External transportability of bacterial- and viral-associated host-response modules across public transcriptomic cohorts

Objectives To determine whether bacterial- and viral-associated whole-blood host-response modules discovered in GSE211567 retain their prespecified directions when applied unchanged to two external cohorts, including a second cross-platform cohort, and to quantify their robustness to analytical choices and gene deletion. Methods Five biologically guided modules were defined and fixed in GSE211567 before external analysis. The same modules were applied unchanged to GSE73461 and GSE72810 using an unweighted mean gene-wise z-score rule. Primary contrasts included 52 bacterial and 94 viral samples in GSE73461 and 23 definite bacterial and 28 definite viral samples in GSE72810. Wilcoxon tests were adjusted across the five modules using the Benjamini-Hochberg method. Hodges-Lehmann shifts, rank-biserial effects and 10,000-replicate bootstrap confidence intervals were calculated. Sensitivity analyses assessed z-score reference populations, probable-case inclusion, probe summarisation, GSVA scoring and exhaustive leave-one/two-gene deletion. Results All five modules retained their expected directions in both projection cohorts. BACT_M2, VIR_M1a, VIR_M1b and VIR_M2 had confidence intervals excluding zero and passed false-discovery-rate correction in both cohorts, whereas BACT_M1 remained directionally concordant but borderline in GSE73461 and GSE72810 (adjusted P = 0.0799 in each). All 30 z-reference, case-definition and probe-collapse sensitivity estimates retained the expected direction. Under GSVA, BACT_M1 gained statistical support, whereas VIR_M2 retained its viral-higher direction but lost confidence-interval and adjusted-P-value support. Across 29,826 leave-one/two-gene variants, every variant retained the expected direction and the minimum Pearson correlation with its complete-module score was 0.9940. Conclusions The predefined host-response modules retained their expected directions across two external cohorts measured on different Illumina platforms, with the strongest reproducible support for BACT_M2 and the three viral-associated modules. BACT_M1 and the GSVA behaviour of VIR_M2 demonstrate that direction preservation does not imply uniform statistical support across cohorts or scoring algorithms.