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BACKGROUND: Reliance on complex, custom-built bioinformatics pipelines is a barrier to the implementation of whole-genome sequencing (WGS) of Mycobacterium tuberculosis in high-burden settings in some low-income and middle-income countries (LMICs). Automated analysis pipelines could address this inequity in access to WGS-based diagnostics and surveillance. This study aimed to systematically evaluate the performance and usability of publicly available WGS pipelines for M tuberculosis. METHODS: We identified automated M tuberculosis WGS analysis pipelines through searches of PubMed and GitHub from database inception up to Aug 31, 2024. Accuracy, cost, accessibility, and scalability were assessed for each pipeline. We evaluated the accuracy of genotypic drug susceptibility testing (gDST) using publicly available sequences with phenotypic susceptibility data for 12 antituberculosis drugs. We estimated pooled sensitivity and specificity for each pipeline, across all drugs, by conducting a bivariate meta-analysis, with random effects representing between-drug variability. Lineage classifications were compared, and a previously epidemiologically well-characterised dataset was used to compare measures of genomic relatedness. FINDINGS: Among 28 candidate pipelines, 16 were excluded as they were unmaintained and inexecutable. 12 pipelines (11 compatible with Illumina and four compatible with Nanopore), all free to use, were included for evaluation. Six pipelines processed and stored data remotely, but for five of these six, scalability was limited by the need to upload sequences through web portals. For local processing pipelines, scalability was dependent on substantial local computational resources, data storage capacity, and command-line interfaces that limited user-friendliness. Only one of six remote-processing pipelines removed human DNA sequences before server upload. gDST was similarly accurate across ten of 11 Illumina-compatible pipelines and three of four Nanopore-compatible pipelines. All pipelines classified the main lineages consistently, although there were differences at sublineage resolution. Outputs from three of four pipelines reporting genomic relatedness were compatible with commonly cited single nucleotide polymorphism difference thresholds. INTERPRETATION: Numerous automated analysis pipelines capable of enhancing equity in M tuberculosis WGS are available. Given the overall similarities between the pipelines evaluated in this study in terms of gDST performance, lineage classification, and genomic relatedness inference, non-functional attributes such as availability, accessibility, scalability, and privacy could represent the point of difference for prospective users in LMICs with a high burden of tuberculosis. FUNDING: The Rhodes Trust, Wellcome, Ellison Institute of Technology, and the UK National Institute for Health and Care Research Oxford Biomedical Research Centre.
HIV-1 drug susceptibility testing by genotypic methods is now widespread in the UK and it is thus important to determine the reproducibility of such investigations. A pilot study was carried out to determine the reproducibility between laboratories of genotypic testing by nucleotide sequencing of patient samples, and to compare the interpretations of the results provided to the clinicians. Samples were distributed between the participating laboratories. Eight laboratories, using three different methods, sequenced four samples. Nucleotide sequence and reports were collated and compared by one laboratory. Where sequencing data were obtained > 99% concordance was observed for nucleotide designations. No discordances in nucleotide sequencing were found in positions associated with drug resistance. However, there were considerable differences between the laboratories in the interpretation of some of the mutations with respect to their effect on drug susceptibility. This study emphasises the variability in the available interpretation systems, and the importance of joint laboratory-clinical discussion in making decisions about treatment options.
