Estimation of country-specific tuberculosis resistance antibiograms using pathogen genomics and machine learning.

BACKGROUND: Global tuberculosis (TB) drug resistance (DR) surveillance focuses on rifampicin. We examined the potential of public and surveillance Mycobacterium tuberculosis (Mtb) whole-genome sequencing (WGS) data, to generate expanded country-level resistance prevalence estimates (antibiograms) using in silico resistance prediction. METHODS: We curated and quality-controlled Mtb WGS data. We used a validated random forest model to predict phenotypic resistance to 12 drugs and bias-corrected for model performance, outbreak sampling and rifampicin resistance oversampling. Validation leveraged a national DR survey conducted in South Africa. RESULTS: Mtb isolates from 29 countries (n=19 149) met sequence quality criteria. Global marginal genotypic resistance among mono-resistant TB estimates overlapped with the South African DR survey, except for isoniazid, ethionamide and second-line injectables, which were underestimated (n=3134). Among multidrug resistant (MDR) TB (n=268), estimates overlapped for the fluoroquinolones but overestimated other drugs. Globally pooled mono-resistance to isoniazid was 10.9% (95% CI: 10.2-11.7%, n=14 012). Mono-levofloxacin resistance rates were highest in South Asia (Pakistan 3.4% (0.1-11%), n=111 and India 2.8% (0.08-9.4%), n=114). Given the recent interest in drugs enhancing ethionamide activity and their expected activity against isolates with resistance discordance between isoniazid and ethionamide, we measured this rate and found it to be high at 74.4% (IQR: 64.5-79.7%) of isoniazid-resistant isolates predicted to be ethionamide susceptible. The global susceptibility rate to pyrazinamide and levofloxacin among MDR was 15.1% (95% CI: 10.2-19.9%, n=3964). CONCLUSIONS: This is the first attempt at global Mtb antibiogram estimation. DR prevalence in Mtb can be reliably estimated using public WGS and phenotypic resistance prediction for key antibiotics, but public WGS data demonstrates oversampling of isolates with higher resistance levels than MDR. Nevertheless, our results raise concerns about the empiric use of short-course fluoroquinolone regimens for drug-susceptible TB in South Asia and indicate underutilisation of ethionamide in MDR treatment.

Phenotype versus genotype discordant rifampicin susceptibility testing in tuberculosis: implications for a diagnostic accuracy.

IMPORTANCE: An accurate diagnosis of drug resistance in clinical isolates is an important step for better treatment outcomes. The current study observed a higher discordance rate of rifampicin resistance on Mycobacteria Growth Indicator Tube (MGIT) drug susceptibility testing (DST) than Lowenstein-Jenson (LJ) DST when compared with the rpoB sequencing. We detected a few novel mutations and their combination in rifampicin resistance isolates that were missed by MGIT DST and may be useful for the better management of tuberculosis (TB) treatment outcomes. Few novel deletions in clinical isolates necessitate the importance of rpoB sequencing in large data sets in geographic-specific locations, especially high-burden countries. We explored the discordance rate on MGIT and LJ, which is important for the clinical management of rifampicin resistance to avoid the mistreatment of drug-resistant TB. Furthermore, MGIT-sensitive isolates may be subjected to molecular methods of diagnosis for further confirmation and treatment options.

The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis.

Extrapulmonary tuberculosis often poses a diagnostic challenge. This study aimed to assess the value of histological examination in diagnosing tuberculous lymphadenitis (LNTB) when performed simultaneously with rapid molecular assay (Xpert MTB/RIF) testing. People presumed to have LNTB were prospectively enrolled in a tertiary care hospital. Excision biopsy was performed and tested by histology, Xpert, and culture. Of 390 lymph nodes, 11 (2.8%) were positive by AFB microscopy, 124 (31.8%) by Xpert, 137 (35.1%) by culture, and histopathology was consistent with TB in 208 (53.3%). Altogether, LNTB was diagnosed in 228 and bacteriologically confirmed TB in 178 cases. Against culture, histopathology versus Xpert had higher sensitivity (93 vs. 62%) but lower specificity (68 vs. 83%). In patients with short clinical history, a significantly higher number of Xpert-positive specimens were culture-positive. Among patients with histology suggestive of TB, no difference was seen in response to treatment between bacteriology positive and negative, but a significant slow response was noted in bacteriology confirmed TB with nonspecific histology. In a country like Pakistan, with high TB and low HIV prevalence, diagnosis is possible for more than 95% of LNTB when Xpert and histopathology examination is used in combination, compared to less than 60% by Xpert alone.

