2022 TB programme review in Pakistan: strengthening governance, with better patient diagnosis and treatment.

BACKGROUND: In Pakistan, 84% of healthcare is provided by the private sector. We conducted an epidemiological and programme review for TB to document progress and guide further efforts. METHODS: Surveillance and data systems were assessed before analysing epidemiological data. We reviewed the programme at federal, provincial and peripheral levels and compiled national data along with WHO estimates to describe the evolution of epidemiological and programme indicators. RESULTS: In 2021, of the estimated number of TB cases, 55% of overall cases and 18% of drug-resistant cases were diagnosed and treated respectively. The contribution of the private sector in case detection increased from 30% in 2017 to 40% by 2021. For newly diagnosed pulmonary TB cases, the overall proportion of confirmed cases was 52%. In 2021, testing for rifampicin resistance among confirmed cases was 66% for new and 84% for previously treated patients. The treatment success rate exceeded 90% for drug susceptible TB. The main challenges identified were a funding gap (60% in 2021-2023), fragmented electronic systems for data collection and suboptimal coordination among provinces. CONCLUSIONS: The main challenges prevent further progress in controlling TB. By addressing these, Pakistan could improve coverage of interventions, including diagnosis and treatment. Bacteriological confirmation using recommended diagnostics also requires further optimisation.

CONTEXTE: Au Pakistan, le secteur privé assure 84% des services de santé. Une étude épidémiologique et programmatique a été réalisée sur la TB afin de recueillir des informations sur les avancées réalisées et de guider les actions à venir. MÉTHODES: Les systèmes de surveillance et de données ont été évalués préalablement à l'analyse des données épidémiologiques. Nous avons examiné le programme aux niveaux fédéral, provincial et local et compilé les données nationales ainsi que les estimations de l'OMS afin de décrire l'évolution des indicateurs épidémiologiques et du programme. RÉSULTATS: En 2021, environ 55% de l'ensemble cas de TB et 18% des cas résistants aux médicaments ont été diagnostiqués et traités respectivement. La contribution du secteur privé dans la détection des cas est passée de 30% en 2017 à 40% en 2021. La proportion totale de cas confirmés pour les nouveaux diagnostics de TB pulmonaire s'élevait à 52%. En 2021, les tests de résistance à la rifampicine parmi les cas confirmés s'élevaient à 66% pour les nouveaux patients et de 84% pour les patients déjà traités. Le taux de réussite du traitement a dépassé 90% pour la TB sensible aux médicaments. Les défis majeurs comprennent un manque de financement (60% pour la période 2021­2023), des systèmes électroniques de collecte de données fragmentés et une coordination insuffisante entre les provinces. CONCLUSIONS: Les défis majeurs entravent les avancées dans la lutte contre la TB. En les mettant en évidence, le Pakistan pourrait améliorer la portée des interventions, y compris le diagnostic et le traitement. Il est également essentiel d'optimiser la confirmation bactériologique en utilisant les diagnostics recommandés.

Experiences from 4 Years of Organization of an External Quality Assessment for Mycobacterium tuberculosis Whole-Genome Sequencing in the European Union/European Economic Area.

Here, we report the development and key features of the first external quality assessment (EQA) scheme for Mycobacterium tuberculosis whole-genome sequencing (WGS). The results of four rounds (2017 to 2020) of implementation within the European tuberculosis reference laboratories network (ERLTB-Net-2) are presented and discussed. EQA panels comprising 10 genomic DNAs were distributed to ERLTB-Net 2 laboratories volunteering to participate in this exercise. Since 2018, five FASTQ files were added to better assess the dry WGS processes, and in 2020, three of the five files were replaced by synthetic files (providing additional flexibility for the mutations included in the panels). Ten National tuberculosis reference laboratories participated in all four EQA rounds, and seven participated in at least one. High-confidence resistance mutations were correctly identified by all laboratories, but challenges remained with respect to the identification of mixed loci and interpretation of rare mutations. M. tuberculosis genotyping and clustering analysis was >90% accurate for pure samples with the main challenges being related to the analysis of mixed genotypes and DNA FASTQ files. The development and implementation of this WGS EQA scheme has contributed to the continuous improvement in performance of participating laboratories in M. tuberculosis WGS and data analysis. This scheme can serve as a model of comprehensive quality assessment for M. tuberculosis WGS that can be replicated in different settings worldwide. IMPORTANCE The wider availability of whole-genome sequencing (WGS) coupled to new developments in bioinformatic tools and databases to interpret Mycobacterium tuberculosis complex WGS data has accelerated the adoption of this method for the routine prediction of antimycobacterial drug resistance and genotyping, thus necessitating the establishment of a comprehensive external quality control system. Here, we report 4 years of development and results from such a panel.

