Enhanced Virus Detection and Metagenomic Sequencing in Patients with Meningitis and Encephalitis

Figures

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  FIG 1. Overview of methods for subject selection and mNGS. Enrollment (A), laboratory methods (B), and analysis methods (C) are shown. Enhanced laboratory methods for methylated DNA depletion and hybrid capture (dashed lines) were included for a subset of the samples as shown. Abbreviations: CSF, cerebrospinal fluid; WBC, white blood cell.

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  FIG 2. Number of infectious disease tests ordered and lengths of stay among subjects. (A) Distributions showing the number of infectious disease (ID)-related tests ordered per subject, stratified by clinical diagnosis category. ID tests were counted if ordered between hospital admission day 1 and hospital discharge. Box plots with horizontal bars represent medians and interquartile ranges for ID tests. Diamonds represent data points greater than 1.5× the IQR. (B) Scatterplot showing the number of ID tests versus length of stay per subject. Colors indicate clinical diagnosis categories. The LOS correlated with the number of total ID tests ordered (Spearman’s ρ = 0.65; P < 0.01). The final clinical diagnosis for viral pathogens is stated for cases whose number of ID tests or LOS was an outlier above the 3rd quartile.

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  FIG 3. Viral taxa identified in cerebrospinal fluid using mNGS with or without enhanced methods. A heat map shows viral taxa identified in each sample. Rows are viral taxa, and columns are samples, some with enhanced sequencing methods (HC and/or MDD). Only classifications with over 100 unique kmers, with at least 1 BLAST-confirmed read, and manually reviewed as noncontaminants are shown. Rows are grouped by RNA viruses (top section) or DNA viruses (bottom section). The color intensity corresponds to the RPM of the taxa. Red boxes correspond to detection in RNA libraries, while blue boxes correspond to detection in DNA libraries. Some DNA viruses were detected in RNA libraries (e.g., adenovirus in subject M121). Gray-shaded columns represent samples that did not undergo DNA or RNA sequencing. Samples in which a contaminant was found are included here as blank columns, and the contaminants are shown in Fig. S4 in the supplemental material. X’s represent the clinical diagnosis. Yellow bars indicate the CSF nucleated cell count for each subject. The four groupings of columns from the top left to the bottom right correspond to infections diagnosed by positive PCR, infections diagnosed by…

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  FIG 4. Enhanced methods for mNGS. (A) Comparison of specific viral abundances among the non-computationally depleted reports for HC, MDD, and HC plus MDD for RNA samples (orange) and DNA samples (blue). (B to D) Hybrid capture improved the overall coverage for DNA and RNA viruses such as EBV (B), enterovirus (C), and JC polyomavirus 2 (D). Methylated DNA depletion improved the coverage for some DNA viruses such as JC polyomavirus 2 (D) but not others such as EBV, which utilizes host methylation in its life cycle (B).