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.

Predictors of slow clinical response and extended treatment in patients with extra-pulmonary tuberculosis in Pakistan, A hospital-based prospective study.

The optimal duration of treatment in different forms of extrapulmonary tuberculosis (EPTB) is not clearly defined. This study aimed to identify predictors of slow clinical response and extended anti-TB treatment in EPTB patients. Socio-demographic, clinical, and microbiological characteristics of EPTB patients registered for anti-TB treatment at a tertiary care hospital, were analysed for identification of predictors of extended treatment. A total of 251 patients (137 lymphadenitis, and 114 pleuritis) were included in the analysis. Treatment was extended to more than 6 months in 58/251 (23%) patients. In the multivariate regression analysis, culture-positive EPTB (p = 0.007) [OR (95% CI) = 3.81 (1.43, 10.11)], history of diabetes (p = 0.014) [OR (95% CI) = 25.18 (1.94, 325.83)], smokeless tobacco use (p = 0.002) [OR (95% CI) = 17.69 (2.80, 111.72)], and slow regression of local signs and symptoms after 2 months of treatment (p < 0.001) [OR (95% CI) = 17.09 [(5.79, 50.39)] were seen to be significantly associated with treatment extension. Identification of predictors of extended treatment can help clinical decisions regarding optimal duration of treatment. Further studies are needed to identify subgroups of EPTB patients who can benefit from a shorter or longer treatment regimen.

Population structure, biogeography and transmissibility of Mycobacterium tuberculosis.

Mycobacterium tuberculosis is a clonal pathogen proposed to have co-evolved with its human host for millennia, yet our understanding of its genomic diversity and biogeography remains incomplete. Here we use a combination of phylogenetics and dimensionality reduction to reevaluate the population structure of M. tuberculosis, providing an in-depth analysis of the ancient Indo-Oceanic Lineage 1 and the modern Central Asian Lineage 3, and expanding our understanding of Lineages 2 and 4. We assess sub-lineages using genomic sequences from 4939 pan-susceptible strains, and find 30 new genetically distinct clades that we validate in a dataset of 4645 independent isolates. We find a consistent geographically restricted or unrestricted pattern for 20 groups, including three groups of Lineage 1. The distribution of terminal branch lengths across the M. tuberculosis phylogeny supports the hypothesis of a higher transmissibility of Lineages 2 and 4, in comparison with Lineages 3 and 1, on a global scale. We define an expanded barcode of 95 single nucleotide substitutions that allows rapid identification of 69 M. tuberculosis sub-lineages and 26 additional internal groups. Our results paint a higher resolution picture of the M. tuberculosis phylogeny and biogeography.

Role of Epistasis in Amikacin, Kanamycin, Bedaquiline, and Clofazimine Resistance in Mycobacterium tuberculosis Complex.

Antibiotic resistance among bacterial pathogens poses a major global health threat. Mycobacterium tuberculosis complex (MTBC) is estimated to have the highest resistance rates of any pathogen globally. Given the low growth rate and the need for a biosafety level 3 laboratory, the only realistic avenue to scale up drug susceptibility testing (DST) for this pathogen is to rely on genotypic techniques. This raises the fundamental question of whether a mutation is a reliable surrogate for phenotypic resistance or whether the presence of a second mutation can completely counteract its effect, resulting in major diagnostic errors (i.e., systematic false resistance results). To date, such epistatic interactions have only been reported for streptomycin that is now rarely used. By analyzing more than 31,000 MTBC genomes, we demonstrated that the eis C-14T promoter mutation, which is interrogated by several genotypic DST assays endorsed by the World Health Organization, cannot confer resistance to amikacin and kanamycin if it coincides with loss-of-function (LoF) mutations in the coding region of eis. To our knowledge, this represents the first definitive example of antibiotic reversion in MTBC. Moreover, we raise the possibility that mmpR (Rv0678) mutations are not valid markers of resistance to bedaquiline and clofazimine if these coincide with an LoF mutation in the efflux pump encoded by mmpS5 (Rv0677c) and mmpL5 (Rv0676c).

