{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T09:03:52Z","timestamp":1767690232598,"version":"3.48.0"},"reference-count":31,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T00:00:00Z","timestamp":1767657600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"New Quality Clinical Specialty Program of High-end Medical Disciplinary Construction in Shanghai Pudong New Area","award":["2024-PWXZ-04"],"award-info":[{"award-number":["2024-PWXZ-04"]}]},{"name":"Shanghai Municipal Health Commission, Key Discipline of Shanghai Health System, Cardiology","award":["2024ZDXK0019"],"award-info":[{"award-number":["2024ZDXK0019"]}]},{"name":"Scientific Research Program of Shanghai Pudong New Area Health Commission","award":["PW2025D-01"],"award-info":[{"award-number":["PW2025D-01"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Plasma cell-free RNA (cfRNA) metagenomics is increasingly explored for blood-based pathogen detection, but the structure of the shared background \u201cblood microbiome\u201d, the reproducibility of reported signals, and the practical limits of this approach remain unclear. We performed a critical re-analysis and benchmarking (\u201cstress test\u201d) of host-filtered blood RNA sequencing data from two cohorts: a bacteriologically confirmed tuberculosis (TB) cohort (n = 51) previously used only to derive host cfRNA signatures, and a coronary artery disease (CAD) cohort (n = 16) previously reported to show a CAD-shifted \u201cblood microbiome\u201d enriched for periodontal taxa. Both datasets were processed with a unified pipeline combining stringent human read removal and taxonomic profiling using the latest versions of specialized tools Kraken2 and MetaPhlAn4. Across both cohorts, only a minority of non-host reads were classifiable; under strict host filtering, classified non-host reads comprised 7.3% (5.0\u201312.0%) in CAD and 21.8% (5.4\u201331.5%) in TB, still representing only a small fraction of total cfRNA. Classified non-host communities were dominated by recurrent, low-abundance taxa from skin, oral, and environmental lineages, forming a largely shared, low-complexity background in both TB and CAD. Background-derived bacterial signatures showed only modest separation between disease and control groups, with wide intra-group variability. Mycobacterium tuberculosis-assigned reads were detectable in many TB-positive samples but accounted for \u22640.001% of total cfRNA and occurred at similar orders of magnitude in a subset of TB-negative samples, precluding robust discrimination. Phylogeny-aware visualization confirmed that visually \u201cenriched\u201d taxa in TB-positive plasma arose mainly from background-associated clades rather than a distinct pathogen-specific cluster. Collectively, these findings provide a quantitative benchmark of the background-dominated regime and practical limits of plasma cfRNA metagenomics for pathogen detection, highlighting that practical performance is constrained more by a shared, low-complexity background and sparse pathogen-derived fragments than by large disease-specific shifts, underscoring the need for transparent host filtering, explicit background modeling, and integration with targeted or orthogonal assays.<\/jats:p>","DOI":"10.3390\/pathogens15010055","type":"journal-article","created":{"date-parts":[[2026,1,6]],"date-time":"2026-01-06T08:42:58Z","timestamp":1767688978000},"page":"55","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Host-Filtered Blood Nucleic Acids for Pathogen Detection: Shared Background, Sparse Signal, and Methodological Limits"],"prefix":"10.3390","volume":"15","author":[{"given":"Zhaoxia","family":"Wang","sequence":"first","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guangchan","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mei","family":"Yang","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Saihua","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiahui","family":"Fang","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ce","family":"Shi","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yuying","family":"Gu","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zhongping","family":"Ning","sequence":"additional","affiliation":[{"name":"Department of Cardiology, Shanghai Pudong New Area Zhoupu Hospital (Shanghai Health Medical College Affiliated Zhoupu Hospital), Shanghai 201318, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1038\/s41576-019-0113-7","article-title":"Clinical metagenomics","volume":"20","author":"Chiu","year":"2019","journal-title":"Nat. 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