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. 2023 Nov 29;18(11):e0288674. doi: 10.1371/journal.pone.0288674

Comparison of new and old BacT/ALERT aerobic bottles for detection of Candida species

Kye Won Choe 1, Yong Kwan Lim 1, Mi-Kyung Lee 1,*
Editor: Ali Amanati2
PMCID: PMC10686453  PMID: 38019833

Abstract

Purpose

A new version of aerobic blood culture media has been developed for the BacT/ALERT (bioMérieux) blood culture system. We evaluated the time to detection and yeast cell counts in positive blood cultures for each Candida spp. according to changes in media.

Methods

Isolates from defibrinated horse blood were inoculated into three types of bottles: the old version of aerobic bottle, new version of aerobic bottle, and anaerobic bottle. All bottles were incubated in the BacT/ALERT Virtuo blood culture system. The time to detection was monitored for each bottle, and yeast cell counts were performed immediately after testing positive, determined via the plate count method. Clinical retrospective data of the candidemia samples before and after aerobic bottle change also were analyzed.

Results

The median time to detection was 52.47 hours in the old aerobic bottles versus 19.92 hours in the new aerobic bottles (P < 0.001) for Candida glabrata, and standard and clinical strains showed similar results. C. albicans (27.6 to 24.95 hours) and C. guilliermondii (28.92 to 26.9 hours) had shorter time to detection. However, C. auris (25.43 to 28.25 hours) had a longer time to detection in the new aerobic bottle. The retrospective clinical analysis showed a significant decrease in time to detection (45.0 to 19.4 hours) for C. glabrata, which is consistent with our simulated study result for C. glabrata. As a result of analysis including all blood specimens, C. tropicalis showed a significant delay in time to detection in new aerobic bottles. In an analysis limited to peripheral blood specimens, the time to detection of C. parapsilosis was longer in new aerobic bottles than in old aerobic bottles.

Conclusion

Most Candida species did not show remarkable TTD differences, but TTD of C. glabrata was markedly reduced in the New FA Plus bottle. The reduction of time to detection enables faster detection and therapeutic approach for C. glabrata infections.

Introduction

Candidemia is an increasingly major healthcare-associated fungal infection associated with high morbidity and mortality [1, 2]. The epidemiology of candidemia varies according to the geographical region, period, and population; however, recent studies have reported a global shift towards other Candida spp. from Candida albicans, although C. albicans is still the predominant species [2, 3].

C. glabrata (also named as Nakaseomyces glabratus) is a common pathogen whose proportions are increasing among non-C. albicans infections and is causing bloodstream infections worldwide, including South Korea [4–6]. Candidemia caused by C. glabrata has clinical significance due to antifungal resistances, including intrinsic decreases in susceptibility to azole agents and resistances to amphotericin B [6]. It has been reported that time to detection (TTD) of C. glabrata was more affected by the blood culture system and medium than C. albicans [7, 8].

The new version of aerobic blood culture media has been developed for the BacT/ALERT (bioMérieux) blood culture system. Although the exact formulation of the bottle media is proprietary and not disclosed, it was reported that trace elements were added and several components unrelated to microbial recovery were removed. The primary purpose of the formulation changes was to increase the growth performance of several microorganisms particularly C. glabrata. The new BacT/ALERT aerobic media (New FA Plus) has been shown to have equivalent performance compared with the old version, except for the reduced TTD of some microorganisms.

There have been no studies on the effects that the change in aerobic blood culture bottle has on the TTD and growth performance of various Candida spp. Therefore, we aimed to analyze the changes in TTD and yeast cell count for several Candida spp. according to media changes through a simulated blood spiking study and clinical retrospective data analysis.

Materials and method

Study design

The study was performed at Chung-Ang University Medical Center laboratory in Seoul, South Korea. The study was conducted in two ways: one is an experiment to check TTD and yeast cell count by inoculating isolates into horse blood, and the other is a retrospective comparative analysis of TTD in blood culture data obtained from outpatient and hospitalized patients.

