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.

FIND Tuberculosis Strain Bank: a Resource for Researchers and Developers Working on Tests To Detect Mycobacterium tuberculosis and Related Drug Resistance.

The spread of multidrug-resistant (MDR) tuberculosis (TB) and extensively drug-resistant (XDR) TB hampers global efforts in the fight against tuberculosis. To enhance the development and evaluation of diagnostic tests quickly and efficiently, well-characterized strains and samples from drug-resistant tuberculosis patients are necessary. In this project, the Foundation for Innovative New Diagnostics (FIND) has focused on the collection, characterization, and storage of such well-characterized reference materials and making them available to researchers and developers. The collection is being conducted at multiple centers in Southeast Asia, South America, Eastern Europe, and soon the sub-Saharan Africa regions. Strains are characterized for their phenotypic resistances and MICs to first-line drugs (FLDs) and second-line drugs (SLDs) using the automated MGIT 960 system following validated procedures and WHO criteria. Analysis of resistance-associated mutations is done by whole-genome sequencing (WGS) using the Illumina NextSeq system. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and WGS are used to determine strain lineages. All strains are maintained frozen at -80°C ± 10°C as distinct mother and daughter lots. All strains are extensively quality assured. The data presented here represent an analysis of the initial part of the collection. Currently, the bank contains 118 unique strains with extracted genomic DNA and matched sputum, serum, and plasma samples and will be expanded to a minimum of 1,000 unique strains over the next 3 years. Analysis of the current strains by phenotypic resistance testing shows 102 (86.4%), 10 (8.5%), and 6 (5.1%) MDR, XDR, and mono/poly resistant strains, respectively. Two of the strains are resistant to all 11 drugs that were phenotypically tested. WGS mutation analysis revealed FLD resistance-associated mutations in the rpoB, katG, inhA, embB, embA, and pncA genes; SLD resistance in the gyrA, gyrB, rrs, eis, and tlyA genes; and ethionamide resistance in the ethA genes. Most important lineages are represented in the bank, and further collections have been initiated to increase geographic and lineage diversity. The bank provides highly characterized and high-quality strains as a resource for researchers and developers in support of the development and evaluation of new diagnostics and drug resistance detection tools.

Target Product Profile for a Diagnostic Assay to Differentiate between Bacterial and Non-Bacterial Infections and Reduce Antimicrobial Overuse in Resource-Limited Settings: An Expert Consensus.

Acute fever is one of the most common presenting symptoms globally. In order to reduce the empiric use of antimicrobial drugs and improve outcomes, it is essential to improve diagnostic capabilities. In the absence of microbiology facilities in low-income settings, an assay to distinguish bacterial from non-bacterial causes would be a critical first step. To ensure that patient and market needs are met, the requirements of such a test should be specified in a target product profile (TPP). To identify minimal/optimal characteristics for a bacterial vs. non-bacterial fever test, experts from academia and international organizations with expertise in infectious diseases, diagnostic test development, laboratory medicine, global health, and health economics were convened. Proposed TPPs were reviewed by this working group, and consensus characteristics were defined. The working group defined non-severely ill, non-malaria infected children as the target population for the desired assay. To provide access to the most patients, the test should be deployable to community health centers and informal health settings, and staff should require <2 days of training to perform the assay. Further, given that the aim is to reduce inappropriate antimicrobial use as well as to deliver appropriate treatment for patients with bacterial infections, the group agreed on minimal diagnostic performance requirements of >90% and >80% for sensitivity and specificity, respectively. Other key characteristics, to account for the challenging environment at which the test is targeted, included: i) time-to-result <10 min (but maximally <2 hrs); ii) storage conditions at 0-40°C, ≤90% non-condensing humidity with a minimal shelf life of 12 months; iii) operational conditions of 5-40°C, ≤90% non-condensing humidity; and iv) minimal sample collection needs (50-100µL, capillary blood). This expert approach to define assay requirements for a bacterial vs. non-bacterial assay should guide product development, and enable targeted and timely efforts by industry partners and academic institutions.

Analytical and Clinical Evaluation of the Epistem Genedrive Assay for Detection of Mycobacterium tuberculosis.

The Epistem Genedrive assay rapidly detects the Mycobacterium tuberculosis omplex from sputum and is currently available for clinical use. However, the analytical and clinical performance of this test has not been fully evaluated. The analytical limit of detection (LOD) of the Genedrive PCR amplification was tested with genomic DNA; the performance of the complete (sample processing plus amplification) system was tested by spiking M. tuberculosismc(2)6030 cells into distilled water andM. tuberculosis-negative sputum. Specificity was tested using common respiratory pathogens and nontuberculosis mycobacteria. A clinical evaluation enrolled adults with suspected pulmonary tuberculosis, obtained three sputum samples from each participant, and compared the accuracy of the Gene drive to that of the Xpert MTB/RIF assay using M. tuberculosiscultures as the reference standard. The Genedrive assay had an LOD of 1 pg/µl (100 genomic DNA copies/reaction). The LODs of the system were 2.5 × 10(4)CFU/ml and 2.5 × 10(5)CFU/ml for cells spiked into water and sputum, respectively. False-positiverpoBprobe signals were observed in 3/32 (9.4%) of the negative controls and also in few samples containing Mycobacterium abscessus,Mycobacterium gordonae, o rMycobacterium thermoresistibile In the clinical study, among 336 analyzed participants, the overall sensitivities for the tuberculosis case detection of Gene drive, Xpert, and smear microscopy were 45.4% (95% confidence interval [CI], 35.2% to 55.8%), 91.8% (95% CI, 84.4% to 96.4%), and 77.3% (95% CI, 67.7% to 85.2%), respectively. The sensitivities of Gene drive and Xpert for the detection of smear-microscopy-negative tuberculosis were 0% (95% CI, 0% to 15.4%) and 68.2% (95% CI, 45.1% to 86.1%), respectively. The Genedrive assay did not meet performance standards recommended by the World Health Organization for a smear microscopy replacement tuberculosis test. Epistem is working on modifications to improve the assay.