Uptake of the lateral flow urine LAM test in Europe and Central Asia.

INTRODUCTION: Since 2015 (updated in 2019), the WHO has recommended to include the commercial lateral flow urine lipoarabinomannan TB test (LF-LAM), AlereLAM, in the diagnostic toolkit for severely ill people living with HIV.METHODS: To assess the current use and barriers to the implementation of the test, we conducted an electronic survey among national focal points and managers of TB and HIV programmes in the 53 Member States of the WHO European Region and a European network of clinicians working in TB and HIV medicine.RESULTS: In all, 45 individual responses (37 countries) were received from programme managers and focal points and 17 responses (14 countries) from clinicians. Only eight countries reported adopting LF-LAM policies, with only four currently using the AlereLAM (Armenia, Belarus, Ukraine and Uzbekistan). The most commonly reported barriers to implementing the test were the small number of eligible patients (with HIV-TB co-infections), the test not being included in the TB or HIV programme´s mandate and lack of budget allocation.CONCLUSION: Consistent with findings from high TB burden countries in Africa and Asia, the survey demonstrated that uptake of AlereLAM is almost non-existent. Addressing the identified barriers and the intrinsic limitations of the test could help to increase the use of the test.

Decreasing trend of drug-resistant TB in Italy.

BACKGROUND: TB caused by rifampicin-resistant (RR) and multidrug-resistant (MDR) Mycobacterium tuberculosis strains is a major concern to TB control globally. However, in the European Union, MDR-TB notifications among all bacteriologically confirmed TB cases with available drug susceptibility testing (DST) results decreased over the last years.METHODS: We conducted a retrospective analysis on DST results reported from 2011 to 2020 by 46 laboratories in 19 out of 20 regions in Italy in order to evaluate resistance trends to first- and second-line drugs in MDR/RR-TB strains isolated from Italian-born persons (IBPs) and foreign-born persons (FBPs).RESULTS: Of 23,972 M. tuberculosis strains examined (15,519 from FBPs and 8,453 from IBPs), MDR-TB decreased from 3.2% in 2011 to 2.2% in 2020. High MDR/RR-TB rates occurred mostly in FBPs from former Soviet Union countries. In 2017, a MDR/RR-TB increase was detected in FBPs from sub-Saharan Africa. MDR-TB strains showed consistent increase in resistance to pyrazinamide (PZA), slight increase in resistance to fluoroquinolones and a decrease in resistance to other drugs.CONCLUSION: While MDR/RR-TB cases slightly decreased, a worrisome increase of resistance to PZA and fluoroquinolones among MDR/RR-TB patients was seen. This implies that a fast and efficient diagnosis aligned with therapy is crucial for TB control.

Clinical standards for the dosing and management of TB drugs.

BACKGROUND: Optimal drug dosing is important to ensure adequate response to treatment, prevent development of drug resistance and reduce drug toxicity. The aim of these clinical standards is to provide guidance on 'best practice´ for dosing and management of TB drugs.METHODS: A panel of 57 global experts in the fields of microbiology, pharmacology and TB care were identified; 51 participated in a Delphi process. A 5-point Likert scale was used to score draft standards. The final document represents the broad consensus and was approved by all participants.RESULTS: Six clinical standards were defined: Standard 1, defining the most appropriate initial dose for TB treatment; Standard 2, identifying patients who may be at risk of sub-optimal drug exposure; Standard 3, identifying patients at risk of developing drug-related toxicity and how best to manage this risk; Standard 4, identifying patients who can benefit from therapeutic drug monitoring (TDM); Standard 5, highlighting education and counselling that should be provided to people initiating TB treatment; and Standard 6, providing essential education for healthcare professionals. In addition, consensus research priorities were identified.CONCLUSION: This is the first consensus-based Clinical Standards for the dosing and management of TB drugs to guide clinicians and programme managers in planning and implementation of locally appropriate measures for optimal person-centred treatment to improve patient care.