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan.

INTRODUCTION: Rifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan. METHOD: A total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations. RESULT: Among the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert. CONCLUSION: Mutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.

Detection of active mycobacterium tuberculosis at 6th month exit among declared successfully treated cases in Pakistan.

OBJECTIVES: Tuberculosis (TB) is a significant public health concern, and the basis of successful anti-tuberculosis treatment (ATT) rests on the complete eradication of live bacilli from a patient. This study was conducted to detect the live TB bacilli in Lowenstein Jensen culture media among exit cases of TB who were declared successfully treated, either cured or treatment completed. METHODS: This cross-sectional study was conducted across Pakistan. Fifty-eight active TB DOTS centers were selected. The sample size of 3355 TB cases were equally distributed in all DOTS facilities. A detailed questionnaire was developed to record the information from TB DOTS and patients. After successful treatment, the sputum was taken from TB cases and examined to detect live bacilli on L-J culture. RESULTS: A total of 3355 TB cases were enrolled in the study. The male to female proportion was 1704(50.9%) and 1651(49.2%). Initially, 1993(59.4%) cases were cured, and 1362(40.6%) were declared as treatment completed cases. At exit, 324(9.65%) cases were again ZN smear-positive, and 328(9.77%) were positive on L-J culture, after being declared successfully treated for TB. CONCLUSIONS: To eradicate live TB bacilli, all TB cases should be subjected to L-J culture at the end of ATT.

Isolation of non-tuberculous mycobacteria among tuberculosis patients, a study from a tertiary care hospital in Lahore, Pakistan.

BACKGROUND: There is scarce knowledge on the prevalence of diseases caused by non-tuberculous mycobacteria (NTM) in Pakistan. In the absence of culture and identification, acid-fast bacilli (AFB) causing NTM disease are liable to be misinterpreted as tuberculosis (TB). Introduction of nucleic acid amplification testing for Mycobacterium tuberculosis complex (MTBC) offers improved diagnostic accuracy, compared with smear microscopy, and also assists in differentiating MTBC from other mycobacteria. This study aimed to investigate the prevalence of NTM among patients investigated for TB and describe NTM disease and treatment outcomes at a tertiary care hospital in Pakistan. METHODS: This is a retrospective study, data on NTM isolates among culture-positive clinical samples over 4 years (2016-19) was retrieved from laboratory records. Information on clinical specimens processed, AFB smear results, and for the AFB positive isolates, results of species identification for MTBC, and for NTM isolates, results of species characterization and drug susceptibility testing was collected. Additional clinical data including patient characteristics, treatment regimens, and outcomes were collected for patients with NTM disease treated at Gulab Devi Hospital, Lahore. RESULTS: During the study period, 12,561 clinical specimens were processed for mycobacterial culture and 3673 (29%) were reported positive for AFB. Among these 3482 (95%) were identified as MTBC and 191 (5%) as NTM. Among NTM, 169 (88%) were isolated from pulmonary and 22 (12%) from extrapulmonary specimens. Results of NTM speciation were available for 60 isolates and included 55% (n = 33) M. avium complex and 25% (n = 15) M. abscesses. Among these patients, complete clinical records were retrieved for 12 patients with pulmonary disease including nine infected with M. avium complex and three with M. abscessus. All 12 patients had a history of poor response to standard first-line anti-TB treatment. Ten patients were cured after 18 months of treatment, whereas, one with M. abscessus infection died and another was lost to follow up. CONCLUSION: In TB endemic areas, NTM can be misdiagnosed as pulmonary TB leading to repeated failed anti-TB treatment and increased morbidity, emphasizing the need for improved diagnosis.

