# Revisiting the time to positivity in blood cultures as a prediagnostic tool: an in vitro experiment and retrospective study

Melanie Bouillon, Damasie Malandain, Sylvie Dargere, Vincent Cattoir, Simon Le Hello, François Gravey

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## To cite this version:

Melanie Bouillon, Damasie Malandain, Sylvie Dargere, Vincent Cattoir, Simon Le Hello, et al. Revisiting the time to positivity in blood cultures as a prediagnostic tool: an in vitro experiment and retrospective study. Diagnostic Microbiology and Infectious Disease, 2026, 114 (3), pp.117224. 10.1016/j.Diagmicrobio.2025.117224. hal-05454698

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https://hal.science/hal-05454698v1

Submitted on 21 Jan 2026

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# Revisiting the time to positivity in blood cultures as a prediagnostic tool: an in vitro experiment and retrospective study

Mélanie Bouillon<sup>a</sup>, Damasie Malandain<sup>a</sup>, Sylvie Dargère<sup>b</sup>, Vincent Cattoir<sup>c</sup>, Simon Le Hello<sup>d</sup>,
François Gravey<sup>d, * ID</sup>

## ARTICLE INFO

### Keywords:

- Blood cultures

- Bacteremia

- Sepsis

- Clinical microbiology

- Time-to-positivity

- Preanalytical

- Diagnostic tool

## ABSTRACT

**Background:** Sepsis is an acute response to infection; rapid detection of pathogens is essential. In this work, the time to positivity (TTP) of blood culture (BC), a key factor for species diagnosis, was studied.

**Methods:** In the experimental phase, six bacterial reference strains were tested under varying conditions, and three years of clinical blood culture data were reviewed. A total of 4,924 first positive BC bottles from 3864 patients hospitalized from 2019 to 2021 were analysed.

**Results:** Experimental results revealed that reducing the inoculum by a factor of 10 increased the TTP by an average of 1.5 hours. A blood sample volume between 1 and 3 mL per vial significantly improved bacterial detection. Each hour of delay before incubation reduced the TTP by 21 minutes; however, the overall TTP was still prolonged. Bacterial growth and viability were preserved at room temperature for up to 24 h. TTP could be exploited as a first diagnostic tool, as it significantly varied between species. *Staphylococcus aureus* can be differentiated from coagulase-negative staphylococci when the TTP is <12 hours (Specificity (Sp) = 0.95, positive predictive value (PPV) = 62 %, negative predictive value (NPV) = 76 %), and Enterobacteriaceae can be differentiated from *Pseudomonas aeruginosa* under the same threshold (Sensibility (Se) = 0.70, Sp = 0.93, PPV = 99 %). A TTP >15 hours exclude clinically relevant pathogens such as *S. pneumoniae*, *S. agalactiae*, and *S. pyogenes*.

**Conclusion:** This work shows how preanalytical factors play a major role in the time required for diagnosis, and highlights the potential of the TTP as a prediagnostic tool to improve sepsis management.

## 1. Introduction

Sepsis represents a significant global public health challenge because of its high incidence, associated morbi-mortality, and costs [1–5].

A diagnosis of bacteremia is made by collecting blood cultures (BC). The volume of blood collected is the most critical parameter for determining the sensitivity of BC [6,7]. During bacteremia, the bacterial load is very low, with up to 54 % of patients having bacterial concentrations between 0.01 and 1 CFU/mL in blood [8]. For adults, the recommended blood volume for culture is between 40 and 60 mL [9]. However, 53 % of hospitalized patients have less than 20 mL of blood inoculated [10],

while every additional millilitre of blood cultured linearly increases the probability of a positive test by 3 % to 13 % [6,7,11]. This point is particularly important, as study found that only 18 % of vials were filled within the recommended 8–10 mL range, whereas 47 % contained less than 8 mL. This study also found that mean volume was significantly higher in bottles that became positive (9.09 ± 0.15 mL) than in those that remained negative (8.47 ± 0.07 mL) (p < 0.001) [11]. Moreover, previous studies have shown that false-negative blood cultures increase mortality risk and prolong hospital stays [12]. Early diagnosis combined with appropriate therapy is the cornerstone for patient outcomes; if the appropriate antibiotic is administered within an hour of hypotension,

