Sequencing Workflows Evaluated for Pathogen Detection in Central Nervous System Infections

Central nervous system infections represent serious diagnostic challenges in clinical medicine due to the wide diversity of potential infectious etiologies and their frequently ambiguous clinical manifestations. These overlapping symptom profiles routinely hinder rapid, definitive identification of the causative microorganism. To address these diagnostic hurdles, the study examined a multifaceted strategy integrating high-throughput preliminary screening via TaqMan Array Cards alongside two advanced sequencing approaches: Illumina-based unbiased metagenomic next-generation sequencing and Oxford Nanopore Technologies-based targeted next-generation sequencing.

The initial phase of the study employed TaqMan Array Cards utilizing quantitative polymerase chain reaction technology to conduct high-throughput preliminary pathogen screening across all 316 clinical cerebrospinal fluid specimens. Through this molecular screening approach, the overall pathogen-positive detection rate reached 24.68 percent across the entire patient cohort. Furthermore, polymicrobial infections, in which multiple distinct pathogens were identified within a single cerebrospinal fluid sample, accounted for 4.11 percent of all specimens tested. Together with subsequent sequencing, the diagnostic workflows identified a broad spectrum of central nervous system pathogens, capturing representative viral, fungal, and bacterial etiological agents responsible for central nervous system infections.

To directly compare the diagnostic accuracy and analytical capabilities of modern sequencing technologies, the investigators selected a representative cohort of 48 cerebrospinal fluid specimens from the larger study population. This targeted evaluation set comprised 25 specimens that had tested positive during preliminary quantitative polymerase chain reaction screening and 23 specimens that had tested negative. The researchers then evaluated these 48 representative samples across two relatively independent next-generation sequencing workflows: an unbiased metagenomic next-generation sequencing pipeline operated on an Illumina platform and a targeted next-generation sequencing pipeline executed on an Oxford Nanopore Technologies platform.

Comparative analysis of pathogen detection among the 25 quantitative polymerase chain reaction-positive cerebrospinal fluid specimens demonstrated comparable overall diagnostic sensitivity between the two sequencing platforms. Unbiased metagenomic next-generation sequencing achieved a pathogen detection sensitivity of 68.57 percent, while targeted next-generation sequencing achieved a sensitivity of 65.71 percent. These findings established that both sequencing workflows exhibited similar baseline sensitivity for identifying central nervous system pathogens in confirmed positive clinical specimens, confirming that both unbiased and targeted amplification approaches can successfully detect targeted pathogens in patient cerebrospinal fluid.

While overall sensitivity remained comparable between the two sequencing methods, the platforms exhibited marked differences in analytical specificity across the evaluated patient samples. Unbiased metagenomic next-generation sequencing achieved a specificity of 82.35 percent, distinctly outperforming the targeted next-generation sequencing workflow, which recorded a lower specificity of 72.73 percent. This reduced specificity in targeted sequencing was attributed to a heightened risk of false-positive detections. Specifically, the targeted amplification process generated elevated detection signals from environmental contaminant bacteria, thereby complicating the interpretation of true positive pathogen signals in clinical specimens.

As a notable example, unbiased metagenomic sequencing successfully detected Human parvovirus B19 in several cerebrospinal fluid samples where the virus was not covered by the initial array card panel, with all metagenomic detections subsequently confirmed through targeted quantitative polymerase chain reaction testing.

Conversely, targeted next-generation sequencing demonstrated substantial technical advantages in target enrichment efficiency, host background depletion, and overall testing turnaround time. Benefiting from targeted amplification, the Oxford Nanopore targeted sequencing workflow generated approximately 692-fold higher pathogen reads per million than the unbiased Illumina metagenomic workflow. Additionally, targeted sequencing dramatically suppressed human genomic background, yielding only 9.2 percent host reads compared to the 89.0 percent host reads present in the unbiased metagenomic datasets. Oxford Nanopore targeted sequencing also substantially reduced the sample-to-answer turnaround time, delivering results in 10 hours compared to the 30 hours required for unbiased metagenomic sequencing.

In their concluding assessment, the authors emphasized that each evaluated diagnostic workflow offers distinct operational strengths and specific limitations for managing central nervous system infections. The TaqMan Array Card strategy facilitates efficient, high-throughput preliminary screening across large patient cohorts, while unbiased metagenomic next-generation sequencing provides essential diagnostic reach for identifying unexpected or rare pathogens outside established panels. Meanwhile, targeted next-generation sequencing delivers robust target enrichment and dramatically accelerated turnaround times, leading the investigators to conclude that the optimal sequencing approach should be selected on a case-by-case basis according to specific clinical urgency and suspected pathogens.

Subject of Research: Diagnostic performance of sequencing and array card workflows for central nervous system infections

Article Title: High-throughput screening for central nervous system infection-associated pathogens using TaqMan array cards, metagenomic next-generation sequencing, and targeted next-generation sequencing: a retrospective study

Article References: Jia, X., Hu, L., Wu, M., Chen, J., Tian, D., Yi, Z., Ling, Y., Shen, Y., & Zhang, X. (2026). High-throughput screening for central nervous system infection-associated pathogens using TaqMan array cards, metagenomic next-generation sequencing, and targeted next-generation sequencing: a retrospective study. Frontiers in Microbiology, 17, Article 2001491. https://doi.org/10.3389/fmicb.2026.2001491

Image Credits: AI Generated

DOI: 10.3389/fmicb.2026.2001491

Keywords: Central nervous system infections, Cerebrospinal fluid, TaqMan Array Cards, Metagenomic next-generation sequencing, Targeted next-generation sequencing, Pathogen detection, Oxford Nanopore Technologies, Illumina sequencing, Diagnostic sensitivity, Diagnostic specificity, Host read depletion, Human parvovirus B19, Polymicrobial infections, Turnaround time, Environmental bacterial contamination, Reads per million, Etiological diagnosis, Quantitative PCR