BACKGROUND: The results of animal studies suggest that isoniazid-resistant strains of Mycobacterium tuberculosis are less pathogenic than isoniazid-susceptible strains. Here, we assess the relative pathogenicity of drug-resistant and drug-susceptible strains, in a human population. METHODS: We linked IS6110 genotype patterns of M. tuberculosis strains with drug-susceptibility test results and epidemiologic information for 85% of culture-positive incident cases of tuberculosis (TB) in San Francisco during 1991-1999. We assumed that drug-susceptible and drug-resistant strains were transmitted to secondary case patients if the drug-resistance and genotype patterns were identical. We calculated the number of secondary cases for each drug-resistance pattern and determined the relative secondary-case rate ratio (SR) of drug-resistant TB to drug-susceptible TB. RESULTS: There were 1800 patients with culture-positive TB, drug-susceptibility test results, and genotyping results. The overall SR of drug-resistant to drug-susceptible TB cases was 0.51 (95% confidence interval [CI], 0.37-0.69). The SR was 0.29 (95% CI, 0.15-0.57) for isoniazid-resistant strains, 0.10 (95% CI, 0.02-0.42) for strains resistant to both isoniazid and streptomycin, and 0.88 (95% CI, 0.53-1.47) for streptomycin-resistant strains. There were no secondary cases caused by multidrug-resistant (MDR) TB. The SR for rifampin-resistant cases was 2.33 (95% CI, 1.04-5.25). Seventy-eight percent (7/9) of the patients with rifampin-resistant secondary cases of TB were seropositive for human immunodeficiency virus. CONCLUSION: In the context of an effective TB program in San Francisco, strains that were resistant to isoniazid either alone or in combination with other drugs were less likely to result in secondary cases than were drug-susceptible strains. In this setting, isoniazid-resistant and MDR TB cases were not likely to produce new, incident drug-resistant TB cases.
Two commercially available DNA line probe assays, Genotype MTBDR and INNO-LiPA Rif. TB, were evaluated for their abilities to detect resistance to isoniazid (INH) and rifampin (RIF) in 52 Mycobacterium tuberculosis isolates. The test results were compared to those obtained by phenotypic drug susceptibility testing and sequencing. Compared to the results of phenotypic drug susceptibility testing, the Genotype MTBDR test results were concordant for INH for 47 of the 52 (90.4%) isolates, and both the Genotype MTBDR and the INNO-LiPA Rif. TB test results were concordant for RIF for 51 of the 52 (98.1%) isolates. The Genotype MTBDR test results correlated with the sequencing results for 48 of the 52 (92.3%) isolates and the INNO-LiPA Rif. TB results for 50 of the 52 (96.2%) isolates. Both assays are useful for the rapid screening of M. tuberculosis isolates obtained from patients suspected of having multidrug-resistant tuberculosis, but the GenoType MTBDR assay has the advantage of being able to detect resistance to both INH and RIF simultaneously.
BACKGROUND: Directly observed therapy (DOTS) is the main strategy for prevention and control of tuberculosis worldwide. However, its effect on tuberculosis transmission in populations with moderate rates of drug-resistant disease is not known. METHODS: This population-based prospective study in southern Mexico between March, 1995, and February, 2000, was based on passive case finding and detection of acid-fast bacilli in sputum samples to diagnose pulmonary tuberculosis. We also used cultures, drug-susceptibility testing, bacterial genotyping, and monitoring of treatment outcomes. FINDINGS: We enrolled 436 patients; the HIV seroprevalence rate was 2%. We used three indicators to monitor continuing tuberculosis transmission: the incidence rate of pulmonary tuberculosis, which decreased by 54.4% between 1995 and 2000, from 42.1 to 19.2 per 10(5) population (p=0.00048); the percentage of clustered pulmonary tuberculosis cases, which decreased by 62.6% from 22% to 8% (p=0.02); and the rate of primary drug resistance, which decreased by 84.0% from 9.4 to 1.5 per 10(5) population (p=0.004). Rates of multidrug-resistant (MDR) tuberculosis also decreased (p<0.0001). The case-fatality ratio was 12% for MDR tuberculosis (five of 41), 7% for strains resistant to at least one drug after exclusion of MDR (four of 55), and 3% for pansusceptible strains (nine of 272). There were 13 treatment failures (11%) in 1995 and one (2%) in 2000 (p=0.012). INTERPRETATION: Even in settings with moderate rates of MDR tuberculosis, DOTS can rapidly reduce the transmission and incidence of both drug-susceptible and drug-resistant tuberculosis. However, further interventions, such as drug-susceptibility testing and standardised or individualised treatment regimens, are needed to reduce mortality rates for MDR tuberculosis.