<footnote><sup>a</sup> Department of Bacteriology, University Hospital of Caen, Normandie Univ, UNICAEN, 14000 Caen, France</footnote>

<footnote><sup>b</sup> Department of Infectious Diseases, University Hospital of Caen, Normandie Univ, UNICAEN, 14000 Caen, France</footnote>

<footnote><sup>c</sup> Department of Bacteriology, University Hospital of Rennes, 35000 Rennes, France</footnote>

<footnote><sup>d</sup> Department of Bacteriology, INSERM, Normandie Univ, DYNAMICURE UMR 1311, Université de Caen Normandie, Univ Rouen Normandie, University Hospital of Caen, 14000 Caen, France</footnote>

<footnote>* Corresponding author at: professional address: CHU Caen Normandie, 14000 Caen.</footnote>

<footnote>E-mail addresses: bouillon-m@chu-caen.fr (M. Bouillon), malandain-d@chu-caen.fr (D. Malandain), dargere-s@chu-caen.fr (S. Dargère), vincent.cattoir@chu-rennes.fr (V. Cattoir), lehello-s@chu-caen.fr (S.L. Hello), gravey-f@chu-caen.fr (F. Gravey).</footnote>
the survival rate is approximately 80 %, but this rate decreases by 8 % after 6 hours each subsequent hour [13].

Time to positivity (TTP) is typically defined as the time between the incubation of the BC bottles in the incubator and the detection of a positive signal and is referred to as “TTP sensu strictu” (TTPss). Consequently, the TTP mentioned in most articles does not account for certain preanalytical factors. Indeed, the "TTP sensu latu" (TTPsl) includes transport and registration times in addition to the TTPss. Shorter time to transport (16 vs. 23 hours) saved up to 6.6 hours in reporting positive blood cultures [14].

Described and routinely used since the 2000s, TTP analysis has proven to be effective in various situations.

If the TTP is at least 120 minutes faster from the catheter-drawn BC than from the peripheral BC, it indicates catheter-related infections, with sensitivity of 94 % and specificity of 91 % [15,16]. Data from the literature also suggest that coagulase-negative staphylococci (CoNS) with a TTP greater than 20 hours are associated with sampling contaminations with a specificity of 83 % [17–19]. Recent studies have shown that a TTP lower than nine hours can assist in the diagnosis of S. aureus endocarditis [20], and direct examination of Gram-positive cocci in clusters within the first 14 hours of culture can predict the presence of S. aureus with sensitivity of 65 % and specificity of 74 % [21]. A TTP greater than 21 hours excludes the possibility that a Gram-positive coccus in the chain is a β-haemolytic streptococcus or S. pneumoniae with 100 % sensitivity [21]. Additionally, a TTP lower than 12 hours with a Gram-negative bacillus (GNB) direct examination suggests an Enterobacterales species with sensitivity of 77 % and specificity of 72 % [21].

The aim of this work was to analyse the BC TTP under controlled conditions and to compare it with microbiological data, with the ultimate goal of assessing TTP as a predictive factor and diagnostic tool for identifying the bacterial species associated with sepsis.

## 2. Materials and methods

### 2.1. Experimental phase

Bacterial species were selected on the basis of several criteria: their prevalence in bacteremia, their clinical impact, and the availability of standardized reference strains. Six strains were studied: *Escherichia coli* (EC) ATCC 25922; *Pseudomonas aeruginosa* (PAER) ATCC 27853; *Enterococcus faecalis* (EFS) ATCC 29212; *Staphylococcus aureus* (SAU) ATCC 29213; *Staphylococcus epidermidis* (SEP) ATCC 9491; and *Streptococcus pneumoniae* (SPN) ATCC 49766.

A bacterial suspension equivalent to 0.5 McFarland (ca. $1.5 \times 10^8$ CFU/mL) was prepared, after which 10-fold dilutions were performed to reach 10 CFU/mL (Fig. 1). The defibrinated horse blood, used as an equivalent to human blood, was preheated to 37°C [22,23]. For each dilution, the inocula were verified on agar plates by colony counting. The experiments were conducted in technical and biological triplicates using three different equivalent 0.5 McFarland values. The vials used were BACT/ALERT® FA and FN PLUS incubated for 5 days. Positive vials were subcultured on agar, and bacterial identification was verified using MALDI-TOF® (Brucker, Germany). The choice of the studied inoculum was determined according to previous studies [24–27].

