Specimen-specific differences in clinical metagenomic sequencing reporting patterns in hospitalized patients: a single-center retrospective observational study

Clinical metagenomic next-generation sequencing (mNGS) is increasingly used in hospitalized patients, but finalized reporting patterns vary across specimen types in routine practice. We conducted a single-center retrospective observational study using routine clinical mNGS data from January 1, 2024, to December 31, 2025. A specimen-specific first-order design retained only the first eligible mNGS order per patient within each specimen category during the study window. Orders were grouped as bronchoalveolar lavage fluid (BALF), blood, cerebrospinal fluid (CSF), and tissue for primary comparisons; heterogeneous "Other" specimens were described separately. The primary endpoint was report-interpreted any-positive at the order level. We summarized specimen-specific report-interpreted positivity, pathogen-group detection, the most frequently reported organisms ranked by order-level report presence, and mixed detections among positive orders. ICU-associated analyses were included as contextual descriptive stratification only. The cohort included DNA-only orders and a subset of PMseq-RNA-tested orders; RNA virus analyses were restricted to PMseq-RNA-tested orders, and DNA-only orders were treated as not tested for RNA virus fields. Among 1, 981 included specimen-specific first orders, BALF accounted for 973, blood 473, CSF 240, and tissue 122. Report-interpreted any-positive differed by specimen type, with BALF highest (876/973, 90.0%; 95% CI, 88.0-91.8%), followed by tissue (95/122, 77.9%; 95% CI, 69.7-84.3%), blood (343/473, 72.5%; 95% CI, 68.3-76.3%), and CSF (63/240, 26.2%; 95% CI, 21.1-32.2%). Among positive orders, at least 2 distinct standardized pathogens were reported in 672/876 BALF orders (76.7%), 182/343 blood orders (53.1%), 39/95 tissue orders (41.1%), and 8/63 CSF orders (12.7%). Across the four primary specimen groups, the most frequently reported organisms included Epstein-Barr virus (n = 485), Candida albicans (n = 285), and cytomegalovirus (n = 262), together with Klebsiella pneumoniae and Acinetobacter baumannii; these rankings reflect report-level frequency rather than adjudicated pathogenic roles, particularly for latency- or reactivation-prone viruses. Of included orders, 277 (14.0%) underwent PMseq-RNA testing. These findings characterize specimen-specific differences in clinical mNGS reporting patterns and provide a specimen-context-aware reference for interpreting routine inpatient reports.