Diagnosing Neonatal Sepsis: A Comprehensive Review of Conventional Culture, Molecular Methods, and Host-Response Biomarkers

Neonatal sepsis remains a leading cause of morbidity and mortality in the first month of life, yet its presentation is nonspecific, and its microbiological confirmation is uniquely difficult. Low-circulating bacterial loads, small obtainable blood volumes, frequent intrapartum antibiotic exposure, and the ambiguity of skin-commensal isolates erode the sensitivity and specificity of blood culture, the long-standing reference standard, so that most evaluated infants are treated empirically despite sterile cultures. This review synthesizes current evidence on the integrated microbiological evaluation of the newborn: the host- and specimen-related diagnostic challenge; the etiologic spectrum and its antimicrobial-resistance burden; conventional culture; rapid and molecular methods, including mass spectrometry and syndromic and broad-range PCR; metagenomic and cell-free-DNA sequencing; host-response biomarkers; and antimicrobial-susceptibility determination. The evidence indicates that newer methods accelerate or supplement culture but do not yet replace it, each addressing one limitation while introducing caveats of its own. Notably, much of the evidence for direct-from-blood molecular testing, microbial cell-free DNA, and metagenomic sequencing is extrapolated from adults, whereas neonatal data are stronger for several biomarkers. The greatest benefit arises from integrating complementary modalities within diagnostic- and antimicrobial-stewardship frameworks. Neonatal-specific validation, standardized thresholds, and outcome-driven trials remain the principal priorities.