An External Evaluation of a Metagenomic Next Generation Sequencing Workflow for Infection Diagnosis Using Oxford Nanopore Sequencing.

Diagnostic microbiology has traditionally involved culture of organisms to diagnose infection, which is time consuming, insensitive for organisms that are difficult to grow, and compromised by prior antimicrobial therapy. Molecular diagnostics, predominantly in the form of nucleic acid amplification tests (NAAT), e.g. PCR, overcome some of these limitations and are now in widespread and increasing use. NAAT-based tests are however limited by only being able to detect a small number of pre-specified organisms and can offer limited to no antimicrobial susceptibility information.

Metagenomic next generation sequencing (mNGS) works by directly sequencing all of the nucleic acid in a microbiological sample, thus allowing identification of all microorganisms that are present in sufficient quantity, along with the potential to infer antimicrobial susceptibility patterns based on the presence or absence of relevant genes. Unlike NAAT, no pre-specification of target pathogen(s) is required, so mNGS has the potential to identify important pathogens that may have not been tested for otherwise. Host (human) sequences will also be present in the sample, so are removed from the analysis either by preventing them from being sequenced, or deleting them during the initial analysis steps.

mNGS therefore has the ability to overcome the limitations of both culture-based and NAAT-based infection diagnosis, with the potential to offer rapid diagnostics with greater levels of antimicrobial susceptibility detail, which is less affected by whether the organism is viable/culturable. Rapid infection diagnostics has the ability to significantly improve patient care, whereby appropriately targeted antimicrobial therapy can be instituted promptly (or ceased if e.g. a viral pathogen is identified). This is of particular importance given ongoing global increases in antimicrobial resistance. Rapid diagnostics with mNGS may also reduce the need for multiple other lines of investigation. There are likely to be certain groups of patients where this technology can be particularly targeted for maximal benefit either due to the rapidity of the results or the ability to diagnose infections that may not have been clinically suspected or detected with standard processes. In the investigators' department, several cases have been seen recently where patients have had very poor outcomes due to delays in diagnosis, where mNGS would have had the potential to markedly improve their outcomes. There are also potential benefits on a population level, such as reducing exposure of the population to overly broad-spectrum antibiotics, rapid identification and surveillance of communicable diseases that may require a public health response, and expediting appropriate management and flow of patients through an already congested hospital system. mNGS also has the ability to detect novel pathogens. As an example, the rapid identification and dissemination of information relating to SARS-CoV-2 was due to the availability of rapid 'agnostic' sequencing technologies.

Next generation sequencing has typically been too expensive to be used as a front-line diagnostic test, with its use confined to larger research-affiliated institutions. However, nanopore sequencing (Oxford Nanopore Technologies [ONT]), now offers a relatively inexpensive option, with a small physical footprint and an ability to generate a large amount of sequence data rapidly, making it a potentially viable option for front-line diagnostic microbiology laboratories. As such, there is considerable interest in the use of nanopore sequencing for mNGS. A number of publications have reported on its use in clinical diagnostics, and it is already in use in a number of healthcare settings overseas

. Continuous Quality Improvement (QI) via the evaluation of new diagnostic assays is a critically important component of clinical laboratory medicine. In line with this, the investigators are interested in evaluating the use of mNGS in their laboratory as a QI initiative to enhance the diagnostic service, increase the sensitivity of infection diagnostic testing, and compare existing standard diagnostic procedures against mNGS. The investigators plan to undertake this in the form of an external evaluation, whereby samples from Wellington Southern Community Laboratories (WSCL) would be forwarded to the Institute of Environmental Sciences and Research (ESR) for mNGS testing. ESR has existing expertise in sequencing and bioinformatics and have already developed mNGS capability, however have not comprehensively evaluated it on real patient samples. The initial evaluation would occur at ESR, with the aim of producing a workflow that could be usable at WSCL.