Preparing a simulated blood culture study

The Candida isolates evaluated in this study (n = 14) are listed in Table 1. These isolates included nine strains (American Type Culture Collection, ATCC; National Culture Collection for Pathogens of Korea, NCCP) and five clinical isolates from the blood of patients at Chung-Ang University Hospital who were diagnosed with candidemia. All the isolates were received and analyzed by the Chung-Ang University Medical Center laboratory. This study was approved by the IRB at the Chung-Ang University Hospital (IRB No.2307-018-19480). The IRB waived the need for informed consent and additional medical ethics review for this study, because the clinical isolates were properly de-identified and anonymized, according to institutional policy. Isolates were stored as 50% glycerol stocks at -70°C. Before testing, the isolates were subcultured on Sabouraud dextrose agar (SDA) plates at 35°C. After 24 to 48 hours of incubation, 0.5 McFarland standard suspensions, which contain 1–5×106 CFU/mL depending on the Candida spp. were prepared [9], and serial dilutions were performed. Approximately 125 CFU of each isolate, in 0.5% sterile saline, was inoculated in 8 mL of defibrinated horse blood and was incubated in three types of blood culture bottles: the old version of the aerobic culture bottle (Old FA Plus), New FA Plus, and the anaerobic culture bottle (FN Plus). Old FA Plus include 30 ml of complex medium and 1.6 g of adsorbent polymeric beads. New FA Plus has similar formulation except addition of essential elements and removal of trace elements. FN Plus is composed of 40 ml of complex medium and 1.6 g of adsorbent polymeric beads. The isolates were inoculated in triplicate. All the bottles were incubated in the BacT/ALERT Virtuo blood culture system until positivity was detected or for a maximum of five days.

Table 1. Candida spp. tested under the simulation study.

Candida species Isolate strain designationsa
C. albicans ATCC 14053
C. auris NCCP 32684
C. dubliniensis ATCC MYA-646
C. guilliermondii ATCC 6260
C. krusei ATCC 6258
C. lusitaniae ATCC 34449
C. parapsilosis ATCC 22019
C. tropicalis ATCC 750
C. glabrata ATCC 2950
CL1
CL2
CL3
CL4
CL5
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a ATCC, American Type Culture Collection; NCCP, National Culture Collection for Pathogens of Korea; CL, Clinical isolates.

Time to detection

TTD is defined as the time from the start of the incubation to when the blood culture vial tests positive. The TTD was monitored for each bottle, and yeast cell counts were performed immediately after testing positive via the plate count method using SDA as the culture medium. The blood culture medium was also subcultured on blood agar plates to exclude contamination and to confirm the true-positive or true-negative detection results.

Yeast cell count

The positive blood culture was mixed and serially diluted in saline to dilutions of 10−3, 10−4, and 10−5, the dilutions were vortexed, and 100 uL of each dilution was spread onto a separate SDA plate. After overnight incubation, the plates were examined for colonies. The number of CFU per milliliter was calculated based on the dilution factor [10].

Retrospective data analysis for clinical blood culture results

We performed blood culture positive data analysis on the Candida spp. to confirm whether the data verified through the procedures using horse blood can be applied to clinical candidemia cases between 1 September 2020 and 15 June 2022. We divided the data into two periods: before changing to the New FA Plus bottles (1 September 2020 to 31 May 2021) and after changing to the New FA Plus bottles (1 June 2021 to 15 June 2022). During the above periods, only C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis were detected; therefore, the four species were included in the final analysis. Blood culture bottles with polymicrobial growth were excluded from the analysis since the polymicrobial culture could have influenced the TTD.

Statistical analysis

Statistical analysis was performed to compare the TTD between the blood culture bottles using Mann-Whitney U test and Kruskal-Wallis test. Differences with p-values of greater than 0.05 were regarded as statistically significant. Datasets were entered into Microsoft Excel (Microsoft, Washington, USA) and analyzed using R version 4.1.0 (R Foundation for Statistical Computing, Vienna, Austria).