The effects of the inoculum were evaluated with concentrations of 100-1000 CFU/mL, 10-100 CFU/mL, 1-10 CFU/mL, and 0.1-1 CFU/mL in 10 mL of blood incubated one hour after inoculation (Fig. 1).

Effects of transport times from the clinical ward to the microbiology laboratory and/or time to incubate the BC bottles were evaluated for all strains with an inoculum of 0.1–1 CFU/mL incubated with 10 mL of blood. Bottles were placed in the automated blood culture system (BACT/ALERT® VIRTUO®, bioMérieux) after inoculation at 1, 2.5, 6, and 24 hours. Before incubation, the bottles were kept at room temperature.

The effects of volume were tested for all strains with an inoculum concentration of 0.1–1 CFU/mL and 100-1000 CFU/mL incubated after 2.5 hours with volumes of 1 mL, 3 mL, and 6 mL of blood.

A total of 456 blood culture bottles were inoculated.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** A bacterial suspension equivalent to 0.5 McFarland was 10-fold diluted to reach $10^4$, $10^3$, $10^2$ and 10 CFU/mL. Blood cultures then were inculated with several inocula into defibrinated horse blood preheated to 37°C.</figcaption></figure>
## 2.2. Retrospective clinical data analysis

The study covered three continuous years of microbiological data from January 1, 2019, to December 31, 2021, from a 1,432-bed French teaching hospital in Caen Normandie, France. Information about BC was collected and merged from the automated blood culture system (BACT/ ALERT® VIRTUO®, bioMérieux) and laboratory software (TD Next-Lab, Technidata medical software). From the 270,315 metadata details about the vials, including patient's names, age, sex, blood collection date, volume, transport time, bacterial identification, and susceptibility, data selection was performed, excluding BCs from external hospitals, non-monobacterial infections, bottles with missing data, and vials without corresponding patient information linking the two datasets (Fig. 2). From two separate data extractions one table was created using a common identifier corresponding to each bottle. When this data was missing the vial was removed from the dataset.

For clinical data analyses, the TTPss values used were based on the

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Flowchart of the 10 steps used to select the complete data. From 270 315 vials, 151 439 were included in this study.</figcaption></figure>

first positive blood culture per patient per species and per hospital stay.

To differentiate contaminants from true infections, following the French guidelines, CoNS were considered contaminants when they were isolated from one or two blood culture bottles and true infections when they were recovered from three or more bottles.

## 2.3. Statistical analysis

Statistical analyses and graphical representations were conducted using R software (version 4.4.1; June 14, 2024). Quantitative variables are expressed as the mean, median, and 95 % confidence interval (CI), all reported in hours. Comparisons between groups were performed using Student's t test for quantitative variables, implemented with the rstatix package in R. We used parametric tests because our data followed a normal distribution and met the assumptions required for this type of analysis.

A p value <0.05 was considered to indicate statistical significance.

# 3. Results

## 3.1. Experimental phase

### 3.1.1. Impact of inoculum

For *E. coli*, the differences in the average TTPss between the different dilutions were 1.4 hours in the aerobic bottles (AB) and 1.3 hours in the anaerobic bottles (ANB). *P. aeruginosa* showed an average difference of 1.9 hours in the AB. For *S. aureus*, the average differences were 1.8 hours in AB and 1.1 hours in ANB. In the case of *S. epidermidis*, the observed differences were 1.2 hours for AB and 2.8 hours for ANB. *E. faecalis* presented average TTPss differences of 1.7 hours in AB and 1.1 hours in ANB. Finally, for *S. pneumoniae*, only one TTPss difference could be determined because of negative results for the other dilutions (1–10 and 0.1–1 CFU/mL), with values of 1.1 hours for AB and 1 hour for ANB (Fig. 3).

Thus, reducing the inoculum by a factor of 10 extended the TTPss by an average of 1.5 h. The differences in growth times between various inocula for the following species were significant: *E. coli* (p = 0.03), *S. aureus* (p = 0.03), *S. epidermidis* (p = 0.03–<0.05), and *E. faecalis* (p = 0.03) (Fig. 3). The results were not significant for *P. aeruginosa* or *S. pneumoniae*, probably because of the small sample sizes.

