sequencing of the INH-R isolates identified three novel katG mutations resulting in R146P, W341R, and L398P KatG proteins, respectively. To investigate the correlation between the observed INH-R phenotypes of the clinical isolates and these katG mutations, wild-type katG from H37Rv was expressed on a plasmid (pMN437-katG) in the isolates, and their susceptibilities to INH were determined. The plasmid expressing H37Rv katG restored INH susceptibility in the two INH-R isolates encoding the W341R KatG and L398P KatG proteins. In contrast, no phenotypic change was observed in the KatG R146P isolate harboring pMN437-katG. H37Rv isogenic mutant with W341R KatG or L398P KatG was further generated. Both showed resistant to INH. In conclusion, W341R KatG and L398P KatG conferred resistance to INH in M. tuberculosis, whereas R146P KatG did not affect the INH susceptibility of M. tuberculosis.

Genomic Analysis of Mycobacterium tuberculosis Isolates and Construction of a Beijing Lineage Reference Genome.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis, kills over 1 million people worldwide annually. Development of drug resistance (DR) in the pathogen is a major challenge for TB control. We conducted whole-genome analysis of seven Taiwan M. tuberculosis isolates: One drug susceptible (DS) and five DR Beijing lineage isolates and one DR Euro-American lineage isolate. Developing a new method for DR mutation identification and applying it to the next-generation sequencing (NGS) data from the 6 Beijing lineage isolates, we identified 13 known and 6 candidate DR mutations and provided experimental support for 4 of them. We assembled the genomes of one DS and two DR Beijing lineage isolates and the Euro-American lineage isolate using NGS data. Moreover, using both PacBio and NGS sequencing data, we obtained a high-quality assembly of an extensive DR Beijing lineage isolate. Comparative analysis of these five newly assembled genomes and two published complete genomes revealed a large number of genetic changes, including gene gains and losses, indels and translocations, suggesting rapid evolution of M. tuberculosis. We found the MazEF toxin-antitoxin system in all the seven isolates studied and several interesting mutations in MazEF proteins. Finally, we used the four assembled Beijing lineage genomes to construct a high-quality Beijing lineage reference genome that is DS and contains all the genes in the four genomes. It contains 212 genes not found in the standard reference H37Rv, which is Euro-American. It is therefore a better reference than H37Rv for the Beijing lineage, the predominant lineage in Asia.

Effect of ventilation improvement during a tuberculosis outbreak in underventilated university buildings.

The role of ventilation in preventing tuberculosis (TB) transmission has been widely proposed in infection control guidance. However, conclusive evidence is lacking. Modeling suggested the threshold of ventilation rate to reduce effective reproductive ratio (ratio between new secondary infectious cases and source cases) of TB to below 1 is corresponding to a carbon dioxide (CO2 ) level of 1000 parts per million (ppm). Here, we measured the effect of improving ventilation rate on a TB outbreak involving 27 TB cases and 1665 contacts in underventilated university buildings. Ventilation engineering decreased the maximum CO2 levels from 3204 ± 50 ppm to 591-603 ppm. Thereafter, the secondary attack rate of new contacts in university dropped to zero (mean follow-up duration: 5.9 years). Exposure to source TB cases under CO2 >1000 ppm indoor environment was a significant risk factor for contacts to become new infectious TB cases (P < .001). After adjusting for effects of contact investigation and latent TB infection treatment, improving ventilation rate to levels with CO2 <1000 ppm was independently associated with a 97% decrease (95% CI: 50%-99.9%) in the incidence of TB among contacts. These results show that maintaining adequate indoor ventilation could be a highly effective strategy for controlling TB outbreaks.

Impact of universal drug susceptibility testing and effective management of multidrug-resistant tuberculosis in Taiwan.

BACKGROUND: The treatment outcomes of multidrug-resistant tuberculosis (MDR-TB) patients in the 1990s in Taiwan was not satisfactory. To strengthen programmatic management of drug-resistant tuberculosis (PMDT), Taiwan MDR-TB Consortium (TMTC) was established in 2007. We assess the performance and epidemiologic impact of TMTC. METHODOLOGY/PRINCIPLE FINDINGS: We analyzed the trends of proportion of TB cases with drug susceptibility testing, enrollment of MDR-TB patients into TMTC and outcomes of treatment of all MDR-TB patients in Taiwan from 2007-2016. We computed the trends of both incidence and prevalence of MDR-TB from 2007-2016. We assessed the trends of MDR-TB among both new and recurrent TB cases. The proportion of TB cases with drug susceptibility testing results increased from 24.2% in 2007 to 97.9% in 2016. Of the 1,452 MDR-TB patients who were eligible for TMTC care, 1,197 (82.4%) were enrolled in TMTC, in whom 82.9% had treatment success. MDR-TB incidence was 9.0 cases per million in 2007, which declined to 4.6 cases per million in 2016 (p<0.0001). MDR-TB prevalence decreased from 19.4 cases per million in 2007 to 8.4 cases per million in 2016 (p<0.0001). The proportion of MDR-TB among new TB cases decreased from 1.4% in 2010 to 1.0% in 2016 (p = 0.039); and that among recurrent TB cases from 9.0% in 2010 to 1.8% in 2016 (p<0.0001). CONCLUSIONS: We concluded that effective PMDT have had a significant impact on the epidemic of drug-resistant TB in Taiwan.