Results

Simulated study data

The hematocrit of the defibrinated horse blood was 41.4%. The TTD values were compared among the 14 isolates performed in triplicate for each bottle type (Table 2). The median TTD of C. glabrata was 52.47 hours using the Old FA Plus bottles versus 19.92 hours using the New FA Plus bottles (P < 0.001). The overall and clinical samples of C. glabrata showed similar results. C. albicans (27.6 versus 24.95 hours; P = 0.2) and C. guilliermondii (28.9 versus 26.9 hours; P = 0.1) also showed relatively shorter TTDs between the Old and New FA Plus bottles, however, the difference was not statistically significant. On the contrary, the TTD was longer for C. auris (25.43 versus 28.25 hours; P = 0.0765) in the New FA Plus bottle compared with the Old FA Plus bottle, although statistical significance of the difference could not be clarified.

Table 2. Time to detection and yeast cell count for Candida spp. in the simulated study.

Time to detection (hr)
Median (25th percentile, 75th percentile)		 P valuea Yeast cell count (CFU/mL)
Median (25th percentile, 75th percentile)		 P valuea
Strainb Old New FN Old New FN
C. albicans ATCC 14053 27.6 (27.5, 27.6) 24.95 (24.7, 25.1) 29.6* 0.2 2.05 (1.58, 2.53)×103 11.8 (9.75, 12.3)×104 500* 0.2
C. auris NCCP 32684 25.43 (25.36, 25.77) 28.25 (28.09, 28.25) 17.1* 0.08 3.2 (3.05, 3.20)×106 7.20 (7.20, 7.85)×106 1.40×104* 0.07
C. dubliniensis ATCC MYA-646 26.13 (25.80, 26.89) 25.30 (24.81, 25.80) 27.0(26.1, 26.99) 0.2 2.8 (1.8, 4.15)×106 2.2 (1.75, 3.60)×106 2.30 (2.00, 5.10)×106 0.2
C. guilliermondii ATCC 6260 28.92 (28.69, 29.00) 26.9(26.85, 27.50) - 0.1 1.9 (1.75, 3.0)×106 1.22 (1.12, 1.86)×106 - 0.4
C. krusei ATCC 6258 21.14 (19,65, 22.64) 17.68 (17.66, 18.0) - 0.4 6.09 (3.33, 8.84)×105 5.7 (4.95, 7.85)×105 - 1
C. lusitaniae ATCC 34449 18.6(17.85, 18.77) 16.78 (16.53, 16.85) 23.52 (23.22, 23.82) 0.1 2.6 (1.7, 3.07)×107 1.69 (1.69, 1.70)×107 5.20 (4.85, 5.55) ×105 0.7
C. parapsilosis ATCC 22019 25.75 (25.49, 27.34) 26.72 (26.22, 27.8) - 1 2.2 (2.0, 3.6)×105 1.60 (1.33, 1.68)×106 - 0.1
C. tropicalis ATCC 750 17.3(17.09, 17.32) 18.17 (17.74, 18.5) - 0.268 1.5 (1.25, 1.5)×105 4.0 (3.75, 6.25)×105 - 0.0765
C. glabrata Total 52.47 (48.42, 56.02) 19.92 (18.81, 20.48) 16.4(15.69, 16.77) 0.000000735 3 (1.7, 3.98)×105 6.5 (5.0, 10.0)×105 9.5 (6.73, 16.75)×105 0.000395
ATCC 2950 46.45 (44.55, 46.96) 17.93 (17.53, 17.94) 14.28 (13.41, 14.47) 0.1 2.00 (1.35, 2.50)×105 6.00 (6.00, 7.80)×105 9.00 (7.65, 13.5)×105 0.0765
CL1 52.65 (52.49, 53.07) 18.52 (17.81, 19.22) 16.50 (16.06, 16.67) 0.2 2.40 (1.40, 2.70)×105 3.90 (2.85, 4.95)×105 9.00 (7.50, 10.5) ×105 0.8
CL2 53.1(53.00, 54.15) 20.4(19.75, 20.55) 16.40 (16.40, 16.40) 0.1 3.90 (2.45, 4.70)×105 7.00 (6.00, 8.50)×105 8.00 (6.50, 9.00)×105 0.2
CL3 51.4(51.25, 52.00) 19.9(19.65, 20.35) 16.60 (16.00, 16.95) 0.1 4.00 (3.50, 4.00)×105 7.00 (5.00, 8.50)×105 8.00 (6.50, 14.5)×105 0.5
CL4 51.90 (49.71, 53.96) 20.64 (20.51, 20.77) 16.17 (16.03, 16.70) 0.1 3.40 (2.15, 4.60)×105 7.00 (5.50, 8.50)×105 2.10 (1.70, 2.20)×106 0.4
CL5 52.90 (50.21, 53.79) 20.18 (19.93, 20.63) 17.03 (16.62, 17.35) 0.1 2.00 (1.80, 7.00)×105 1.20 (0.85, 1.35)×106 1.30 (0.75, 2.00)×106 0.268
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a Comparison of Old FA Plus and New FA Plus bottles using Mann-Whitney U test.

