Predicting stringent QuantiFERON-TB Gold Plus conversions in contacts of tuberculosis patients.

OBJECTIVES: To assess associations between disease severity in index TB patients and QuantiFERON-TB Gold Plus (QFT-Plus) results in contacts, and predictors for QFT-Plus conversion in contacts over 6-12 months. METHODS: TB patients (n = 295) and the contacts (n = 1051) were enrolled during 2018-2021 with QFT-Plus performed at baseline and months 6 and 12. A strong CD8 response was defined as TB2 interferon gamma (IFN-γ) response minus TB1 >0.6 IU/ml and stringent conversion as change from QFT-plus negative to high-positive QFT-Plus (TB1 or TB2 IFN-γ responses >0.7 IU/ml). RESULTS: Contacts with index TB patients with sputum smear >1+ was associated with positive QFT-Plus compared to those without (p < 0.001). Contacts with index TB patients with bilateral lung disease were more likely to have strong CD8 responses than those without (p = 0.038). QFT-Plus stringent conversion occurred in 9.7% of contacts over 6-12 months. A TB1 IFN-γ response ≥0.03 IU/ml combined with a TB2 ≥0.06 IU/ml was predictive of a 19-fold increased risk for QFT-Plus stringent conversion in contacts (odd ratio 19.565 [8.484-45.116], p < 0.001). CONCLUSION: Bacterial burden and bilateral lung disease of index TB patients were associated with positive QFT-Plus and strong CD8 responses in contacts. TB1 and TB2 IFN-γ responses were synergistically predictive of stringent conversion in contacts.

Updating the WHO target product profile for next-generation Mycobacterium tuberculosis drug susceptibility testing at peripheral centres.

There were approximately 10 million tuberculosis (TB) cases in 2020, of which 500,000 were drug-resistant. Only one third of drug-resistant TB cases were diagnosed and enrolled on appropriate treatment, an issue partly driven by a lack of rapid, accurate drug-susceptibility testing (DST) tools deployable in peripheral settings. In 2014, World Health Organization (WHO) published target product profiles (TPPs) which detailed minimal and optimal criteria to address high-priority TB diagnostic needs, including DST. Since then, the TB community's needs have evolved; new treatment regimens, changes in TB definitions, further emergence of drug resistance, technological advances, and changing end-users requirements have necessitated an update. The DST TPP's revision was therefore undertaken by WHO with the Stop TB Partnership New Diagnostics Working Group. We describe the process of updating the TPP for next-generation TB DST for use at peripheral centres, highlight key updates, and discuss guidance regarding technical and operational specifications.

Designing molecular diagnostics for current tuberculosis drug regimens.

Diagnostic development must occur in parallel with drug development to ensure the longevity of new treatment compounds. Despite an increasing number of novel and repurposed anti-tuberculosis compounds and regimens, there remains a large number of drugs for which no rapid and accurate molecular diagnostic option exists. The lack of rapid drug susceptibility testing for linezolid, bedaquiline, clofazimine, the nitroimidazoles (i.e pretomanid and delamanid) and pyrazinamide at any level of the healthcare system compromises the effectiveness of current tuberculosis and drug-resistant tuberculosis treatment regimens. In the context of current WHO tuberculosis treatment guidelines as well as promising new regimens, we identify the key diagnostic gaps for initial and follow-on tests to diagnose emerging drug resistance and aid in regimen selection. Additionally, we comment on potential gene targets for inclusion in rapid molecular drug susceptibility assays and sequencing assays for novel and repurposed drug compounds currently prioritized in current regimens, and evaluate the feasibility of mutation detection given the design of existing technologies. Based on current knowledge, we also propose design priorities for next generation molecular assays to support triage of tuberculosis patients to appropriate and effective treatment regimens. We encourage assay developers to prioritize development of these key molecular assays and support the continued evolution, uptake, and utility of sequencing to build knowledge of tuberculosis resistance mechanisms and further inform rapid treatment decisions in order to curb resistance to critical drugs in current regimens and achieve End TB targets.Trial registration: ClinicalTrials.gov identifier: NCT05117788..

Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Circulating mitochondrial cell-free DNA dynamics in patients with mycobacterial pulmonary infections: Potential for a novel biomarker of disease.