To gain information about the genetic variation of the protease and reverse transcriptase gene-coding regions in HIV-1 strains belonging to CRF04_cpx, genotyping and drug susceptibility testing were performed on serum samples derived from seven patients carrying sequences belonging to this circulating recombinant form. Substitutions classically associated with resistance to antiretroviral drugs were observed in six of seven samples, including G48V, V82A, L90M, M46I in the protease protein, and K70R, D69D/N, M184V, T215F, K103N in the reverse transcriptase protein. Genotypic resistance patterns of CRF04_cpx samples were found to be similar to those identified in subtype B viruses, suggesting that the drug-selective pressure has similar effects on both subtype B and CRF04_cpx.
UNLABELLED: Discordant drug susceptibility testing (DST) results between the Bactec MGIT 960 system (MGIT) and the GenoType MTBDRplus assay (MTBDRplus) for isoniazid (INH) complicate clinical decision-making. In this study, we performed minimum inhibitory concentration (MIC) assays and whole-genome sequencing (WGS) on 53 Mycobacterium tuberculosis strains identified as INH-resistant by MGIT but INH-susceptible by MTBDRplus. The variants conferring INH resistance were evaluated by the WHO mutation catalogue. Our results showed that only five strains carried variants classified as "associated with resistance" (Group 1/2), including katG Trp39STOP, katG Ser315Asn, inhA -154G>A, and inhA Ser94Ala. In addition, 44 strains carried 70 variants classified as "Group 3: Uncertain significance" across nine genes, including katG, ahpC, inhA, Rv0010c, Rv1129c, Rv2752c, mshA, dnaA, and Rv1258c. The remaining four strains carried no variants (Groups 1-3) linked to INH resistance. No significant difference in the prevalence of high-level INH resistance was observed between lineage 2 and lineage 4 strains (χ² = 0.232, P = 0.630). Our findings indicate that the variants classified as "uncertain significance" may be the main genetic determinants causing discordant results, highlighting their associations with INH resistance that need to be further investigated. IMPORTANCE: This study addresses a critical challenge in drug susceptibility testing (DST): the discrepancies in DST results for isoniazid (INH) between the Bactec MGIT 960 system and the GenoType MTBDRplus assay. These discordant results significantly complicate treatment decisions, potentially leading to suboptimal patient outcomes. Using MIC assays and WGS on 53 clinical Mycobacterium tuberculosis strains, we provide valuable insights into the genetic basis of INH resistance. Our findings showed that only a small fraction of strains carried variants definitively linked to INH resistance, while a larger number harbored variants of uncertain significance across multiple genes, underscoring the complexity of INH resistance mechanisms. This study highlights the urgent need to refine our understanding of these "Group 3: uncertain significance" variants, as they appear to be a primary driver of the discrepancies. Additionally, this study emphasizes the importance of integrating advanced sequencing tools into DST to improve the accuracy of INH resistance detection.
The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.
BACKGROUND: Childhood drug-resistant tuberculosis is often underdiagnosed and inadequately characterized due to the paucibacillary nature of the disease. This study aimed to assess resistance to first-line anti-tuberculosis drugs in children using phenotypic drug susceptibility testing and whole-genome sequencing. METHODS: A retrospective-prospective study was conducted on culture-confirmed childhood tuberculosis cases in Ethiopia (2017–2023). Phenotypic drug susceptibility testing was performed on 110 Mycobacterium tuberculosis complex isolates. Whole-genome sequencing was completed for 85 of these isolates, which were analyzed using the TB-Profiler and MTBSeq pipelines. We assessed the sensitivity, specificity, predictive values, and kappa agreement of whole-genome sequencing compared with phenotypic drug susceptibility testing. RESULTS: Phenotypic resistance to at least one first-line anti-TB drug was observed in 26/110 (23.6%) of the examined isolates, with isoniazid resistance being the most frequent, 23/110 (20.9%), followed by rifampicin resistance, 18/110 (16.4%). TB-Profiler showed almost perfect agreement with phenotypic drug susceptibility testing for rifampicin (sensitivity 94.4%, kappa = 0.96) and isoniazid (sensitivity 91.3%, kappa = 0.91), whereas MTBSeq showed slightly lower performance. Both pipelines demonstrated moderate to weak agreement with phenotypic drug susceptibility testing for detecting resistance to ethambutol, pyrazinamide, and streptomycin. The most frequently observed resistance mutations among phenotypically resistant isolates were rpoB (Ser450Leu), katG (S315Thr), embB (Met306Ile), and pncA (C-11 A > G) for rifampicin, isoniazid, ethambutol, and pyrazinamide, respectively. Discrepancies between genotypic and phenotypic drug susceptibility testing were observed across all first-line anti-TB drug-resistant isolates, particularly for ethambutol and pyrazinamide. CONCLUSION: We found a high prevalence of isoniazid resistance, along with rifampicin resistance, underscoring the need for early detection in vulnerable groups. Whole-genome sequencing showed good accuracy for these drugs, with TB-Profiler performing best. CLINICAL TRIAL NUMBER: Not applicable.