Second-line injectable drugs for rifampicin-resistant tuberculosis: better the devil we know?

In its 2020 guidelines for the treatment of rifampicin-resistant TB (RR-TB), the WHO recommends all-oral fluoroquinolone-based regimens, with bedaquiline replacing the second-line injectable drugs (SLIDs). SLIDs were used for their strong acquired resistance-preventing activity. Data from three cohorts showed acquired bedaquiline resistance ranging between 2.5% and 30.8%, with no protection from a SLID in most cases. If bedaquiline resistance is that easily acquired, it will fail to protect fluoroquinolones and other drugs from acquiring resistance. Until evidence on resistance-preventing activity shows that SLIDs can safely be replaced, we call for more prudent use of the few potent second-line TB drugs available. Studies on new treatment regimens need to prioritize the prevention of acquired resistance along with treatment success. Meanwhile, reducing the dosing of SLIDs to thrice weekly from Day 1, and their replacement for any degree of audiometry abnormalities before or during treatment will largely avoid serious ototoxicity.

Isoniazid resistance profile and associated levofloxacin and pyrazinamide resistance in rifampicin resistant and sensitive isolates/from pulmonary and extrapulmonary tuberculosis patients in Pakistan: A laboratory based surveillance study 2015-19.

BACKGROUND: Pakistan is among top five high burden countries for tuberculosis and drug resistant TB. Among rifampicin sensitive new pulmonary TB (PTB), prevalence of isoniazid resistance is 8.3% (95%CI: 7.0-10.7) and resistance to fluoroquinolone is higher (11·1%, 95%CI: 7·8-14·3) than isoniazid resistance. METHOD: Five year retrospective data (2015-2019) of drug susceptibility testing (DST) for Mycobacterium tuberculosis isolates, performed using recommended phenotypic (pDST) and/or genotypic (gDST) methods was analyzed stratified by rifampicin results for isoniazid resistance profiles and associated levofloxacin and pyrazinamide resistance. FINDINGS: DST data was analyzed from 11045 TB patients. Isolates were tested using pDST (87%), gDST (92%) and both methods (79.5%). For both rifampicin and isoniazid, a significant difference (P < .001) was noted between resistance detected by pDST and gDST. Among isolates, tested by both methods (8787), 49% were resistant to rifampicin and 51.7% to isoniazid with discordance in resistant results of 15.8% for each, with 13.2% (570) of rifampicin resistance reported sensitive by pDST and 14.2% (660) of isoniazid resistance missed by gDST. Estimated isoniazid resistance among rifampicin sensitive new PTB, extrapulmonary TB and previously treated PTB was 9.8% (95%CI: 8.7-11.1), 6.8% (95%CI: 5.4-8.5) and 14.6% (95%CI: 11.8-17.9) respectively. Significant differences were reported between the genotypic profile of isoniazid resistance associated with rifampicin-resistant and sensitive isolates including detectable mutations (87% vs 71.6%), frequency of inhA (7.6% and 30.2%) and katG mutations (76.1% vs 41.2%) respectively. Among rifampicin resistant and sensitive isolates, a significantly higher level of resistance to levofloxacin and pyrazinamide was seen associated with isoniazid resistance. CONCLUSION: There are risks and many challenges in implementing WHO recommended treatment for isoniazid resistant tuberculosis. The laboratory based surveillance can complement random surveys in country specific planning for TB diagnostics and appropriate treatment regimens.

Extrapulmonary tuberculosis in Pakistan- A nation-wide multicenter retrospective study.