b ATCC, American Type Culture Collection; NCCP, National Culture Collection for Pathogens of Korea; CL, Clinical isolates.

* Positive only in one bottle among triplicates.

The number of yeast in the bottles spanned a 3.78-log range, from 3.80×103 to 2.31×107 CFU/mL. The median yeast cell count for C. glabrata in the New FA bottle was approximately 2.2-fold higher than in the Old FA bottle (P < 0.001). Considering that the TTD of C. glabrata in the New FA Plus bottle was shorter than in the Old FA Plus bottle by 20 hours, the yeast cell count confirmed that the growth rate of the yeast was increased in the new bottle. In addition, although there was no significant difference, median yeast cell counts of New FA Plus was higher than Old FA Plus for C. auris, C. parapsilosis, and C. tropicalis.

Aerobic versus anaerobic bottle

Although most Candida strains had no growth in the FN Plus bottles, C. dubliniensis, C. lusitaniae, and C. glabrata grew in two or more bottles. There was no significant difference in the TTD of C. dubliniensis, but there was an increase in TTD of C. lusitaniae compared with the FA bottles (Old FA Plus 18.6 hours and New FA Plus 16.78 hours versus FN Plus 23.52 hours; P = 0.044). The yeast cell count showed no significant difference between the aerobic and anaerobic bottles for C. dubliniensis and C. lusitaniae.

The TTD of C. glabrata in the FN Plus bottle was shorter than that of the Old and New FA Plus bottles (Old FA Plus 52.47 hours and New FA Plus 19.92 hours versus FN Plus 16.4 hours; P < 0.001). The increased proliferation of C. glabrata in the anaerobic bottle was consistent with several previous studies [11–13]. The yeast cell count was significantly higher in the FN Plus bottle compared with the Old FA Plus (P<0.001), but yeast cell count difference was not statistically significant with New FA Plus (P = 0.1581).

Retrospective review for clinical blood culture results

In total, 99 isolates of candidemia were analyzed (Fig 1). S1 Table summarizes the number of specimens of candidemia included in two periods. The TTD of C. glabrata was significantly shorter in the New FA Plus bottles (45.0 versus 19.4 hours; P < 0.05), which was consistent with the results from our horse blood simulation study. The detection time difference between the old and new bottles of C. glabrata from peripheral blood (40.6 versus 23.9 hours; P < 0.05) was similar to the total blood sample data. For the other three species, overall, the mean TTD was increased in the new bottles. In particular, when analyzing all blood samples for C. tropicalis (Old FA Plus 9.58 versus New FA Plus 20.28 hours; P = 0.0361) and only peripheral blood samples for C. parapsilosis (Old FA Plus 21.33 versus New FA Plus 27.17 hours; P = 0.02), New FA Plus showed a statistically significant increase of TTD compared to Old FA Plus.

Fig 1. Time to detection (TTD) of Candida spp. for the clinical blood culture bottles with different types of aerobic bottles; the New FA Plus (New) and Old FA Plus (Old).

Fig 1

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Box plots indicate the medians and the 25th and 75th percentiles. P value indicates the comparison of New and Old bottles using Mann-Whitney U test. (A) All the clinical blood samples were obtained from central line access and peripheral blood. (B) Only peripheral blood samples were included.

Discussion

Fast and accurate detection of candidemia is crucial in order to choose and start the appropriate antifungal therapy. This study evaluated the TTD and yeast cell counts in positive blood bottles for each Candida spp. according to the changes in aerobic bottle media. This study shows that the new aerobic blood culture medium allows for faster detection of C. glabrata in the horse blood simulation study and the retrospective clinical sample detection study. The yeast cell counts confirmed that the faster growth of yeast resulted in faster detection of C. glabrata.