Drug resistance among children with culture-confirmed tuberculosis (TB) provides an accurate measure of transmitted drug resistance within the community. We describe the genotype diversity in children with culture-confirmed TB and investigate the relationship between genotype and drug resistance. A prospective study was conducted from March 2003 through August 2005 at Tygerberg Children's Hospital, in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed TB were included. Genotype analysis and phenotypic drug susceptibility testing were performed on the first culture-positive isolate from each patient. Mutation analysis was performed on all drug-resistant isolates. Spoligotyping was successfully performed on isolates from 391/399 (98%) children diagnosed with culture-confirmed TB. Drug susceptibility testing was also performed on 391 isolates; 49 (12.5%) were resistant to isoniazid, and 20 (5.1%) of these were resistant to both isoniazid and rifampin. Beijing was the most common genotype family, identified in 130/391 (33.2%) cases, followed by LAM in 114/391 (29.2%) cases. The presence of both Beijing and Haarlem genotype families was significantly associated with drug resistance (26/49 [53.1%] versus 113/342 [33.0%]; odds ratio, 1.7; 95% confidence interval, 1.0 to 2.9). The high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrates their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.
BACKGROUND AND PURPOSE: We tested whether antibiotic susceptibilities of drug-resistant Acinetobacter baumannii isolates could be used to predict the clinically important genotypes bla(TEM-1), AmpC-type bla, and integrase 1 gene (IntI1). METHODS: We analyzed 401 A. baumannii isolates obtained at Changhua Christian Hospital between April 2001 and March 2002. The isolates were all from blood cultures, and identification of A. baumannii was confirmed by API-20NE. Antibiotic susceptibility testing (phenotype) was performed by disk diffusion method. Polymerase chain reaction was used to detect the genes bla(TEM-1), AmpC-type bla and IntI1. RESULTS: Of 32 A. baumannii isolates, 10 possessed bla(TEM-1), 21 AmpC-type bla, and 26 IntI1. Resistance to ceftazidime (CAZ) predicted bla(TEM-1) genotype with 63.6% sensitivity, 100% specificity, 55.6% positive predictive value (PPV) and 0% negative predictive value (NPV). Trimethoprim-sulfamethoxazole (SXT) and gentamicin (GM) resistance predicted IntI1 genotype with 83.3% sensitivity, 71.4% specificity, 95.2% PPV and 45.4% NPV. No resistance phenotype could predict the AmpC-type bla genotype. CONCLUSIONS: CAZ resistance predicted the bla(TEM-1) genotype with 100% specificity, and SXT and GM resistance predicted the IntI1 genotype with 92.5% PPV. Therefore, antibiotic susceptibilities to CAZ, SXT, and GM can be utilized clinically to detect critical genotypes in A. baumannii.