Next-generation sequencing-based user-friendly platforms for drug-resistant tuberculosis diagnosis: A promise for the near future.

Since 2002, there has been a gradual worldwide 1.3% annual decrease in the incidence of tuberculosis (TB). This is an encouraging statistic; however, it will not achieve the World Health Organization's goal of eliminating TB by 2050, and it is being compounded by the persistent global incidence of drug-resistant tuberculosis (DR-TB) acquired by transmission and by treatment pressure. One key to effectively control tuberculosis and the spread of multiresistant strains is accurate information pertaining to drug resistance and susceptibility. Next-generation sequencing (NGS) has the potential to effectively change global health and the management of TB. Industry has focused primarily on using NGS for oncology diagnostics and human genomics, but the area in which NGS can rapidly impact health care is in the area of infectious disease diagnostics in low- and middle-income countries. To date, there has been a failure as a community to capitalize on the potential of NGS, especially at the reference laboratory level where it can provide actionable information pertaining to treatment options for patients. The rapid evolution of knowledge about the genetic foundations of tuberculosis drug resistance makes sequencing a versatile technology platform for providing rapid, accurate, and actionable results for treating this disease. No "plug-and-play" and "end-to-end" NGS solutions exist that provide clinically relevant sequence data from the Mycobacterium tuberculosis complex genome from primary clinical samples (e.g., sputum) in high-burden country reference laboratories, which is where they are most needed. However, such a system-based solution is underdeveloped by Foundation for Innovative Diagnostics (FIND), in collaboration with partners from academia, nongovernmental organizations, and industry. The solution is modular and is designed and developed to perform targeted amplicon sequencing directly from a patient's primary sputum sample. This solution will initially allow reference laboratories to perform reflex NGS that provides a rapid and comprehensive analysis of a patient's M. tuberculosis complex drug resistance profile, thereby facilitating optimization of a patient's treatment, improving treatment outcomes, and reducing the spread of DR-TB. Such a system could also enable countries to implement culture-free drug resistance surveillance programs, which could bypass the need for expensive culture facilities, decrease a country's dependence on external laboratories, and significantly expand the map of global surveillance capabilities. In addition, the introduction of such a system will provide a foundation for NGS to be used for genotypic testing for human immunodeficiency virus-infected patients, surveillance of other diseases, in-country capability for outbreak discovery and management, and a host of other diagnostic benefits that are currently limited to high-income countries.

Collaborative Effort for a Centralized Worldwide Tuberculosis Relational Sequencing Data Platform.

Continued progress in addressing challenges associated with detection and management of tuberculosis requires new diagnostic tools. These tools must be able to provide rapid and accurate information for detecting resistance to guide selection of the treatment regimen for each patient. To achieve this goal, globally representative genotypic, phenotypic, and clinical data are needed in a standardized and curated data platform. A global partnership of academic institutions, public health agencies, and nongovernmental organizations has been established to develop a tuberculosis relational sequencing data platform (ReSeqTB) that seeks to increase understanding of the genetic basis of resistance by correlating molecular data with results from drug susceptibility testing and, optimally, associated patient outcomes. These data will inform development of new diagnostics, facilitate clinical decision making, and improve surveillance for drug resistance. ReSeqTB offers an opportunity for collaboration to achieve improved patient outcomes and to advance efforts to prevent and control this devastating disease.

Integration of published information into a resistance-associated mutation database for Mycobacterium tuberculosis.

Tuberculosis remains a major global public health challenge. Although incidence is decreasing, the proportion of drug-resistant cases is increasing. Technical and operational complexities prevent Mycobacterium tuberculosis drug susceptibility phenotyping in the vast majority of new and retreatment cases. The advent of molecular technologies provides an opportunity to obtain results rapidly as compared to phenotypic culture. However, correlations between genetic mutations and resistance to multiple drugs have not been systematically evaluated. Molecular testing of M. tuberculosis sampled from a typical patient continues to provide a partial picture of drug resistance. A database of phenotypic and genotypic testing results, especially where prospectively collected, could document statistically significant associations and may reveal new, predictive molecular patterns. We examine the feasibility of integrating existing molecular and phenotypic drug susceptibility data to identify associations observed across multiple studies and demonstrate potential for well-integrated M. tuberculosis mutation data to reveal actionable findings.