BACKGROUND: Pakistan is fifth among high burden countries for tuberculosis. A steady increase is seen in extrapulmonary tuberculosis (EPTB), which now accounts for 20% of all notified TB cases. There is very limited information on the epidemiology of EPTB. This study was performed with the aim to describe the demographic characteristics, clinical manifestations and treatment outcomes of EPTB patients in Pakistan. METHOD: We performed descriptive analysis on routinely collected data for cohorts of TB patients registered nationwide in 2016 at health facilities selected using stratified convenient sampling. FINDINGS: Altogether 54092 TB including 15790 (29.2%) EPTB cases were registered in 2016 at 50 study sites. The median age was 24 years for EPTB and 30 years for PTB patients. The crude prevalence of EPTB in females was 30.5% (95%CI; 29.9-31.0) compared to 27.9% (95%CI; 27.3-28.4) in males. The likelihood of having EPTB (OR), was 1.1 times greater for females, 2.0 times for children, and 3.3 times for residents of provinces in the North-West. The most common forms of EPTB were pleural (29.6%), lymphatic (22.7%) and abdominal TB (21.0%). Pleural TB was the most common clinical manifestation in adults (34.2%) and abdominal TB in children (38.4%). An increase in the prevalence of pleural and osteoarticular and decline in lymphatic and abdominal TB was observed with advancing age. Diversity in demography and clinical manifestations were noted between provinces. The treatment success rate for all type EPTB was significantly high compared to bacteriology confirmed PTB with the exception of EPTB affecting CNS with a high mortality rate. CONCLUSIONS: The study provides an insight into demography, clinical manifestations and treatment outcomes of EPTB. Further studies are needed to explain significant diversities observed between provinces, specific risk factors and challenges concerning EPTB management.

Prevalence and genetic profiles of isoniazid resistance in tuberculosis patients: A multicountry analysis of cross-sectional data.

BACKGROUND: The surveillance of drug resistance among tuberculosis (TB) patients is central to combatting the global TB epidemic and preventing the spread of antimicrobial resistance. Isoniazid and rifampicin are two of the most powerful first-line anti-TB medicines, and resistance to either of them increases the risk of treatment failure, relapse, or acquisition of resistance to other drugs. The global prevalence of rifampicin resistance is well documented, occurring in 3.4% (95% CI 2.5%-4.4%) of new TB patients and 18% (95% CI 7.6%-31%) of previously treated TB patients in 2018, whereas the prevalence of isoniazid resistance at global and regional levels is less understood. In 2018, the World Health Organization (WHO) recommended a modified 6-month treatment regimen for people with isoniazid-resistant, rifampicin-susceptible TB (Hr-TB), which includes rifampicin, pyrazinamide, ethambutol, and levofloxacin. We estimated the global prevalence of Hr-TB among TB patients and investigated associated phenotypic and genotypic drug resistance patterns. METHODS AND FINDINGS: Aggregated drug resistance data reported to WHO from either routine continuous surveillance or nationally representative periodic surveys of TB patients for the period 2003-2017 were reviewed. Isoniazid data were available from 156 countries or territories for 211,753 patients. Among these, the global prevalence of Hr-TB was 7.4% (95% CI 6.5%-8.4%) among new TB patients and 11.4% (95% CI 9.4%-13.4%) among previously treated TB patients. Additional data on pyrazinamide and levofloxacin resistance were available from 6 countries (Azerbaijan, Bangladesh, Belarus, Pakistan, the Philippines, and South Africa). There were no cases of resistance to both pyrazinamide and levofloxacin among Hr-TB patients, except for the Philippines (1.8%, 95% CI 0.2-6.4) and Belarus (5.3%, 95% CI 0.1-26.0). Sequencing data for all genomic regions involved in isoniazid resistance were available for 4,563 patients. Among the 1,174 isolates that were resistant by either phenotypic testing or sequencing, 78.6% (95% CI 76.1%-80.9%) had resistance-conferring mutations in the katG gene and 14.6% (95% CI 12.7%-16.8%) in both katG and the inhA promoter region. For 6.8% (95% CI 5.4%-8.4%) of patients, mutations occurred in the inhA promoter alone, for whom an increased dose of isoniazid may be considered. The main limitations of this study are that most analyses were performed at the national rather than individual patient level and that the quality of laboratory testing may vary between countries. CONCLUSIONS: In this study, the prevalence of Hr-TB among TB patients was higher than the prevalence of rifampicin resistance globally. Many patients with Hr-TB would be missed by current diagnostic algorithms driven by rifampicin testing, highlighting the need for new rapid molecular technologies to ensure access to appropriate treatment and care. The low prevalence of resistance to pyrazinamide and fluoroquinolones among patients with Hr-TB provides further justification for the recommended modified treatment regimen.

