Abstract Background Central nervous system (CNS) infections are diagnostically challenging due to their nonspecific clinical presentations and wide array of potential pathogens. The rising population of immunocompromised patients further complicates this landscape, increasing the prevalence of atypical and opportunistic infections that are often missed by conventional testing. Objective This article provides guidance on the use and clinical interpretation of cerebrospinal fluid (CSF) metagenomic next-generation sequencing (mNGS) in suspected CNS infections. Discussion We highlight the paradigm shift from targeted molecular testing to agnostic mNGS, emphasizing key factors that impact diagnostic utility, including specimen handling, neuroanatomical factors, host inflammatory response, and pathogen kinetics. Using illustrative cases, we demonstrate how these biological and technical variables influence test sensitivity and result adjudication. We further discuss the impact of mNGS on clinical decision-making and current limitations regarding cost and turnaround time. Conclusions Cerebrospinal fluid mNGS is a transformative diagnostic tool, particularly for unusual presentations and in immunocompromised hosts. However, it does not replace clinical judgment and requires careful multidisciplinary interpretation. When integrated thoughtfully with clinical and laboratory data, mNGS can meaningfully reduce the diagnostic gap in CNS infections.
Waldrop et al. (Mon,) studied this question.