Therapeutic strategies and outcomes of MDR and pre-XDR-TB in Italy: a nationwide study.

BACKGROUND: Treatment outcomes in multidrug-resistant TB (MDR-TB) patients are suboptimal in several low-incidence countries.METHODS: The primary outcome measure was the proportion of successfully treated patients in Italy during an 18-year period. Secondary outcomes were treatment outcomes in certain drug-containing regimens and the possibility for the WHO shorter MDR-TB regimen.RESULTS: In the 191 patients included (median age at admission: 33 years; 67.5% male, following drug-resistance patterns were found: MDR-TB in 68.6%, pre-extensively drug-resistant TB (pre-XDR-TB) in 30.4% and XDR-TB in 1.1% patients. The most frequently prescribed drugs were fluoroquinolones in 84.6% cases, amikacin in 48.7%, linezolid in 34.6% and meropenem/clavulanic acid in 29.5%. The median duration of treatment was 18 months. Treatment success was achieved in 71.2% patients, of whom, 44% were cured and 27.2% completed treatment. Treatment success rates did not statistically differ between the MDR- (68.8%) and pre-XDR-TB (77.6%) groups (P = 0.26). Treatment success rates had large variability between North and South of Italy (81.3% vs. 53.3%). Only 22.5% of the cases would have been eligible for shorter MDR-TB regimensCONCLUSION: Our study highlights variability in treatment outcomes in MDR- and pre-XDR-TB patients. Study findings confirmed the potential utility of linezolid and, for patients with limited oral options, meropenem/clavulanic acid and amikacin.

First molecular-based anti-TB drug resistance survey in Eritrea.

BACKGROUND: In the absence of reliable data on drug-resistant TB in Eritrea, a national survey was conducted in 2018 using molecular-based methods, bypassing the need for culture.METHODS: A cross-sectional study was conducted in all 77 TB microscopy centres in the country. All 629 newly registered sputum smear-positive pulmonary TB patients were enrolled over 12 months. Sputum samples were tested using the Xpert® MTB/RIF assay and targeted next-generation sequencing (Deeplex Myc-TB) to identify resistance and explore the phylogenetics of Mycobacterium tuberculosis complex strains.RESULTS: Drug resistance profiles were obtained for 555 patients (502 new, 53 previously treated). The prevalence of rifampicin-resistant TB (RR-TB) was respectively 2.0% and 7.6% among new and previously treated cases. All RR-TB isolates that were susceptible to isoniazid displayed a phylogenetic marker conferring capreomycin resistance, confirming circulation of a previously described resistant TB sub-lineage in the Horn of Africa. Only one case of fluoroquinolone resistance was detected.CONCLUSION: The prevalence of rifampicin resistance among TB patients is encouragingly low. The scarcity of fluoroquinolone resistance bodes well for the success of the recommended all-oral treatment regimen. Surveillance based on molecular approaches enables a reliable estimation of the burden of resistance and can be used to guide appropriate treatment and care.

Characterization of Genomic Variants Associated with Resistance to Bedaquiline and Delamanid in Naive Mycobacterium tuberculosis Clinical Strains.