### 3.1.2. Impact of the bottle atmosphere

Differences in the TTPss were observed on the basis of the bottle atmosphere (Fig. 3). These observations were significant for *S. aureus* and *S. epidermidis* but not for the other species. With respect to the equivalent inocula, *S. aureus* seemed to grow faster under anaerobic conditions than under aerobic conditions, with a mean difference of 1.7 h (Fig. 3).

### 3.1.3. Impact of transportation time

All the bottles left at room temperature and incubated at t = 6 h and t = 24 h were detected as positive, indicating that bacterial viability and growth were maintained at room temperature.

The analysis of the regression lines representing TTPss as a function of TTPsl presented in Fig. 4 revealed high regression coefficients, with R² values ranging from 0.71 to 0.95 for *E. coli*, *E. faecalis*, *P. aeruginosa*, and *S. aureus*, while *S. epidermidis* had an R² of 0.43.

After 2.5 hours, each hour of delay in the introduction of the bottles into the incubator reduced the TTP from approximately 10 to 25 minutes (Fig. 4, Fig. S1). Despite that, this extended the overall or TTPsl (Fig. 5).

Given the relationship between the TTP and the time to incubate the blood bottles, a correction factor for the TTP was proposed to calculate the adjusted TTP by considering preanalytical parameters. The proposed calculation is available in the following formula (Eq. 1), with 0.41 representing the mean slope of the curves observed across all the tested species. However, no significant differences in p-value confidence in-
<figure><img src="image_6.png" /><figcaption>Fig. 3. Blox plots representing the dispersion of time to positivity regarding the inoculum, as well as two atmospheres (red : aerobic and blue : anaerobic) for six reference strains.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>Fig. 4. Correlation between time to positivity *sensu latu* (which correspond to the time between blood collection and positive signal from the automated blood culture system) and time to positivity *sensu strictu* (which correspond to the time between the incubation of blood culture bottle into the machine and positive signal from the automated blood culture system). R² was calculated using R software.</figcaption></figure>

tervals were found between the corrected and uncorrected delay conditions even for longer delays (> 6 hours) for species discrimination.

$$ \text{Adjusted TTP}(h) = \text{TTP}^{\text{sensu strictu}}(h) - (\text{pre} - \text{analytical time}(h) \times 0.41) $$

### Eq. 1: Adjusted TTP formula

#### 3.1.4. Impact of the volume of blood

The positivity rates observed with an inoculum of 0.1–1 CFU/mL were 99 % with a volume of 10 mL, 93 % for 6 and 3 mL, and 52 % with 1 mL (Fig. 6). When the relationship between volume and positivity percentage was linear, between 1 and 3 mL, the positivity rate increased by

20.5 % per additional mL and by 1 % between 3 and 10 mL (Fig. 6).

Therefore, at an inoculum of 100–1000 CFU/mL, every vial exhibited growth for all species, including those inoculated with only 1 mL.

Concerning the delay attributable to varying sample volume, with an inoculum of 0.1–1 UFC/ml we observed no significant differences in time to detection were observed for the 1, 3, and 6 mL volumes compared with 10 mL for *E. coli*, *P. aeruginosa*, and *E. faecalis*. In contrast, a significant increase in time to positivity was noted for *S. aureus* and *S. epidermidis* when using 1, 3, and 6 mL compared with 10 mL. For *S. aureus*, the mean time to detection for the lower volumes was 16.18 h versus 12.15 h for the 10 mL volume (p = 0.003). For *S. epidermidis*, the mean times were 22.6 h for the lower volumes compared with 17.15 h for 10 mL (p = 0.003). This observation was also observed for an inoculum of 100–1000 UFC/ml.

#### 3.2. Clinical data analysis

##### 3.2.1. Epidemiology

The data of a total of 151,439 vials were analysed. The positivity rate was 8 %. The mean age of patients with at least one positive blood culture was 69.3 (69–69.7) years.

The epidemiology with respect to bacteria in the adult population was as follows (Table 1): 37 % staphylococci, 37 % *Enterobacterales*, 8 % streptococci, 5 % enterococci, 3 % nonfermenting bacilli, and 1.4 % *Bacteroides* spp. (Table 1). In the paediatric population, 64 % were staphylococci, 13 % were *Enterobacterales*, 7 % were streptococci, 2 % were nonfermenting bacilli, 1 % were *Bacillus* spp., and 1.1 % were enterococci (Table 1). Details about the epidemiology are available in Table S1.