Altun et al. reported that the median TTD of two Candida species was 24.4 hours for C. albicans and 54 hours for C. glabrata in the Virtuo system using the Old FA Plus bottle with simulated horse blood culture [14]. However, Menchinelli et al. described a marked difference in the TTD; their median TTD of C. glabrata in simulated human blood samples was 32.1 and 29.0 hours using the Virtuo and BACTEC (BD Diagnostics) systems and corresponding culture bottles for each system, for the Virtuo system the Old FA Plus bottle was used [15]. The similarity of the results between this study and the Altun et al. study is possibly due to the use of horse blood, which contrasts with the use of human blood in the Menchinelli et al. study. Compared with this study’s clinical retrospective data, the median TTD of the Old FA Plus bottle was 45 hours. This result suggests that the human clinical blood culture samples had a shorter detection time in the Virtuo device compared with the simulated data.

The simulation study showed lower median values of TTD for C. albicans and C. guilliermondii in the New FA Plus than in the Old FA Plus bottle. However, in the retrospective data analysis of the clinical samples, the TTD of C. albicans was longer in the New FA Plus bottle, although the difference was not statistically significant. The non-significant difference was because the TTD difference in the simulated study was only slightly different (about 2 hours), and the initial CFU, antibiotic use, and inhibitors in the blood samples and the initial inoculated volume were different according to the clinical conditions of the patients.

Retrospective data analysis results showed that some Candida species had an extended TTD due to aerobic culture bottle changes, which can also be related to the simulated study data. In the simulated study, the median TTD values for C. parapsilosis and C. tropicalis were approximately 1 hour longer in the New FA Plus bottle, but the difference was not statistically significant. In the retrospective analysis, it is difficult to generalize because the TTD prolongation was not significantly observed in all sample groups. However, although the existing blood culture bottle formulation change was known to be substantially equivalent except for the detection of C. glabrata, this study suggests the possibility of causing some detection delay for C. parapsilosis and C. tropicalis.

Although C. auris is often known to have multidrug-resistance and several major C. auris outbreaks, including bloodstream infections, are reported worldwide, there are few studies on C. auris detection using automated blood culture systems, especially the Bact/ALERT Virtuo system [16–19]. This study’s simulated blood culture data showed that the C. auris detection time using the New FA Plus bottle was delayed about two hours compared with the Old FA Plus bottle, although statistical significance was limited. This result was contrary to the TTD of C. glabrata. A recent study conducted a simulation study using sheep blood and reported that the mean TTD of C. auris was 29.7 hours in the Old FA Plus Bottle using the Virtuo system [20], which was longer than our data (25.6 hours). The discrepancy in the TTD of C. auris in the Old FA Plus bottle between the previous and this study may be due to the differences between sheep and horse blood. Although the difference in the TTD can be regarded as small, further evaluations are needed to determine whether the change in medium ingredients substantially delayed C. auris detection, especially in human blood.

The difference in TTD between the simulated and clinical blood culture data may be due to various conditions. In the simulated study, the inoculation concentration of the isolates was 125 CFU/bottle or about 15 CFU/mL; however, the median CFU/mL was 1 CFU/mL in the initial positive candidemia blood culture samples [21]. Also, the differences in plasma components and red blood cell enzymes between horse and human blood may affect fungal growth [22]. However, it has been previously reported that using horse blood does not significantly influence the performance of blood culture systems [14]. A comparison study of horse and human blood has been conducted to demonstrate the equivalence of horse blood for use in blood culture bottle evaluation studies, and it supports that horse blood can be used for blood culture validation studies [22].