The role of mutations in genes associated with phenotypic resistance to bedaquiline (BDQ) and delamanid (DLM) in Mycobacterium tuberculosis complex (MTBc) strains is poorly characterized. A clear understanding of the genetic variants' role is crucial to guide the development of molecular-based drug susceptibility testing (DST). In this work, we analyzed all mutations in candidate genomic regions associated with BDQ- and DLM-resistant phenotypes using a whole-genome sequencing (WGS) data set from a collection of 4,795 MTBc clinical isolates from six countries with a high burden of tuberculosis (TB). From WGS analysis, we identified 61 and 163 unique mutations in genomic regions potentially involved in BDQ- and DLM-resistant phenotypes, respectively. Importantly, all strains were isolated from patients who likely have never been exposed to these medicines. To characterize the role of mutations, we calculated the free energy variation upon mutations in the available protein structures of Ddn (DLM), Fgd1 (DLM), and Rv0678 (BDQ) and performed MIC assays on a subset of MTBc strains carrying mutations to assess their phenotypic effect. The combination of structural and phenotypic data allowed for cataloguing the mutations clearly associated with resistance to BDQ (n = 4) and DLM (n = 35), only two of which were previously described, as well as about a hundred genetic variants without any correlation with resistance. Significantly, these results show that both BDQ and DLM resistance-related mutations are diverse and distributed across the entire region of each gene target, which is of critical importance for the development of comprehensive molecular diagnostic tools.

Epidemiology and treatment outcome of MDR and pre-XDR TB in international migrants at two reference centers in the North of Italy: a cross-sectional study coordinated by Stop TB Italia Onlus.

OBJECTIVE: We evaluated the epidemiology and treatment outcomes of multi-drug-resistant (MDR) and pre-extensively-resistant (pre-XDR) tuberculosis (TB) in migrants at two TB reference centers in Italy. STUDY DESIGN: Patient selection criteria for the present study were as follows: age ≥18 years, international migrants (i.e., person who lives in a country other than his/her country of origin), MDR or pre-XDR-TB based on drug-susceptibility test findings, full availability of microbiological, radiological and clinical data. Non-intersecting populations between the two centers were selected. The primary outcome was the proportion of patients with a successful (i.e., cured and treatment completed) treatment outcome. METHODS: A retrospective cross-sectional study was conducted, from 01/Jan/2000 to 01/Jan/2015, at the Regional TB Reference Centre of Lombardy Region, Villa Marelli Institute/ASST Niguarda Ca' Granda (Milan, Italy) and at the Reference Center for MDR-TB and HIV-TB, Eugenio Morelli Hospital ASST (Sondalo, Italy). All data were made anonymous. Qualitative and quantitative variables were collected in an ad hoc electronic database. The statistical software used for all computations was STATA version 15 (StataCorp, Texas, USA). RESULTS: Overall, 116 MDR-TB and pre-XDR-TB cases were recorded: 82 (70.7%) MDR-TB and 34 (29.3%) pre-XDR-TB patients, respectively. The majority (53.5%) were from the World Health Organization European Region (excluding EU/EEA) and 75 (64.5%) were male. Median (interquartile range) age was 32 (26-39) years. TB/HIV coinfection was found in 12 (10.3%) patients. Pulmonary TB was diagnosed in 107/116 (92.2%) patients. Resistance to fluoroquinolones and second-line injectables was detected in 22/116 (19.0%) and 12/107 (11.2%) patients, respectively. Overall treatment success was reached in 95/116 (81.9%) cases. CONCLUSION: Pre-XDR-TB in migrants coming from high-endemic countries represents a matter of concern; therefore, prevention and control activities targeted to high-risk populations are needed to progress toward TB elimination.

Tuberculosis screening in outpatient healthcare workers: lessons from a high-income, low TB burden country.

SETTING: Early diagnosis of latent tuberculous infection (LTBI) should be pursued in healthcare workers (HCWs). While HCWs in hospitals are screened for LTBI, HCWs in outpatient settings are usually not. In 2017, in Italy, a tuberculosis (TB) infected paediatrician working in an outpatient vaccination service infected 15 adults and nine children. The investigation involved 2490 children and 151 adults. Among children, nine were tuberculin skin test-positive, and four developed active TB. Among 123 adult contacts with longer exposure, seven were interferon-gamma release assay (IGRA) positive and none had active TB. Among 28 close contacts, eight had a positive IGRA, and three had pulmonary TB. The total outbreak cost €1 017 903.OBJECTIVE: To compare the outbreak cost with those of potential screening programme strategies.RESULTS: Regular screening of paediatric outpatient HCWs would have cost between €2592 and €11 373. Extending the screening to all outpatient HCWs (caring for adults and children) would have cost between €66 384 and €155 043. Investigating only close contacts would have cost €42 857.CONCLUSION: Each of these screening strategies would have been cost-effective compared with the outbreak investigation occurring in real life with a cut-off of 474 for the maximum number of tested outpatient HCWs needed for the screening strategy to be cost-saving.