The average filling volume of blood culture bottles was 6.7 mL, with a median of 6 mL, and only 27 % of bottles contain a volume equal to or greater than 10 mL. The total pre-analytical delay averaged 2.7 hours, with a median of 2.2 hours.

The comparison of sample volumes between positive and negative blood cultures revealed significant differences, as we observed in
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Illustration of the impact of time to transport upon the total time to positivity. Full orange rectangle represents time to transport and plain orange rectangle represents time to positivity "sensu strictu". This figure represents a decrease of time to positivity sensu strictu associated with an increase in the time to transport. Overall the time to positivity sensu latu increased with the time to transport.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Illustration of the impact of volume inoculated and the percentage of blood culture positivity. This figure shows a quasi-threshold effect at 3 ml.</figcaption></figure>

experimental data. A lower proportion of positive bottles with volumes of 1 mL (3.8 % of positive bottles vs. 4.2 % of negative bottles) and 3 mL (8.3 % of positive bottles vs. 8.8 % of negative bottles) was observed (p = 0.01).

### 3.2.2. TTP differences between species

Compared with the other staphylococci, *S. aureus* exhibited a significantly faster growth rate, with a mean growth delay (MGD) of 17.9 h vs. 23.1 h; *p* < 0.001 (Table 1).

Additional significant differences in TTPs were noted between several bacterial groups: between staphylococci and enterococci (MGD of 21.6 h vs. 17 h; *p* < 0.001), between staphylococci and streptococci (MGD of 21.6 h vs. 14.7 h; *p* < 0.001), and between enterococci and streptococci (MGD of 17 h vs. 14.7 h; *p* = 0.02) (Table 1).

The difference in the TTP between *Enterobacterales* and non-fermenting bacilli was statistically significant, with MGDs of 12.3 h vs. 22.2 h (*p* < 0.001) (Table 1).

In the case of Gram-negative bacilli, TTPs of less than 12 hours was strongly indicative of bacteremia caused by *Enterobacterales*, with a sensitivity (Se) of 0.70, an specificity (Sp) of 0.93, a positive predictive (PPV) value of 99 %, and an negative predictive value (NPV) of 23 % (Fig. 7C). Conversely, a TTP greater than 15 hours is evidence against *Enterobacterales* bacteremia, with a Se of 0.79, Sp of 0.86, PPV of 38 %, and NPV of 97 %.

For streptococci, a TTPs exceeding 15 hours effectively ruled out the presence of *S. agalactiae*, *S. pyogenes*, or *S. pneumoniae*, with Sp of 0.98, Se of 0.34, PPV of 68 %, and NPV of 99 % (Fig. 7B).

With respect to staphylococci, a TTP between 10 and 12 hours was useful for distinguishing *S. aureus* from other staphylococcal species (Fig. 7A), with Sp ranging from 0.92 to 0.95, PPV between 60 % and 62

%, and NPV between 74 % and 76 %; however, the Se remains low, between 0.17 and 0.30. A TTP of 18 hours was the best compromise between Se of 0.66 and Sp of 0.60, with a PPV of 40 % and NPV of 82 %. A TTP of 22 hours yields an excellent Se of 0.80 for identifying *S. aureus* at the cost of a reduced Sp of 0.40, with PPV of 39 % and NPV of 83 %. Thus, in conjunction with direct microscopic examination, TTPs offers valuable diagnostic insights.

### 3.2.3. Evaluation of contaminants with TTP

The median TTPs for BC-positive samples with CoNS classified as contaminants was 21.2 hours. Conversely, for those classified as true infections, the median duration was 15.5 hours. The difference in the TTPs between contaminants and true infections in CoNS was highly significant (*p* < 0.001). We also investigated cases with only two BC positive for CoNS. The results were as follows: the mean TTP for contamination (1 positive vial) was 25 hours, while the mean TTP for true infection (2 or more positive vials) was 18.2 hours, with a p-value < 0.001.

To differentiate contaminations from true infections, different MGD thresholds were evaluated. At a threshold of 16 hours, the Se concentration was 0.53, the Sp was 0.85, the PPV was 56 %, and the NPV was 84 %, indicating high specificity and making MGD thresholds useful for confidently identifying true infections, although the sensitivity was low. In contrast, a threshold of 25 hours maximized the Se (0.85) and NPV (88 %), allowing most true infections to be detected while minimizing missed cases; however, the Sp was 0.37, and the PPV was 32 %.