Objectives: Human mitochondrial cell-free DNA (Mt-cfDNA) may serve as a useful biomarker for infectious processes. We investigated Mt-cfDNA dynamics in patients with pulmonary mycobacterial infections to determine if this novel biomarker could be used to differentiate disease states and severity. Methods: Patients with pulmonary tuberculosis (PTB), latent tuberculosis infection (LTBI), and nontuberculous mycobacterial-lung disease (NTM-LD) were enrolled at a tertiary care hospital in Taiwan between June 2018 and August 2021. Human Mt-cfDNA and nuclear-cfDNA (Nu-cfDNA) copy numbers were estimated by quantitative polymerase chain reaction. Variables associated with PTB and 2-month sputum culture-positivity, indicating poor treatment response, were assessed using logistic regression. Results: Among 97 patients with PTB, 64 with LTBI, and 51 with NTM-LD, Mt-cfDNA levels were higher in patients with PTB than in LTBI (p=0.001) or NTM-LD (p=0.006). In the Mycobacterium tuberculosis-infected population, Mt-cfDNA levels were highest in smear-positive PTB patients, followed by smear-negative PTB (p<0.001), and were lowest in LTBI persons (p=0.009). A Mt-cfDNA, but not Nu-cfDNA, level higher than the median helped differentiate culture-positive PTB from culture-negative PTB and LTBI (adjusted OR 2.430 [95% CI 1.139-5.186], p=0.022) and differentiate PTB from NTM-LD (adjusted OR 4.007 [1.382-12.031], p=0.011). Mt-cfDNA levels decreased after 2 months of treatment in PTB patients (p=0.010). A cutoff Mt-cfDNA level greater than 62.62 x 106 copies/µL-plasma was associated with a 10-fold risk of 2-month culture-positivity (adjusted OR 9.691 [1.046-89.813], p=0.046). Conclusion: Elevated Mt-cfDNA levels were associated with PTB disease and failed sputum conversion at 2 months in PTB patients, and decreased after treatment.

Variants in Bedaquiline-Candidate-Resistance Genes: Prevalence in Bedaquiline-Naive Patients, Effect on MIC, and Association with Mycobacterium tuberculosis Lineage.

Studies have shown that variants in bedaquiline-resistance genes can occur in isolates from bedaquiline-naive patients. We assessed the prevalence of variants in all bedaquiline-candidate-resistance genes in bedaquiline-naive patients, investigated the association between these variants and lineage, and the effect on phenotype. We used whole-genome sequencing to identify variants in bedaquiline-resistance genes in isolates from 509 bedaquiline treatment naive South African tuberculosis patients. A phylogenetic tree was constructed to investigate the association with the isolate lineage background. Bedaquiline MIC was determined using the UKMYC6 microtiter assay. Variants were identified in 502 of 509 isolates (98.6%), with the highest (85%) prevalence of variants in the Rv0676c (mmpL5) gene. We identified 36 unique variants, including 19 variants not reported previously. Only four isolates had a bedaquiline MIC equal to or above the epidemiological cut-off value of 0.25 µg/mL. Phylogenetic analysis showed that 14 of the 15 variants observed more than once occurred monophyletically in one Mycobacterium tuberculosis (sub)lineage. The bedaquiline MIC differed between isolates belonging to lineage 2 and 4 (Fisher's exact test, P = 0.0004). The prevalence of variants in bedaquiline-resistance genes in isolates from bedaquiline-naive patients is high, but very few (<2%) isolates were phenotypically resistant. We found an association between variants in bedaquiline resistance genes and Mycobacterium tuberculosis (sub)lineage, resulting in a lineage-dependent difference in bedaquiline phenotype. Future studies should investigate the impact of the presence of variants on bedaquiline-resistance acquisition and treatment outcome.

The 2021 WHO catalogue of Mycobacterium tuberculosis complex mutations associated with drug resistance: A genotypic analysis.