Fluoroquinolone heteroresistance in Mycobacterium tuberculosis: detection by genotypic and phenotypic assays in experimentally mixed populations.

Heteroresistance - the simultaneous presence of drug-susceptible and -resistant organisms - is common in Mycobacterium tuberculosis. In this study, we aimed to determine the limit of detection (LOD) of genotypic assays to detect gatifloxacin-resistant mutants in experimentally mixed populations. A fluoroquinolone-susceptible M. tuberculosis mother strain (S) and its in vitro selected resistant daughter strain harbouring the D94G mutation in gyrA (R) were mixed at different ratio's. Minimum inhibitory concentrations (MICs) against gatifloxacin were determined, while PCR-based techniques included: line probe assays (Genotype MTBDRsl and GenoScholar-FQ + KM TB II), Sanger sequencing and targeted deep sequencing. Droplet digital PCR was used as molecular reference method. A breakpoint concentration of 0.25 mg/L allows the phenotypic detection of ≥1% resistant bacilli, whereas at 0.5 mg/L ≥ 5% resistant bacilli are detected. Line probe assays detected ≥5% mutants. Sanger sequencing required the presence of around 15% mutant bacilli to be detected as (hetero) resistant, while targeted deep sequencing detected ≤1% mutants. Deep sequencing and phenotypic testing are the most sensitive methods for detection of fluoroquinolone-resistant minority populations, followed by line probe assays (provided that the mutation is confirmed by a mutation band), while Sanger sequencing proved to be the least sensitive method.

Role and value of whole genome sequencing in studying tuberculosis transmission.

BACKGROUND: Tuberculosis (TB) remains a serious public health threat worldwide. Theoretically ultimate resolution of whole genome sequencing (WGS) for Mycobacterium tuberculosis complex (MTBC) strain classification makes this technology very attractive for epidemiological investigations. OBJECTIVES: To summarize the evidence available in peer-reviewed publications on the role and place of WGS in detection of TB transmission. SOURCES: A total of 69 peer-reviewed publications identified in Pubmed database. CONTENT: Evidence from >30 publications suggests that a cut-off value of fewer than six single nucleotide polymorphisms between strains efficiently excludes cases that are not the result of recent transmission and could be used for the identification of drug-sensitive isolates involved in direct human-to-human TB transmission. Sensitivity of WGS to identify epidemiologically linked isolates is high, reaching 100% in eight studies with specificity (17%-95%) highly dependent on the settings. Drug resistance and specific phylogenetic lineages may be associated with accelerated mutation rates affecting genetic distances. WGS can be potentially used to distinguish between true relapses and re-infections but in high-incidence low-diversity settings this would require consideration of epidemiological links and minority alleles. Data from four studies looking into within-host diversity highlight a need for developing criteria for acceptance or rejection of WGS relatedness results depending on the proportion of minority alleles. IMPLICATIONS: WGS will potentially allow for more targeted public health actions preventing unnecessary investigations of false clusters. Consensus on standardization of raw data quality control processing criteria, analytical pipelines and reporting language is yet to be reached.

Latent tuberculous infection among foreign-born individuals applying to shelters in the metropolitan area of Milan.