## 4. Discussion

Preanalytic parameters, TTP, and bacterial epidemiology are readily accessible and useful for improving the microbial diagnosis of sepsis. Studies published over the past decades have already addressed the question of TTP and microbial diagnosis of infection, such as the link between a short time to positivity and urinary tract-related *E. coli* bacteremia [21], or the integration of TTP into the diagnostic approach for *S. aureus* infective endocarditis [28,29].

The impact of the inoculum on the TTP is undeniable. By comparing the TTPs for each inoculum diluted by a factor of 10, we established a correlation between time and biomass expressed in CFU/mL and observed that a 10-fold dilution resulted in an average increase of 1.5 hours in TTPs. In the context of catheter-associated infection, our work suggests that the bacterial density in the bottle drawn from the catheter is at least 10 times greater than that observed in the sample taken from the peripheral vein.

Regarding the volume inoculated into the vials, we concluded that the effect is likely linked to a threshold phenomenon: the higher the patient's bacterial load, the less the sample volume matters; conversely, when the bacterial load is low, volume becomes increasingly important.
<table><caption>**Table 1**
Epidemiology of germs found in adults from January 1, 2019, to December 31, 2021 in a large university hospital in France.</caption><thead><tr><th>Bacteria</th><th>Counts</th><th>Mean (h)</th><th>Mean interval confidence 95% (h)</th><th>Percentage among positives (%)</th><th>Percentage within the family (%)</th><th>Mean TTP of aerobic bottle (%)</th><th>Mean TTP of anaerobic bottle (%)</th></tr></thead><tbody><tr><td><strong>Staphylococci</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Staphylococcus epidermidis</td><td>612</td><td>21</td><td>(20,6-22)</td><td>13,3</td><td>36,1</td><td>18.8</td><td>26.1</td></tr><tr><td>Staphylococcus aureus</td><td>528</td><td>17.9</td><td>(16,7-18,5)</td><td>11,5</td><td>31,2</td><td>14.7</td><td>23.1</td></tr><tr><td>Staphylococcus hominis</td><td>252</td><td>23.7</td><td>(22,3-26,1)</td><td>5,5</td><td>14,9</td><td>19.9</td><td>35.2</td></tr><tr><td>Staphylococcus capitis</td><td>137</td><td>30</td><td>(27,6-32,2)</td><td>3</td><td>8,1</td><td>24.3</td><td>39.6</td></tr><tr><td>Staphylococcus haemolyticus</td><td>93</td><td>18.7</td><td>(17,2-22,7)</td><td>2</td><td>5,5</td><td>15.5</td><td>33.5</td></tr><tr><td><strong>Enterococci</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Enterococcus faecalis</td><td>139</td><td>18.3</td><td>(15,9-21)</td><td>3</td><td>60</td><td>16.4</td><td>20.4</td></tr><tr><td>Enterococcus faecium</td><td>78</td><td>14.7</td><td>(13,6-15,7)</td><td>1,7</td><td>33,2</td><td>14.6</td><td>14.7</td></tr><tr><td><strong>Streptococci</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Streptococcus