An institutional investigation of false-positive signals in an automated blood culture system: evaluating clinical predictors and molecular etiologies.

UNLABELLED: In bloodstream infections, initiating appropriate antimicrobial therapy promptly is crucial to reduce morbidity and mortality. Automated blood culture monitoring systems efficiently screen out negative specimens, allowing clinical laboratories to prioritize true-positive cultures. However, unconfirmed positive signals (false positives) present diagnostic challenges. This study aimed to perform an institutional evaluation of the false-positivity rate within the BD BACTEC FX system, assess pre-analytic and clinical variables, and investigate the potential presence of bacterial and fungal nucleic acids using MALDI-TOF MS, polymerase chain reaction, and next-generation sequencing (NGS). Over a 15-month period, 80 false-positive automated blood culture (ABC) bottles (positive growth signal but no growth on routine subculture) and 30 negative-signal controls were evaluated. Direct identification was attempted using MALDI-TOF MS (BD MALDI Biotyper Sirius, Bruker Daltonics). Bacterial and fungal DNA presence was investigated via amplification of bacterial 16S rRNA, fungal 28S rRNA, and ITS gene regions, followed by NGS targeting amplicons. Clinical parameters, time-to-positivity (TTP), and blood gas parameters were recorded. The institutional false-positivity rate was 0.2% (80/41,073 bottles), fully aligning with manufacturer specifications and published benchmarks. Bacterial DNA (16S rRNA) was not detected in any false-positive or negative control bottle. Fungal DNA (28S rRNA D1/D2 region) was amplified in 9 of 80 false-positive samples (11.25%); however, ITS region amplification was negative across all samples. Sequence analysis identified diverse environmental mold taxa (Aspergillus melleus, Alternaria infectoria, Amorphotheca resinae, Alternaria conjuncta, Eremomyces bilateralis, Pseudomicrostroma glucosiphilum, and Aspergillus steynii) in low relative abundance. No statistically significant differences were observed between fungal DNA-positive and negative groups regarding age, WBC count, or CRP levels (P > 0.05). All direct MALDI-TOF MS scores remained below 1.70, consistent with the absence of intact microbial protein biomass. The median TTP for the fungal DNA-positive false-positive bottles was 68.0 h, with several bottles taking up to 109 h to emit a signal. The BD BACTEC FX system demonstrated high operational specificity, with an institutional false-positivity rate of 0.2%. Comprehensive molecular analysis confirmed that false-positive signals did not conceal missed true-positive clinical pathogens. The sporadic identification of disparate fungal DNA in a minor subset of vials represents clinically insignificant environmental background contamination. Extending incubation periods of routine blood culture bottles is not recommended, as standard bottles are not formulated for mold recovery. In cases where mold infection is clinically suspected, direct subculturing onto dedicated fungal media or ordering dedicated fungal diagnostic testing is advised. IMPORTANCE: False-positive blood culture signals present a significant clinical and economic challenge, often leading to unnecessary antimicrobial treatment, extended hospital stays, and increased laboratory workloads. In this study, we investigated the underlying molecular etiology of these unexplained false-positive signals specifically occurring within the widely used BD FX automated blood culture system (BD Diagnostic Systems, USA). We believe this article is highly relevant to the readership of Microbiology Spectrum because it provides concrete molecular evidence of hidden fungal DNA contamination within commercial blood culture components. Since the BD BACTEC system is a global standard in clinical diagnostics, our findings offer highly actionable insights for clinical microbiologists worldwide to distinguish true clinical anomalies from technical artifacts, thereby reducing laboratory-driven diagnostic errors and improving antimicrobial stewardship.