This study had several limitations. First, simulated candidemia blood cultures may have led to the differences in growth and detection in Candida spp., compared with the clinical specimens. Second, the number of repetitions of the simulation experiment was small, making it difficult to claim any statistical significance. Third, the simulation study only included ATCC strains for the majority of species and clinical isolates were included only for C. glabrata. Fourth, in the clinical retrospective blood culture data, only a single institution data was used, and the number of cases was small; therefore, the total number of analyzed cases was insufficient to secure statistical power. In addition, since the clinical retrospective data analysis was conducted only for several Candida strains that frequently cause candidemia, it was impossible to confirm whether the delay in TTD of C. auris in the New FA Plus bottle during the simulated study was similar to the clinical blood cultures. Fifth, the specific demographic characteristics of the patient group and the blood volume differences among the blood cultures were not considered. The clinical data analysis only included Candida spp. positive bottles; therefore, there was a bias in the analysis. Last, in retrospective study, the substantial patient population might be different between two time periods. Although the specimen collections from both periods were obtained from within same institution, patient characteristics may differ between two periods. Furthermore, the number and distribution of isolates included in the analysis in each period were different, therefore, it is possible that the differences may have affected the results.

Despite these limitations, this study is significant because it showed a reduced TTD compared with previous studies on C. glabrata in aerobic bottles. The clinical blood culture results supported the TTD of C. glabrata from the simulation study. In addition, our study compared various Candida spp., which can result in candidemia, and semi-quantitatively evaluated the growth performance of Candida spp. through yeast cell counts, adding to the novelty of this study. In some Candida species such as C. parapsilosis and C. tropicalis, the result that there may be a slight detection delay due to a change in the blood culture bottle is different from what was previously known and seems to require further evaluation.

In conclusion, most Candida species did not show remarkable TTD differences, but TTD of C. glabrata was markedly reduced in the New FA Plus bottle. This result is meaningful because faster detection and treatment of C. glabrata, which is gradually increasing in clinical importance, is possible due to the changes in the media in the blood culture bottle.

Supporting information

S1 Table. A summary of the Candida specimen included in retrospective analysis.

(DOCX)

Click here for additional data file. (13.6KB, docx)

Acknowledgments

We thank bioMérieux for providing the BacT/ALERT blood culture bottles used in this study.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (No.2020R1A5A1018052). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

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Decision Letter 0

Ali Amanati

29 Aug 2023

PONE-D-23-19815Comparison of New and Old BacT/Alert Aerobic Bottles for Detection of Candida spp.PLOS ONE

Dear Dr. Lee,

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

Reviewer #4: Yes

Reviewer #5: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: No

**********

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**********

5. Review Comments to the Author

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Reviewer #1: Choe et al. report on an interesting study concerning a new blood culture bottle from Bioemerieux in the Bact/Alert series, which should speed up the detection of C. glabrata. The study is well presented, that data are appropriately related to the literature, and there is a good discussion and explanation on the weaknesses of the study, e.g., the inoculum used as compared to the clinical consiiton, and the use of horse blood instead of human blood.

I have a couple of comments:

1. The aurhors need to review their statistical analysis of their data. From the CFUs reported in Table 2, it seems that the data may be skewed in direction of Poisson-distibutions - SD is larger than mean in several cases, and this also appears to be true for the TDD in Figure 1, where medians and and percentiles are shown. Here, data in Figure 2, the authors use T-tests for comparisons, which should instead be non-parametric tests such as the Mann-Whitney test. I wonder, whether the same counts for the CFU data in Table 2, CFUs are usually Poisson distributed. Also, since some of the NF-bottles were negative, again it is more correct to use medians and percentiles. It may not change the results but is more correct.

2. The hematocrit data for horse blood should be moved to the Results section.

Reviewer #2: Mi-Kyung Lee presents a laboratory based study comparing the updated BacT/Alert blood culture bottles to the previous version of the bottle.

Abstract: TTD is not written in full in the abstract. This should be done as readers will likely read the abstract prior to the full paper.

Introduction:

- Line 55- 56: what are the changes in the new bottle compared to the old bottle?

Methods:

- Suggest using the new names (based on taxonomic changes e.g. Nakaseomyces glabrata)

- Table 1 - spelling error on "guilliermondii"

- Why were clinical isolates included only for C.glabrata, not the other species

Retrospective data analysis: were there any diagnostic or management differences between the 2 time periods which could have affected the TTD of the 2 bottles. Were the 2 periods collecting specimens from the same patient population?

Results:

- Line 150: how many of each species was included in each period? Overall were there a similar number of isolates in both periods?

- greater variability in TTD as per figure 1. These findings are inconsistent with the conclusion of the paper which suggests that the newer bottle performs better. It only performed better for 1 species.