mitis</td><td>95</td><td>14.4</td><td>(12,8-16,2)</td><td>2,1</td><td>24,1</td><td>13.8</td><td>14.7</td></tr><tr><td>Streptococcus dysgalactiae</td><td>61</td><td>12.2</td><td>(9,8-14,6)</td><td>1,3</td><td>15,8</td><td>8.5</td><td>16.5</td></tr><tr><td>Streptococcus gallolyticus</td><td>55</td><td>11.8</td><td>(10,2-13,5)</td><td>1,2</td><td>14,3</td><td>13.4</td><td>10.8</td></tr><tr><td>Streptococcus pneumoniae</td><td>47</td><td>9.8</td><td>(9,2-10,7)</td><td>1</td><td>12,2</td><td>9.6</td><td>11.3</td></tr><tr><td>Streptococcus milleri</td><td>43</td><td>33</td><td>(27,5-38,5)</td><td>0,9</td><td>11,2</td><td>30.4</td><td>37.8</td></tr><tr><td>Streptococcus agalactiae</td><td>24</td><td>9.6</td><td>(8,6-10,5)</td><td>0,5</td><td>6,2</td><td>8.3</td><td>10.4</td></tr><tr><td>Streptococcus pyogenes</td><td>20</td><td>10.6</td><td>(7,8-13,3)</td><td>0,4</td><td>5,2</td><td>9.5</td><td>14.7</td></tr><tr><td><strong>Gram-negative bacilli</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Escherichia coli</td><td>941</td><td>11.3</td><td>(10,9-11,8)</td><td>20,4</td><td>58,2</td><td>12.2</td><td>10.9</td></tr><tr><td>Enterobacter cloacae</td><td>212</td><td>12.8</td><td>(11,6-14,4)</td><td>4,6</td><td>13,1</td><td>13.8</td><td>12.2</td></tr><tr><td>Klebsiella pneumoniae</td><td>145</td><td>12.4</td><td>(10,6-14,3)</td><td>3,1</td><td>8,9</td><td>13.3</td><td>11.8</td></tr><tr><td>Proteus mirabilis</td><td>53</td><td>15.9</td><td>(11,8-20)</td><td>1,2</td><td>3,3</td><td>15.9</td><td>15.8</td></tr><tr><td>Klebsiella oxytoca</td><td>48</td><td>11.9</td><td>(10,1-13,8)</td><td>1</td><td>3</td><td>11</td><td>12.6</td></tr><tr><td>Klebsiella aerogenes</td><td>39</td><td>13.2</td><td>(11,4-15,1)</td><td>0,8</td><td>2,4</td><td>15.7</td><td>11.9</td></tr><tr><td>Serratia marcescens</td><td>41</td><td>17.6</td><td>(12,1-23,5)</td><td>0,9</td><td>2,5</td><td>14.2</td><td>20.9</td></tr><tr><td>Citrobacter koseri</td><td>28</td><td>12.6</td><td>(10,3-14,9)</td><td>0,6</td><td>1,7</td><td>12.1</td><td>12.7</td></tr><tr><td>Klebsiella variicola</td><td>25</td><td>11.3</td><td>(10-12,9)</td><td>0,6</td><td>1,5</td><td>11.4</td><td>11.5</td></tr><tr><td>Morganella morganii</td><td>24</td><td>19.2</td><td>(9,4-29)</td><td>0,5</td><td>1,5</td><td>15</td><td>22.8</td></tr><tr><td><strong>Non-fermenting bacilli</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Pseudomonas aeruginosa</td><td>133</td><td>21</td><td>(18,8-23,3)</td><td>3</td><td>76.4</td><td>20.4</td><td>34.6</td></tr><tr><td><strong>Anaerobes</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Bacteroides fragilis</td><td>45</td><td>30</td><td>(28-32)</td><td>1</td><td>64,7*</td><td>/</td><td>30</td></tr></tbody><tfoot><tr><td><strong>TOTAL</strong></td><td>3918</td><td></td><td></td><td>85,1</td><td></td><td></td><td></td></tr></tfoot></table>