SETTING: Screening for latent tuberculous infection (LTBI) of groups at high risk of active tuberculosis (TB) is a key component of the End TB Strategy. OBJECTIVE: To conduct a retrospective descriptive analysis of LTBI rates among foreign-born individuals applying to shelters in the metropolitan area of Milan, Italy. DESIGN: All foreign-born individuals registering for accommodation centres in the city of Milan from November 2009 to April 2017 were screened for active TB and LTBI. Individuals aged <36 years with a tuberculin skin test (TST) induration of >10 mm were offered confirmatory testing with QuantiFERON®-TB Gold In-Tube (QFT-GIT). RESULTS: Of the 2666 TST-positive migrants aged <36 years who underwent LTBI confirmation testing, 1322 (49.6%) tested negative, 1339 (50.2%) were positive and five (0.2%) had indeterminate results. In the multivariate analysis, TB incidence in the country of origin and age were significantly associated with QFT-GIT positivity. Although estimated TB incidence in Eritrea, Morocco and Romania was 100/100 000 person-years (py), the probability of being QFT-GIT-positive in individuals from these countries were not statistically significantly different from individuals from countries with TB incidence > 250/100 000 person-years. CONCLUSION: Our data showed a high proportion of LTBI among individuals coming from intermediate TB burden countries.

Is sequencing better than phenotypic tests for the detection of pyrazinamide resistance?

SETTING: Phenotypic tests used to detect pyrazinamide (PZA) resistance are slow and have a high rate of false resistance. OBJECTIVE: To evaluate the accuracy of pncA sequencing for the detection of PZA resistance in Mycobacterium tuberculosis strains isolated in Tunisia. DESIGN: A total of 82 isolates, 41 resistant and 41 susceptible to PZA on BACTEC™ MGIT™ 960, were sequenced for pncA. Whole genome sequencing was performed for strains that were phenotypically resistant and had wild-type pncA in addition to MGIT retesting with a modified protocol. RESULTS: Twenty-three strains resistant to PZA with negative pyrazinamidase (PZase) activity harboured a mutation in the promoter or coding region of pncA. However, 18 strains resistant to PZA did not present any mutation. Repeat MGIT 960 showed that 16 of 18 M. tuberculosis isolates were falsely resistant to PZA. Compared with MGIT, PZase activity assay and pncA sequencing both presented a sensitivity of 92.0% (95%CI 73.9-99.0) and a specificity of respectively 96.5% (positive predictive value [PPV] 92.0%, negative predictive value [NPV] 96.5%) and 100.0% (PPV 100.0%, NPV 96.6%). CONCLUSION: The standard MGIT assay showed a high rate of false resistance to PZA, and the PZase activity assay is slow. pncA sequencing could therefore represent a rapid, accurate, alternative test to detect PZA resistance.

Alarming levels of multidrug-resistant tuberculosis in Ukraine: results from the first national survey.

SETTING: The true prevalence of multidrug-resistant tuberculosis (MDR-TB) in Ukraine is not known. Available data are a decade old and limited to only one province. OBJECTIVE: To determine the prevalence of MDR-TB among new and previously treated TB cases in Ukraine and explore the risk factors associated with drug resistance. METHODS: A total of 1550 sputum smear-positive pulmonary TB patients were recruited from 40 clusters throughout Ukraine. Sputum specimens were examined using culture, drug susceptibility testing and pncA gene sequencing. RESULTS: The proportion of MDR-TB among new and previously treated TB cases was respectively 24.1% (95%CI 20.7-27.6) and 58.1% (95%CI 52.1-64.1). More than one third (38.0%) of MDR-TB or rifampicin (RMP) resistant cases showed resistance to either a fluoroquinolone (FQ) or a second-line injectable agent or both. Resistance to pyrazinamide and FQs was low in patients with RMP-susceptible TB. Among new TB cases, the odds of MDR-TB were higher among patients who were younger, female and living in south-eastern provinces, as well as among human immunodeficiency virus-positive patients who belonged to a low socio-economic group. CONCLUSIONS: Our study showed that the burden of MDR-TB in Ukraine was much greater than previously assumed. Urgent actions are needed to prevent further spread of drug-resistant TB in Ukraine.

Evaluation of a novel line probe assay to detect resistance to pyrazinamide, a key drug used for tuberculosis treatment.

OBJECTIVES: The development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan). METHODS: To evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts. RESULTS: For the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing. CONCLUSIONS: The PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.