A new oligonucleotide array for the detection of multidrug and extensively drug-resistance tuberculosis.

Drug-resistant tuberculosis (TB) is a global crisis and a threat to health security. Since conventional drug susceptibility testing (DST) takes several weeks, we herein described a molecular assay to rapidly identify multidrug-resistant (MDR) and extensively drug-resistant (XDR) and reveal transmission associated-mutations of Mycobacterium tuberculosis complex (MTBC) isolates in 6 to 7 hours. An array was designed with 12 pairs of primers and 60 single nucleotide polymorphisms of 9 genes: rpoB, katG, inhA, ahpC, embB, rpsL, gyrA, rrs and eis. We assessed the performance of the array using 176 clinical MTBC isolates. The results of culture-based DST were used as the gold standard, the GenoType MTBDRplus and MTBDRsl tests were used for parallel comparison, and gene sequencing was performed to resolve the discordance. The sensitivities and specificities of the array are comparable to those of the MTBDRplus test for resistance to isoniazid (INH) (100.0%, 96.7%) and rifampicin (RIF) (99.4%, 96.7%) and of the MTBDRsl test for resistance to fluoroquinolones (FQs) (100%, 100%) and second-line injectable drugs (SLIDs) (98.3%, 100%). The sensitivities of the array for detecting resistance to ethambutol and streptomycin were 79.3% and 64.9%, respectively. The array has potential as a powerful tool for clinical diagnosis and epidemiological investigations.

Redefining MDR-TB: Comparison of Mycobacterium tuberculosis clinical isolates from Russia and Taiwan.

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis are global challenges due to the limited number of effective drugs for treatment. Treatment with less than 4-5 effective drugs might lead to the further emergence of drug resistance and poor clinical outcomes. For better prediction of treatment outcomes, we compared drug-resistance profiles of consecutive clinical MDR Mycobacterium tuberculosis isolates from high- and low-burden settings. This was a retrospective cohort study. We analysed 225 and 229 MDR isolates from Moscow (Russia) and Taiwan, respectively, obtained between 2014 and 2015. Drug susceptibility testing was performed by the Bactec MGIT 960 automated system and the agar proportion method. Detection of resistance-associated mutations in the M. tuberculosis genome was carried out by an array and/or sequencing of selected loci. The principal differences between resistance profiles of MDR isolates in the two countries were the percentages of pre-XDR (40.9% vs. 14.8%) and XDR (34.7% vs. 1.7%) isolates, both of which were significantly higher in Moscow isolates. Forty-eight (33%) of 147 MDR and pre-XDR Russian isolates fall into a group with less than four effective drugs, which accounts for 40% (N = 120) of these isolates. The other 60% in this group were XDR strains (N = 72). Consequently, the average number of effective anti-tuberculosis drugs for MDR-TB treatment was lower for Russian isolates (3 vs. 7). Furthermore, a notable percentage (9%) of isolates resistant to kanamycin harboured mutations in the whiB7 locus, which was not detected by molecular tests targeting common mutations in the rrs and eis loci. We found that 98.2% and 45.9% of MDR isolates from Moscow and Taiwan, respectively, were resistant to streptomycin. Molecular tests for detecting resistance to drugs other than rifampicin, isoniazid, fluoroquinolones, and second-line injectable drugs are needed for individualized therapy. The conventional MDR treatment schemes most probably fail in these cases due to the limited number of effective drugs.

Performance of an Xpert-based diagnostic algorithm for the rapid detection of drug-resistant tuberculosis among high-risk populations in a low-incidence setting.

Timely diagnosis of drug-resistant tuberculosis (DR-TB) is beneficial for case treatment and management. We implemented an algorithm to improve molecular diagnostic utilization to intensify DR-TB case findings. The GeneXpert MTB/RIF (Xpert) test was used for initial diagnosis. Samples with Mycobacterium tuberculosis complex (MTBC)-positive and rifampicin resistance (RR) results were subsequently and simultaneously tested using the GenoType MTBDRplus (DRplus) and MTBDRsl (DRsl) tests. This prospective cohort study enrolled 2957 high-risk DR-TB cases. We tested sputum specimens using conventional mycobacteriological and molecular tests. Gene sequencing was performed to resolve discordant results. According to the Xpert test, 33.6% of specimens were MTBC-positive and 5.1% were RR. RR specimens were further analyzed in the DRplus and DRsl tests. We identified 1 extensively drug-resistant (XDR), 8 pre-XDR, 18 simple multidrug-resistant (MDR), 22 mono-RR, and 2 RR cases with concurrent second-line injection DR-TB. Of these, 25 (49%) were relapses, 13 (25.5%) were treatment failures, 10 (19.6%) were from MDR-TB high-incidence areas/countries, 1 was from MDR-TB contact and 2 were unknown. Among culture-positive TB cases, the sensitivities, specificities, and positive predictive values (PPVs) of the Xpert test and RR cases were 73.6% and 100.0%, 85.7% and 98.6%, and 73.5% and 80.0%, respectively. Gene sequencing of discordant results revealed 7 disputed rpoB mutations and 2 silent mutations for RIF, 1 ahpC mutation for isoniazid and 1 gyrA mutation for fluoroquinolone. The algorithm effectively identified approximately 23% of annual MDR-/XDR-TB and 37.5% of RR-TB cases that were enrolled in our DR-TB treatment and management program within 3 days.