World Health Organization 2018 treatment guidelines for rifampicin-resistant tuberculosis: uncertainty, potential risks and the way forward.

The 2018 World Health Organization (WHO) treatment guidelines for multidrug-/rifampicin-resistant tuberculosis (MDR/RR-TB) give preference to all-oral long regimens lasting for 18-20 months. The guidelines strongly recommend combining bedaquiline, levofloxacin (or moxifloxacin) and linezolid, supplemented by cycloserine and/or clofazimine. The effectiveness of this combination in a long regimen has not been tested in any study to date, with corresponding uncertainty. The guidelines indicate that, ideally, all MDR-TB patients should have - as a minimum - the isolate tested for fluoroquinolones, bedaquiline and linezolid susceptibility before the start of treatment. Unfortunately, the capacity for drug susceptibility testing is insufficient in resource-limited settings. The risk of acquired bedaquiline resistance cannot be ignored, especially in patients with undetected resistance to fluoroquinolones. Both linezolid and cycloserine are known for their high frequency of serious adverse events. The combination of bedaquiline, moxifloxacin and clofazimine in the same regimen may excessively increase the QT interval. These expected adverse effects are difficult to monitor and manage in resource-limited settings, and may result in frequent modifications and a less effective regimen. The final STREAM results have confirmed the non-inferiority of the short regimen compared with the long regimen. Before evidence on the all-oral long and modified all-oral short treatment regimens is available, the WHO-recommended short MDR-TB regimens, with monitoring for ototoxicity, remain a better treatment option for the management of MDR/RR-TB patients who are eligible for short regimens in low- and middle-income countries. National tuberculosis programmes should also strengthen their capacity in the detection and management of fluoroquinolone-resistant MDR-TB following the WHO guidelines.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan.

We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential isolates from 30 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing and by the detection of mutations in relevant genes. We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Antibiotic resistance prediction for Mycobacterium tuberculosis from genome sequence data with Mykrobe.

Two billion people are infected with Mycobacterium tuberculosis, leading to 10 million new cases of active tuberculosis and 1.5 million deaths annually. Universal access to drug susceptibility testing (DST) has become a World Health Organization priority. We previously developed a software tool, Mykrobe predictor, which provided offline species identification and drug resistance predictions for M. tuberculosis from whole genome sequencing (WGS) data. Performance was insufficient to support the use of WGS as an alternative to conventional phenotype-based DST, due to mutation catalogue limitations.  Here we present a new tool, Mykrobe, which provides the same functionality based on a new software implementation. Improvements include i) an updated mutation catalogue giving greater sensitivity to detect pyrazinamide resistance, ii) support for user-defined resistance catalogues, iii) improved identification of non-tuberculous mycobacterial species, and iv) an updated statistical model for Oxford Nanopore Technologies sequencing data. Mykrobe is released under MIT license at https://github.com/mykrobe-tools/mykrobe. We incorporate mutation catalogues from the CRyPTIC consortium et al. (2018) and from Walker et al. (2015), and make improvements based on performance on an initial set of 3206 and an independent set of 5845 M. tuberculosis Illumina sequences. To give estimates of error rates, we use a prospectively collected dataset of 4362 M. tuberculosis isolates. Using culture based DST as the reference, we estimate Mykrobe to be 100%, 95%, 82%, 99% sensitive and 99%, 100%, 99%, 99% specific for rifampicin, isoniazid, pyrazinamide and ethambutol resistance prediction respectively. We benchmark against four other tools on 10207 (=5845+4362) samples, and also show that Mykrobe gives concordant results with nanopore data.  We measure the ability of Mykrobe-based DST to guide personalized therapeutic regimen design in the context of complex drug susceptibility profiles, showing 94% concordance of implied regimen with that driven by phenotypic DST, higher than all other benchmarked tools.