This trend differs from what has been reported in previous studies [6,7, 11]. A delay in sample processing is associated with reduced TTPss. These findings suggest that storing BC bottles at room temperature does not affect bacterial viability or growth. However, bacterial growth is slower at room temperature than at 35°C; TTPsl is extended when the incubation of blood collection is delayed. Rapid transport of samples remains essential to minimize the TTPsl, especially since a delay in diagnosis is associated with increased mortality in patients [30]. There was a strong correlation between transportation time and TTPss, with an estimated delay of diagnosis between 10.2 and 25.6 minutes for each hour of delay of bottle incubation.

The proposal of a corrected delay to correct the preanalytical delays did not provide more significant differences in distinguishing one

species from another. Nevertheless, this adjusted delay could confer other benefits: it could serve as a relevant indicator for monitoring bacteremia in the same patient independently of preanalytical delays.

When TTPss was used to detect CoNS contamination, a 20-hour threshold yielded sensitivity of 0.52 and specificity of 0.72, both of which were less than 0.8, which differs from previous reports (Sp reported with TTP > 20 h of 83 %) [15,16]. To improve the sensitivity (i. e., Se > 80 %) and better identify contamination, extending the threshold to 25 hours may be more suitable. To maximize specificity and reliably detect true infections, a threshold of 16 hours is suggested. It should be noted, however, that our definition of contamination may differ from those used in other studies, which could affect direct comparisons.
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Violin plots showing the time to positivity *sensu stricu* dispersion of the first positive blood culture per patient and per stay. Red and pink lines represent respectively the most effective TTP threshold (in hours) specificity and sensibility for distinguishing species. **Fig. A**, threshold used for *Staphylococcus aureus* and coagulase negative staphylococci, **Fig. B**, threshold used for *Streptococcus agalactiae*, *Streptococcus pyogenes*, *Streptococcus pneumoniae* and other streptococci. **Fig. C**, threshold used for *Enterobacterales* and non fermenting bacilli.</figcaption></figure>

We observed similar sensitivity but improved specificity in differentiating Enterobacterales from non-fermenting Gram-negative bacilli compared with previously published studies [21]. This higher specificity suggests that our approach may reduce misclassification, thereby improving the reliability of early microbiological differentiation in

clinical practice.

Second, we observed shorter TTPs values for clinically relevant streptococci, with a median of 15 hours, whereas the literature generally reports approximately 21 hours [21]. We believe that this difference is explained mainly by our study design. Unlike previous reports, which
included all blood culture findings during hospitalization, we restricted our analysis to the first positive blood culture per patient and per hospital stay. This choice was intended to minimize the potential confounding effect of prior antibiotic therapy. By focusing on initial episodes, we likely captured bacterial growth under conditions closer to the untreated state, which could explain the shorter TTP observed.

This study has several limitations. It is monocentric and relies on a single type of automated BC incubator, which may influence the results because of the difference in performance between the instruments [31]. Multicentric studies using different incubators are needed to validate and generalize these findings. Our study was limited by the lack of clinical data, which were not retrieved from patient medical records.

Moreover, the volume of blood introduced into the patient culture bottles was not controlled and may represent a major source of variability, potentially affecting the TTP.

It would be relevant to develop an artificial intelligence (AI) model based on the data extracted in this study to evaluate and optimize its usefulness in clinical practice for interpreting TTP. Our current approach was limited to examining one criterion at a time, whereas AI would allow the simultaneous integration of all the microbiological, demographic, and epidemiological criteria.

We hope that this approach will improve the precision and discriminatory power of the extracted data.

## 5. Conclusion

TTP appears to be an easily exploitable diagnostic tool, as it differs significantly between species. It can be used to differentiate certain clinically relevant organisms, such as *S. aureus*, from other coagulase-negative staphylococci and Enterobacteriaceae from *P. aeruginosa*. It is particularly useful for diagnosing severe infections caused by *S. pneumoniae*, *S. agalactiae*, and *S. pyogenes* and for distinguishing true infections from sample contamination.

This work highlights several avenues for improving practices, particularly by reducing delays in sample processing and adjusting the blood volumes collected while quantifying their impacts experimentally. This study also highlights the importance of TTP and its potential as a diagnostic tool for health care services to optimize the therapeutic management of patients and thereby reduce sepsis-related mortality.

## Glossary

**TTPss:** Time to positivity of blood cultures, measured from the moment of loading into the automated system until the detection of a positive signal.

**TTPsl:** Time to positivity measured from the moment of blood collection until the detection of a positive signal.

## Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Ethics approval and consent to participate

This study was performed in accordance with the Declaration of Helsinki and relevant guidelines and regulations. This study was approved by the Caen University Ethical Committee under decision number 2025091814053200000350003597.

## Informed consent

No consent form was required for this study, as no clinical patient data were used.

## CRediT authorship contribution statement

**Mélanie Bouillon:** Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Data curation, Conceptualization.
**Damasie Malandain:** Writing – review & editing, Methodology, Formal analysis.
**Sylvie Dargère:** Writing – review & editing, Methodology, Data curation.
**Vincent Cattoir:** Writing – review & editing.
**Simon Le Hello:** Writing – review & editing, Conceptualization.
**François Gravey:** Writing – review & editing, Writing – original draft, Methodology, Investigation, Data curation, Conceptualization.

## Declaration of competing interest

All the authors have no conflict of interest I have co conflict of interest and nothing to declare.

## Acknowledgements

Acknowledgment is made to the research teams and clinicians of the University Hospital of Caen for their assistance and commitment throughout this project.

## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.diagmicrobio.2025.117224.

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