Background: Molecular diagnostics are considered the most promising route to achieving rapid, universal drug susceptibility testing for Mycobacterium tuberculosiscomplex (MTBC). We aimed to generate a WHO endorsed catalogue of mutations to serve as a global standard for interpreting molecular information for drug resistance prediction. Methods: A candidate gene approach was used to identify mutations as associated with resistance, or consistent with susceptibility, for 13 WHO endorsed anti-tuberculosis drugs. 38,215 MTBC isolates with paired whole-genome sequencing and phenotypic drug susceptibility testing data were amassed from 45 countries. For each mutation, a contingency table of binary phenotypes and presence or absence of the mutation computed positive predictive value, and Fisher's exact tests generated odds ratios and Benjamini-Hochberg corrected p-values. Mutations were graded as Associated with Resistance if present in at least 5 isolates, if the odds ratio was >1 with a statistically significant corrected p-value, and if the lower bound of the 95% confidence interval on the positive predictive value for phenotypic resistance was >25%. A series of expert rules were applied for final confidence grading of each mutation. Findings: 15,667 associations were computed for 13,211 unique mutations linked to one or more drugs. 1,149/15,667 (7·3%) mutations were classified as associated with phenotypic resistance and 107/15,667 (0·7%) were deemed consistent with susceptibility. For rifampicin, isoniazid, ethambutol, fluoroquinolones, and streptomycin, the mutations' pooled sensitivity was >80%. Specificity was over 95% for all drugs except ethionamide (91·4%), moxifloxacin (91·6%) and ethambutol (93·3%). Only two resistance mutations were classified for bedaquiline, delamanid, clofazimine, and linezolid as prevalence of phenotypic resistance was low for these drugs. Interpretation: This first WHO endorsed catalogue of molecular targets for MTBC drug susceptibility testing provides a global standard for resistance interpretation. Its existence should encourage the implementation of molecular diagnostics by National Tuberculosis Programmes. Funding: UNITAID, Wellcome, MRC, BMGF.

Detecting rifampin and isoniazid resistance in Mycobacterium tuberculosis direct from patient sputum using an automated integrated system.

While there has been progress in detection of drug resistant tuberculosis globally, WHO estimates only about half of the patients with bacteriologically confirmed tuberculosis were tested for rifampicin resistance over the past two years. To close this drug resistance diagnostic gap, an expansion of testing for rifampicin and isoniazid resistance is critically needed. The Akonni Biosystem Integrated System combines DNA extraction and a Lab-on-a-Film assembly (LFA) to perform rapid probe and PCR-based detection of resistance associated mutations to first-line anti-tuberculosis drugs. Using raw sputum samples from 25 tuberculosis patients at risk for drug resistance, we conducted a proof-of-concept study of the Integrated System with an MDR-TB assay. Performance of the Integrated System was compared to liquid Mycobacteria Growth Indicator Tube (MGIT) culture reference phenotypes using 2012 WHO endorsed critical concentrations for rifampicin and isoniazid. The overall percent agreement for rifampicin and isoniazid was 91.7% and 100% respectively, with agreement for rifampicin increasing to 95.7% after low-level resistance mutations in rpoB were excluded. The Integrated System, combining DNA extraction and LFA amplification, is a promising new tool for detection of both rifampicin and isoniazid using liquefied raw sputum.

Prevalence of SARS-CoV-2 infection in Baja California, Mexico: Findings from a community-based survey in February 2021 in the Mexico-United States border.

Between March 2020 and February 2021, the state of Baja California, Mexico, which borders the United States, registered 46,118 confirmed cases of COVID-19 with a mortality rate of 238.2 deaths per 100,000 residents. Given limited access to testing, the population prevalence of SARS-CoV-2 infection is unknown. The objective of this study is to estimate the seroprevalence and real time polymerase chain reaction (RT-PCR) prevalence of SARS-CoV-2 infection in the three most populous cities of Baja California prior to scale-up of a national COVID-19 vaccination campaign. Probabilistic three-stage clustered sampling was used to conduct a population-based household survey of residents five years and older in the three cities. RT-PCR testing was performed on nasopharyngeal swabs and SARS-CoV-2 seropositivity was determined by IgG antibody testing using fingerstick blood samples. An interviewer-administered questionnaire assessed participants' knowledge, attitudes, and preventive practices regarding COVID-19. In total, 1,126 individuals (unweighted sample) were surveyed across the three cities. Overall prevalence of SARS-CoV-2 infection by RT-PCR was 7.8% (95% CI 5.5-11.0) and IgG seroprevalence was 21.1% (95% CI 17.4-25.2). There was no association between border crossing in the past 6 months and SARS-CoV-2 prevalence (unadjusted OR 0.40, 95%CI 0.12-1.30). While face mask use and frequent hand washing were common among participants, quarantine or social isolation at home to prevent infection was not. Regarding vaccination willingness, 30.4% (95% CI 24.4-3 7.1) of participants said they were very unlikely to get vaccinated. Given the high prevalence of active SARS-CoV-2 infection in Baja California at the end of the first year of the pandemic, combined with its low seroprevalence and the considerable proportion of vaccine hesitancy, this important area along the Mexico-United States border faces major challenges in terms of health literacy and vaccine uptake, which need to be further explored, along with its implications for border restrictions in future epidemics.