Antimicrobial susceptibility testing has traditionally been based on measurements of minimal inhibitory concentrations of antimicrobials. Molecular genetic studies on antimicrobial resistance have produced a great deal of genetic information which can be used for diagnosis of antimicrobial resistance determinants. Bacteria can acquire resistance to macrolides, lincosamides and streptogramin antibiotics by modification of the target site of the drugs, by active efflux of the drugs, and by inactivation of the drugs. The genetic backgrounds of these resistance mechanisms are well known and several molecular methods for detection of resistance determinants have been developed. Outbreaks of multidrug-resistant tuberculosis have focused international attention on the emergence of Mycobacterium tuberculosis strains that are resistant to antimycobacterial agents. Knowledge of the antimycobacterial resistance genetics and progress in molecular methods has made it possible to develop rapid molecular methods for susceptibility testing. This review presents the genetic background of drug resistance and introduces some methods for genotypic susceptibility testing.
There are 16 approved human immunodeficiency virus type 1 (HIV-1) drugs belonging to three mechanistic classes: protease inhibitors, nucleoside and nucleotide reverse transcriptase (RT) inhibitors, and nonnucleoside RT inhibitors. HIV-1 resistance to these drugs is caused by mutations in the protease and RT enzymes, the molecular targets of these drugs. Drug resistance mutations arise most often in treated individuals, resulting from selective drug pressure in the presence of incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations, however, may also be transmitted to newly infected individuals. Three expert panels have recommended that HIV-1 protease and RT susceptibility testing should be used to help select HIV drug therapy. Although genotypic testing is more complex than typical antimicrobial susceptibility tests, there is a rich literature supporting the prognostic value of HIV-1 protease and RT mutations. This review describes the genetic mechanisms of HIV-1 drug resistance and summarizes published data linking individual RT and protease mutations to in vitro and in vivo resistance to the currently available HIV drugs.
OBJECTIVE(S): To compare antiretroviral resistance susceptibility testing of patient HIV-1 strains using genotype and phenotype methods. DESIGN: Eighteen plasma samples with viral load > 2000 HIV-1 RNA copies/ml were randomly selected for testing by both methods. Disease and treatment data were available for all patients. METHODS: Samples were analysed genotypically using a kit assay (HIV-1 Genotyping Systems, Applied Biosystems), performed by the Clinical Research Laboratory at Macfarlane Burnet Centre for Medical Research. Samples were analysed phenotypically using a rapid phenotypic assay (PhenoSenseTM HIV, ViroLogic), performed by the manufacturer. Results from both methods were interpreted using a defined protocol. Each susceptibility assay was performed and interpreted by individuals unaware of either the clinical data or the results of the other susceptibility assay. Concordance was defined categorically as either the presence of reduced susceptibility (> 2.5-fold change) in the phenotypic assay and resistance associated mutations in the genotypic assay, or the absence of these findings in both assays. RESULTS: Concordance between phenotypic and genotypic susceptibility testing was 81% for nucleoside reverse transcriptase inhibitors, 91% for non-nucleoside reverse transcriptase inhibitors and 90% for protease inhibitors. Complete concordance between phenotype and genotype for all 14 drugs evaluated was observed in three (17%) patient samples. CONCLUSIONS: Phenotypic and genotypic susceptibility appear to provide similar results. However, interpretation of genotypic results can be complicated, and both methods still require clinical validation.
BACKGROUND: In most low income countries there are twice as many cases of tuberculosis (TB) reported among men than among women, a difference commonly attributed to biological and epidemiological characteristics as well as socioeconomic and cultural barriers in access to health care. The World Health Organization has encouraged gender specific comparisons in TB rates to determine whether women with TB are less likely than men with TB to be diagnosed, reported, and treated. A study was undertaken to identify gender based differences in patients with pulmonary TB and to use this information to improve TB control efforts. METHODS: Individuals with a cough for more than 2 weeks in southern Mexico were screened from March 1995 to April 2003. Clinical and mycobacteriological information (isolation, identification, drug susceptibility testing and IS6110 based genotyping, and spoligotyping) was collected from those with bacteriologically confirmed pulmonary TB. Patients were treated in accordance with official norms and followed to ascertain treatment outcome, retreatment, and vital status. RESULTS: 623 patients with pulmonary TB were enrolled. The male:female incidence rate ratio for overall, reactivated, and recently transmitted disease was 1.58 (95% CI 1.34 to 1.86), 1.64 (95% CI 1.36 to 1.98), and 1.41 (95% CI 1.01 to 1.96), respectively. Men were more likely than women to default from treatment (adjusted OR 3.30, 95% CI 1.46 to 7.43), to be retreated (hazard ratio (HR) 3.15, 95% CI 1.38 to 7.22), and to die from TB (HR 2.23, 95% CI 1.25 to 3.99). CONCLUSIONS: Higher rates of transmitted and reactivated disease and poorer treatment outcomes among men are indicators of gender differentials in the diagnosis and treatment of pulmonary TB, and suggest specific strategies in endemic settings.
