Clinical Metagenomic Next-Generation Sequencing for Diagnosis of Central Nervous System Infections: Advances and Challenges

Abstract

Central nervous system (CNS) infections carry a substantial burden of morbidity and mortality worldwide, and accurate and timely diagnosis is required to optimize management. Metagenomic next-generation sequencing (mNGS) has proven to be a valuable tool in detecting pathogens in patients with suspected CNS infection. By sequencing microbial nucleic acids present in a patient's cerebrospinal fluid, brain tissue, or samples collected outside of the CNS, such as plasma, mNGS can detect a wide range of pathogens, including rare, unexpected, and/or fastidious organisms. Furthermore, its target-agnostic approach allows for the identification of both known and novel pathogens. This is particularly useful in cases where conventional diagnostic methods fail to provide an answer. In addition, mNGS can detect multiple microorganisms simultaneously, which is crucial in cases of mixed infections without a clear predominant pathogen. Overall, clinical mNGS testing can help expedite the diagnostic process for CNS infections, guide appropriate management decisions, and ultimately improve clinical outcomes. However, there are key challenges surrounding its use that need to be considered to fully leverage its clinical impact. For example, only a few specialized laboratories offer clinical mNGS due to the complexity of both the laboratory methods and analysis pipelines. Clinicians interpreting mNGS results must be aware of both false negatives-as mNGS is a direct detection modality and requires a sufficient amount of microbial nucleic acid to be present in the sample tested-and false positives-as mNGS detects environmental microbes and their nucleic acids, despite best practices to minimize contamination. Additionally, current costs and turnaround times limit broader implementation of clinical mNGS. Finally, there is uncertainty regarding the best practices for clinical utilization of mNGS, and further work is needed to define the optimal patient population(s), syndrome(s), and time of testing to implement clinical mNGS.

Figure 1.. Overview of the metagenomic Next-Generation Sequencing (mNGS) workflow.

This figure illustrates the step-by-step utilization of mNGS for central nervous system diagnosis, from the bedside to the interpretation of final results. It underscores the importance of collaborative effort between clinicians and laboratory specialists in achieving a consensus on diagnostic decisions. Abbreviations: NAAT = nucleic acid amplification test; CSF = cerebrospinal fluid; NA = nucleic acid. Figure created using BioRender.

Figure 2.. Current and potential future approaches to pathogen detection.

Whereas mNGS is currently employed to identify rare and unexpected pathogens, it also offers the opportunity to detect pathogens that are currently identified through nucleic acid amplification tests (NAAT), culture, and serology, with the following considerations: To be considered an alternative to NAAT, mNGS needs to be more rapid and cost-effective. To be considered an alternative to culture, mNGS needs to be more able to clearly distinguish true-positives from background/contamination. To be considered an alternative to serology, further investigation is needed to determine whether nucleic acid is truly present in a pathogen-specific, sample-specific, and likely patient-specific manner. Figure created using BioRender.