Detection and quantification of Mycobacterium tuberculosis antigen CFP10 in serum and urine for the rapid diagnosis of active tuberculosis disease.

Outside of the ongoing COVID-19 pandemic, tuberculosis is the leading cause of infectious disease mortality globally. Currently, there is no commercially available point-of-care diagnostic that is rapid, inexpensive, and highly sensitive for the diagnosis of active tuberculosis disease. Here we describe the development and optimization of a novel, highly sensitive prototype bioelectronic tuberculosis antigen (BETA) assay to detect tuberculosis-specific antigen, CFP10, in small-volume serum and urine samples. In this proof-of-concept study we evaluated the performance of the BETA assay using clinical specimens collected from presumptive tuberculosis patients from three independent cohorts. Circulating CFP10 antigen was detected in ALL serum (n = 19) and urine (n = 3) samples from bacteriologically confirmed tuberculosis patients who were untreated or had less than one week of treatment at time of serum collection, successfully identifying all culture positive tuberculosis patients. No CFP10 antigen was detected in serum (n = 7) or urine (n = 6) samples from individuals who were determined to be negative for tuberculosis disease. Additionally, antigen quantification using the BETA assay of paired serum samples collected from tuberculosis patients (n = 8) both before and after treatment initiation, indicate consistently declining within-person levels of CFP10 antigen during treatment. This novel, low-cost assay demonstrates potential as a rapid, non-sputum-based, point-of-care tool for the diagnosis of tuberculosis disease.

Age and sex distribution of Mycobacterium tuberculosis infection and rifampicin resistance in Myanmar as detected by Xpert MTB/RIF.

BACKGROUND: Detection of tuberculosis disease (TB) and timely identification of Mycobacterium tuberculosis (Mtb) strains that are resistant to treatment are key to halting tuberculosis transmission, improving treatment outcomes, and reducing mortality. METHODS: We used 332,657 Xpert MTB/RIF assay results, captured as part of the Myanmar Data Utilization Project, to characterize Mtb test positivity and rifampicin resistance by both age and sex, and to evaluate risk factors associated with rifampicin resistance. RESULTS: Overall, 70% of individuals diagnosed with TB were males. Test positivity was higher among males (47%) compared to females (39%). The highest positivity by age occurred among individuals aged 16-20, with test positivity for females (65%) higher than for males (57%). Although a greater absolute number of males were rifampicin resistant, a greater proportion of females (11.4%) were rifampicin resistant as compared to males (9.3%). In the multivariate model, history of previous treatment, age less than 30, testing in the Yangon region, and female sex were significantly positively associated with rifampicin resistance after adjusting for HIV status and year test was performed. CONCLUSIONS: Our results indicate that young adults were more likely to test positive for TB and be identified as rifampicin resistant compared to older adults.

Rapid Detection of Extensively Drug-Resistant Tuberculosis in Clinical Samples Using a Novel Tabletop Platform: Protocol for a Prospective Clinical Study.

