Blood Culture Collection Strategies for Suspected Bloodstream Infection

Bloodstream infections are associated with substantial morbidity and mortality, particularly in patients with sepsis and septic shock. Timely identification of the causative microorganism enables targeted antimicrobial treatment and supports antimicrobial stewardship. Blood cultures remain the microbiological reference standard, but their performance is influenced by pre-analytical factors, particularly the total blood volume collected, the number of culture bottles, timing of collection, and contamination during venipuncture.

Conventional practice commonly involves collection of blood culture sets from two separate venipuncture sites. However, available evidence suggests that total blood volume may contribute more to pathogen detection than the number of venipunctures. Collecting multiple bottles during one venipuncture may simplify the procedure and improve compliance with the intended blood volume, whereas each additional venipuncture creates another opportunity for contamination. Prospective within-participant evidence from routine clinical settings remains limited.

This is a single-center, prospective, within-participant diagnostic study conducted at Izola General Hospital. Consecutive adults for whom the treating physician orders blood cultures as part of routine care will be assessed for eligibility. The clinical indication for blood culture collection and all treatment decisions will remain under the responsibility of the treating team.

During the initial venipuncture, two complete blood culture sets will be collected, comprising two aerobic and two anaerobic bottles with a target total blood volume of approximately 40 mL. During the same diagnostic episode, generally within 30-60 minutes and according to routine clinical workflow, an additional blood culture set comprising one aerobic and one anaerobic bottle with approximately 20 mL of blood will be collected from a different venipuncture site. The complete study collection will therefore consist of six bottles and approximately 60 mL of blood.

The initial four bottles will define the single-venipuncture strategy. All six bottles will define the sequential two-venipuncture strategy. Because both diagnostic strategies are reconstructed from specimens obtained from the same participant, participant-level clinical characteristics are held constant and each participant serves as their own control. The comparison evaluates the incremental contribution of the additional set and cannot separate the effect of the second venipuncture from the effect of the additional 20 mL of blood.

All bottles will be transported, incubated for five days, and processed according to routine microbiology laboratory procedures. Positive cultures will undergo microorganism identification and antimicrobial susceptibility testing when clinically indicated. Isolates will be classified as clinically significant pathogens or contaminants using predefined criteria based on organism identity, the pattern of positive bottles and sets, and available clinical information. The primary analysis will compare participant-level pathogen detection between the initial four-bottle strategy and the complete six-bottle strategy using paired methods.

The study is intended to quantify the diagnostic performance of a simplified collection strategy and the incremental value of the additional blood culture set. The results may inform local blood culture collection policy and future diagnostic stewardship recommendations.