Line 155: "For the other 3 species...there was no significant difference" This statement is incorrect. For C.parapsilosis, TTD was much longer with the new bottle (p= 0.05)...which is bordering on a significant difference.

Discussion:

- line 181: spelling error again on C.guilliermondii

- Line 188 - Suggest reword to "Although C.auris is resistant to multiple antifungal agents"

- line 189 - blood infections should be bloodstream infections

Additional Limitations:

- only including ATCC strains for the majority of species and including clinical isolates only for C.glabrata

- comparing retrospective clinical data over 2 separate time periods

Reviewer #3: Dear Authors,

In your study, in which you compared the reproduction times of Candida species in your study, it does not include any information or discussion about the blood culture system, except for the time period. In order for your study to have a scientific result, you should reveal the differences in the old and new blood culture bottles, and the issues that can be developed technically or that are recommended to be developed should be expressed. The scientific contribution of the study in this state is very limited. The introduction, material, method and discussion parts of the study contain insufficient or inappropriate information. The work in its current form is unacceptable to me.

Best regards

Reviewer #4: Thanks for your invitation to review the manuscript entitled” Comparison of New and Old BacT/Alert Aerobic Bottles for Detection of Candida spp”. according to my opinion, the number of samples in this study is low, and new and important methods are not used. It is better to publish this manuscript after revision as a report or a short article.

Comments:

- Please don’t use abbreviate words in the Title and Abstract.

- In this sentence” The overall and clinical samples of C. glabrata showed similar results. C. albicans (27.6 versus 25.1 hours; P < 0.001) and C. guilliermondii (28.8 versus 27.3 hours; P = 0.026) also showed significantly shorter TTDs between the Old and New FA Plus bottles”. The P value calculation is not correct because there is no significant difference between the presented numbers.

Reviewer #5: Concerns;

1. For the abstract, please ensure full meanings of words/expressions are written for the first time and then subsequently, abbreviations could be used.

2. Line 53 "It has been reported that C. glabrata was more affected by the blood culture system and

medium than C. albicans" this statement is vague, please clarify, affected in what way.

3. The methods/materials should start by stating clearly what study design this research employed and the study settings. This will help readers to understand the study concept from the start.

4. Without going deep into proprietary issues, the authors should at least describe in basic terms what difference there is among the culture media employed.

5. The conclusion does not bring out the full picture of this study. The absence of significant difference in the other Candida species between the new and old media should at least be acknowledged and then the major finding of interest (i.e C. glabrata) and its importance highlighted.

6. Also, the authors should have explained in the methods or discussion what the new FA Plus bottle differentially has to make this conclusion "This study shows that the TTD of C. glabrata was markedly reduced in the New FA Plus bottle due to the optimized growth performance". Otherwise, "due to the optimized growth performance" could not be concluded from the findings in this study.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes: Parisa Badiee

Reviewer #5: No

**********

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PLoS One. 2023 Nov 29;18(11):e0288674. doi: 10.1371/journal.pone.0288674.r002

Author response to Decision Letter 0

16 Oct 2023

Responses to the Editor’s and Reviewers’ Comments

Dear reviewers and editorial staffs in Plos One

We are sincerely grateful for your thorough consideration and scrutiny of our manuscript, “Comparison of New and Old BacT/Alert Aerobic Bottles for Detection of Candida spp.”, manuscript No. PONE-D-23-19815R1.

We have revised the manuscript according to the reviewer’s suggestions. We hope that our revised manuscript will be considered and accepted for publication in Plos One. We acknowledge that the scientific and clinical quality of our manuscript was improved by the scrutinizing efforts of the reviewers and editors. The changes within the revised manuscript were highlighted (with yellow color). Point-by-point responses to the reviewers’ comments are provided below. We hope that our responses sufficiently address the reviewer’s concerns.

To. Reviewer #1

Thank you for the valuable insights. We present the responses to your comments below.