BACKGROUND: The lack of accurate and efficient diagnostic devices for extensively drug-resistant tuberculosis (XDR-TB) makes it a severe threat to global public health. A prospective clinical study in an intended-use cohort was designed to evaluate the Akonni Biosystems XDR-TB TruArray and lateral flow cell (XDR-LFC) to address this gap in tuberculosis diagnostics. OBJECTIVE: This paper presents the protocol for a study that aims to document the conceptualization and design of this evaluation method for early dissemination while data collection and analysis are ongoing. METHODS: The clinical study was conducted in three phases. The first phase was to observe changes in bacterial load and culture positivity in patient sputa over time and better understand the diversity of prospective clinical samples. The second phase was to prospectively collect clinical samples for sensitivity and specificity testing of the Akonni Biosystems XDR-LFC device. Lastly, the third phase was to explore the anti-TB drug concentrations in serum throughout the drug-resistant tuberculosis treatment. RESULTS: The methodology described includes the study design, laboratory sample handling, data collection, and the protection elements of human subjects of this clinical study to evaluate a potential new XDR-TB diagnostic device. A total of 664 participants were enrolled across the three phases. The implemented complex systems facilitated a thorough clinical data collection for an objective evaluation of the device. The study is closed to recruitment. The follow-up data collection and analysis are in progress. CONCLUSIONS: This paper outlined a prospective cohort study protocol to evaluate a rapid XDR-TB detection device, which may be informative for other researchers with similar goals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/26748.

Laboratory Evaluation of a Lateral-Flow Cell for Molecular Detection of First-Line and Second-Line Antituberculosis Drug Resistance.

Despite the WHO's call for universal drug susceptibility testing for all patients being evaluated for tuberculosis (TB), a lack of rapid diagnostic tests which can fully describe TB resistance patterns is a major challenge in ensuring that all persons diagnosed with drug-resistant TB are started on an appropriate treatment regime. We evaluated the accuracy of the Akonni Biosystems XDR-TB TruArray and lateral-flow cell (XDR-LFC), a novel multiplex assay to simultaneously detect mutations across seven genes that confer resistance to both first- and second-line anti-TB drugs. The XDR-LFC includes 271 discrete three-dimensional gel elements with target-specific probes for identifying mutations in katG, inhA promoter, and ahpC promoter (isoniazid), rpoB (rifampin), gyrA (fluoroquinolones), rrs and eis promoter (kanamycin), and rrs (capreomycin and amikacin). We evaluated XDR-LFC performance with 87 phenotypically and genotypically characterized clinical Mycobacterium tuberculosis isolates. The overall assay levels of accuracy for mutation detection in specific genes were 98.6% for eis promoter and 100.0% for the genes katG, inhA promoter, ahpC promoter, rpoB, gyrA, and rrs The sensitivity and specificity against phenotypic reference were 100% and 100% for isoniazid, 98.4% and 50% for rifampin (specificity increased to 100% once the strains with documented low-level resistance mutations in rpoB were excluded), 96.2% and 100% for fluoroquinolones, 92.6% and 100% for kanamycin, 93.9% and 97.4% for capreomycin, and 80% and 100% for amikacin. The XDR-LFC solution appears to be a promising new tool for accurate detection of resistance to both first- and second-line anti-TB drugs.

Application of Targeted Next-Generation Sequencing Assay on a Portable Sequencing Platform for Culture-Free Detection of Drug-Resistant Tuberculosis from Clinical Samples.

Targeted next-generation sequencing (tNGS) has emerged as a comprehensive alternative to existing methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis from patient sputum samples for clinical diagnosis of drug-resistant tuberculosis (DR-TB). However, the complexity of sequencing platforms has limited their uptake in low-resource settings. The goal of this study was to evaluate the use of the tNGS-based DST solution Genoscreen Deeplex Myc-TB, for use on the compact, low-cost Oxford Nanopore Technologies MinION sequencer. One hundred four DNA samples extracted from smear-positive sputum sediments, previously sequenced using the Deeplex assay on an Illumina MiniSeq, were resequenced on MinION after applying a custom library preparation. MinION read quality, mapping statistics, and variant calling were computed using an in-house pipeline and compared to the reference MiniSeq data. The average percentage of MinION reads mapped to an H37RV reference genome was 90.8%, versus 99.5% on MiniSeq. The mean depths of coverage were 4,151× and 4,177× on MinION and MiniSeq, respectively, with heterogeneous distribution across targeted genes. Composite reference coverage breadth was >99% for both platforms. We observed full concordance between technologies in reporting the clinically relevant drug-resistant markers, including full gene deletions. In conclusion, we demonstrated that the workflow and sequencing data obtained from Deeplex on MinION are comparable to those for the MiniSeq, despite the higher raw error rates on MinION, with the added advantage of MinION's portability, versatility, and low capital costs. Targeted NGS on MinION is a promising DST solution for rapidly providing clinically relevant data to manage complex DR-TB cases.