BACKGROUND: Consequences of drug-resistant tuberculosis (TB) in developing countries using directly observed treatment, short-course (DOTS), are not well defined. OBJECTIVE: To determine the impact of drug resistance on clinical outcome and transmission of TB under programmatic conditions. PATIENTS AND METHODS: A prospective cohort and molecular epidemiologic study was conducted in southern Mexico. Between March 1995 and February 1998 all patients with persistent cough whose sputa had acid-fast bacilli (AFB) underwent clinical and mycobacteriologic evaluation (species identification, drug susceptibility testing, and IS6110-based genotyping). Treatment was provided in accordance with Mexico's National Tuberculosis Program. Clinical and microbiologic outcomes and molecular epidemiologically defined transmission were measured. RESULTS: Mycobacterium tuberculosis was isolated from 238 of the 284 AFB smear-positive persons. The overall rate of resistance was 28.4% (new, 20.7%; retreated, 54.7%), and 10.8% (new, 3.3%; retreated, 35.8%) had multi-drug-resistant TB (ie, resistance to isoniazid and rifampin). After treatment, 75% (new, 81.0%; retreated, 52.8%) were cured, 8% (new, 7.8%; retreated, 7.5%) abandoned therapy, 9% (new, 3.9%; retreated, 28.3%) had treatment failure, and 4% (new, 3.3%; retreated, 7.5%) died. Another 2% of patients relapsed, and 9% died during a median of 24.4 months of follow-up. Drug-resistance was a strong independent risk factor for treatment failure. Being infected with multi-drug-resistant TB was the only factor associated with a decreased likelihood of being in a restriction fragment length polymorphism cluster. CONCLUSIONS: Despite the use of DOTS, patients with drug-resistant TB had a dramatically increased probability of treatment failure and death. Although multi-drug-resistant TB may have a decreased propensity to spread and cause disease, it has a profoundly negative impact on TB control.
OBJECTIVE: To rapidly identify drug-resistant Mycobacterium tuberculosis using phenotypic and genotypic methods and to evaluate the clinical significance of rapid phenotypic susceptibility test by phage amplified biologically assay (PhaB). METHODS: PhaB, DNA sequencing and polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) were performed on the 91 rifampicin (RFP)-resistant strains (including 82 multi-drug resistant Mycobacterium tuberculosis strains), 42 RFP-susceptible strains, 75 ofloxacin (OFLX)-resistant strains and 40 OFLX-susceptible strains at the same time. RESULTS: The results obtained using PhaB assay, DNA sequencing and PCR-SSCP were compared with the absolute concentration method. For RFP susceptibility, the accordance, sensitivity and specificity of PhaB were 93%, 92% and 95% respectively; the accordance, sensitivity and specificity of DNA sequencing were 93%, 90% and 100% respectively; those of PCR-SSCP were 90%, 86% and 100% respectively. For OFLX susceptibility, the accordance, sensitivity and specificity of PhaB assay were 95%, 95% and 95%; those of DNA sequencing were 80%, 71% and 98% respectively; those of PCR-SSCP were 75%, 63% and 98% respectively. CONCLUSIONS: PhaB assay is a low-cost, rapid, and sensitive method and shows high accordance with absolute concentration technology. It can give drug susceptibility test results within 48 - 96 h, and is a promising technology in clinical laboratory.