Review of automated DNA extraction systems for sequencing-based solutions for drug-resistant tuberculosis detection.

Robust clinical specimen nucleic acid extraction instrumentation and methods are critical to the performance of downstream molecular diagnostics for the diagnosis of drug-resistant tuberculosis (DR-TB). Currently, there is a high level of interest in sequencing-based solutions for rapid and comprehensive DR-TB testing from primary specimens (i.e., sputum). However, there is no standardized or fully automated sputum extraction system that has been widely implemented for use with Mycobacterium tuberculosis complex-containing sputum specimens. For sequencing-based technologies to be widely adopted in clinical laboratory settings in low- and middle-income countries, automated extraction technologies will be important to enhance scalability and reliability and to standardize performance of the downstream assays. Additionally, the ease of automatic technologies allows for faster uptake in laboratories currently without the expertise or infrastructure to perform manual extractions at the same automated throughput. This work is intended to provide an initial specification comparison of available automated DNA extraction systems that could serve as front-end components for existing and future sequencing approaches and provide the framework for future evaluations.

Author Correction: Integrating standardized whole genome sequence analysis with a global Mycobacterium tuberculosis antibiotic resistance knowledgebase.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Requiring smartphone ownership for mHealth interventions: who could be left out?

BACKGROUND: Mobile health (mHealth) interventions have the potential to improve health through patient education and provider engagement while increasing efficiency and lowering costs. This raises the question of whether disparities in access to mobile technology could accentuate disparities in mHealth mediated care. This study addresses whether programs planning to implement mHealth interventions risk creating or perpetuating health disparities based on inequalities in smartphone ownership. METHODS: Video Directly Observed Therapy (VDOT) is an mHealth intervention for monitoring tuberculosis (TB) treatment adherence through videos sent by patients to their healthcare provider using smartphones. We conducted secondary analyses of data from a single-arm trial of VDOT for TB treatment monitoring by San Diego, San Francisco, and New York City health departments. Baseline and follow-up treatment interviews were used to assess participant smartphone ownership, sociodemographics and TB treatment perceptions. Univariate and multivariable logistic regression analyses were used to identify correlates of smartphone ownership. RESULTS: Of the 151 participants enrolled, mean age was 41 years (range: 18-87 years) and 41.1% were female. Participants mostly identified as Asian (45.0%) or Hispanic/Latino (29.8%); 57.8% had at most a high school education. At baseline, 30.4% did not own a smartphone, which was similar across sites. Older participants (adjusted odds ratio [AOR] = 1.09 per year, 95% confidence interval [CI]: 1.05-1.12), males (AOR = 2.86, 95% CI: 1.04-7.86), participants having at most a high school education (AOR = 4.48, 95% CI: 1.57-12.80), and those with an annual income below $10,000 (AOR = 3.06, 95% CI: 1.19, 7.89) had higher odds of not owning a smartphone. CONCLUSIONS: Approximately one-third of TB patients in three large United States of America (USA) cities lacked smartphones prior to the study. Patients who were older, male, less educated, or had lower annual income were less likely to own smartphones and could be denied access to mHealth interventions if personal smartphone ownership is required.

Guidance for Studies Evaluating the Accuracy of Rapid Tuberculosis Drug-Susceptibility Tests.

The development and implementation of rapid molecular diagnostics for tuberculosis (TB) drug-susceptibility testing is critical to inform treatment of patients and to prevent the emergence and spread of resistance. Optimal trial planning for existing tests and those in development will be critical to rapidly gather the evidence necessary to inform World Health Organization review and to support potential policy recommendations. The evidence necessary includes an assessment of the performance for TB and resistance detection as well as an assessment of the operational characteristics of these platforms. The performance assessment should include analytical studies to confirm the limit of detection and assay ability to detect mutations conferring resistance across globally representative strains. The analytical evaluation is typically followed by multisite clinical evaluation studies to confirm diagnostic performance in sites and populations of intended use. This paper summarizes the considerations for the design of these analytical and clinical studies.

Whole genome sequencing of Mycobacterium tuberculosis: current standards and open issues.

Whole genome sequencing (WGS) of Mycobacterium tuberculosis has rapidly progressed from a research tool to a clinical application for the diagnosis and management of tuberculosis and in public health surveillance. This development has been facilitated by drastic drops in cost, advances in technology and concerted efforts to translate sequencing data into actionable information. There is, however, a risk that, in the absence of a consensus and international standards, the widespread use of WGS technology may result in data and processes that lack harmonization, comparability and validation. In this Review, we outline the current landscape of WGS pipelines and applications, and set out best practices for M. tuberculosis WGS, including standards for bioinformatics pipelines, curated repositories of resistance-causing variants, phylogenetic analyses, quality control and standardized reporting.

Whole-genome and targeted sequencing of drug-resistant Mycobacterium tuberculosis on the iSeq100 and MiSeq: A performance, ease-of-use, and cost evaluation.

BACKGROUND: Accurate, comprehensive, and timely detection of drug-resistant tuberculosis (TB) is essential to inform patient treatment and enable public health surveillance. This is crucial for effective control of TB globally. Whole-genome sequencing (WGS) and targeted next-generation sequencing (NGS) approaches have potential as rapid in vitro diagnostics (IVDs), but the complexity of workflows, interpretation of results, high costs, and vulnerability of instrumentation have been barriers to broad uptake outside of reference laboratories, especially in low- and middle-income countries. A new, solid-state, tabletop sequencing instrument, Illumina iSeq100, has the potential to decentralize NGS for individual patient care. METHODS AND FINDINGS: In this study, we evaluated WGS and targeted NGS for TB on both the new iSeq100 and the widely used MiSeq (both manufactured by Illumina) and compared sequencing performance, costs, and usability. We utilized DNA libraries produced from Mycobacterium tuberculosis clinical isolates for the evaluation. We conducted WGS on three strains and observed equivalent uniform genome coverage with both platforms and found the depth of coverage obtained was consistent with the expected data output. Utilizing the standardized, cloud-based ReSeqTB bioinformatics pipeline for variant analysis, we found the two platforms to have 94.0% (CI 93.1%-94.8%) agreement, in comparison to 97.6% (CI 97%-98.1%) agreement for the same libraries on two MiSeq instruments. For the targeted NGS approach, 46 M. tuberculosis-specific amplicon libraries had 99.6% (CI 98.0%-99.9%) agreement between the iSeq100 and MiSeq data sets in drug resistance-associated SNPs. The upfront capital costs are almost 5-fold lower for the iSeq100 ($19,900 USD) platform in comparison to the MiSeq ($99,000 USD); however, because of difference in the batching capabilities, the price per sample for WGS was higher on the iSeq100. For WGS of M. tuberculosis at the minimum depth of coverage of 30x, the cost per sample on the iSeq100 was $69.44 USD versus $28.21 USD on the MiSeq, assuming a 2 × 150 bp run on a v3 kit. In terms of ease of use, the sequencing workflow of iSeq100 has been optimized to only require 27 minutes total of hands-on time pre- and post-run, and the maintenance is simplified by a single-use cartridge-based fluidic system. As these are the first sequencing attempts on the iSeq100 for M. tuberculosis, the sequencing pool loading concentration still needs optimization, which will affect sequencing error and depth of coverage. Additionally, the costs are based on current equipment and reagent costs, which are subject to change. CONCLUSIONS: The iSeq100 instrument is capable of running existing TB WGS and targeted NGS library preparations with comparable accuracy to the MiSeq. The iSeq100 has reduced sequencing workflow hands-on time and is able to deliver sequencing results in <24 hours. Reduced capital and maintenance costs and lower-throughput capabilities also give the iSeq100 an advantage over MiSeq in settings of individualized care but not in high-throughput settings such as reference laboratories, where sample batching can be optimized to minimize cost at the expense of workflow complexity and time.