Reviewer Point P1.1

The aurhors need to review their statistical analysis of their data. From the CFUs reported in Table 2, it seems that the data may be skewed in direction of Poisson-distibutions - SD is larger than mean in several cases, and this also appears to be true for the TDD in Figure 1, where medians and and percentiles are shown. Here, data in Figure 2, the authors use T-tests for comparisons, which should instead be non-parametric tests such as the Mann-Whitney test. I wonder, whether the same counts for the CFU data in Table 2, CFUs are usually Poisson distributed. Also, since some of the NF-bottles were negative, again it is more correct to use medians and percentiles. It may not change the results but is more correct.

Reply: We agree with the reviewer on this point about statistical analysis. For statistical analysis of TTD (Time to detection) and CFU data, it may be more appropriate to use the Mann-Whitney U test rather than the t-test because the data is closer to the Poisson-distribution than to the normal distribution. The p-value in Fig 1 was modified to the value using Mann-Whitney U test instead of t test (see Figure R1 below). Also, in Table 2, we performed statistical analysis again using the Mann-Whitney test to determine whether there was a significant difference between new and old aerobic bottles. Additionally, considering the non-parametric distribution of data, the values in table 2 were changed to median, 25 percentile, and 75 percentile values instead of mean and sd.

Figure R 1 (Fig 1): Time to detection (TTD) of Candida spp. for the clinical blood culture bottles with different types of aerobic bottles; the New FA Plus (New) and Old FA Plus (Old). Box plots indicate the medians and the 25th and 75th percentiles. P value indicates the comparison of New and Old bottles using Mann-Whitney U test. (A) All the clinical blood samples were obtained from central line access and peripheral blood. (B) Only peripheral blood samples were included.

Unfortunately, changes in statistical analysis methods have changed the p-values for several Candida species, making it difficult to claim significant differences. This is thought to be due to the small number of repetitions of the simulation experiment (n=3), and changes in statistical results and interpretation have been added to the results (Line 139-144, 156-158) and discussion (Line 209-210) like below:

“The overall and clinical samples of C. glabrata showed similar results. C. albicans (27.6 versus 24.95 hours; P =0.2) and C. guilliermondii (28.9 versus 26.9 hours; P = 0.1) also showed relatively shorter TTDs between the Old and New FA Plus bottles, however, the difference was not statistically significant. On the contrary, the TTD was longer for C. auris (25.43 versus 28.25 hours; P =0.0765) in the New FA Plus bottle compared with the Old FA Plus bottle, although statistical significance of the difference could not be clarified.”

“In addition, although there was no significant difference, median yeast cell counts of New FA Plus was higher than Old FA Plus for C. auris, C. parapsilosis, and C. tropicalis.”

“The simulation study showed lower median values of TTD for C. albicans and C. guilliermondii in the New FA Plus than in the Old FA Plus bottle.”

Reviewer Point P1.2

The hematocrit data for horse blood should be moved to the Results section.

Reply: We totally agreed with this suggestion. The hematocrit data for horse blood was moved to the Results section (Line 136).

To. Reviewer #2

We greatly appreciate your detailed suggestions to improve the quality of our paper. Responses to each comment are below:

Reviewer Point P2.1

Abstract: TTD is not written in full in the abstract. This should be done as readers will likely read the abstract prior to the full paper.

Reply: We agree with you, the abbreviation in the abstract should have been mentioned in full term beforehand, but was omitted. The full term of TTD (time to detection) has been added to the abstract (Line 24).

Reviewer Point P2.2

Introduction:

- Line 55- 56: what are the changes in the new bottle compared to the old bottle?

Reply: It would have been better if we could have mentioned specific compositional changes as you asked. However, this information could not be obtained because it was proprietary information of the manufacturer, and instead, only a sentence about the approximate information mentioned by the company was added after that sentence (Line 61-63) like below:

“Although the exact formulation of the bottle media is proprietary and not disclosed, it was reported that trace elements were added and several components unrelated to microbial recovery were removed.”

Reviewer Point P2.3

Methods:

- Suggest using the new names (based on taxonomic changes e.g. Nakaseomyces glabrata)

Reply: Thanks to your suggestion, we noticed that based on molecular phylogenetic study, Candida glabrata was recently renamed Nakaseomyces glabratus. However, since this paper mainly focuses on the evaluation of candidemia and we considered that the taxonomy of Candida glabrata has not been completely established, especially in the clinical area, we did not replace all C. glabrata with N. glabratus and added the new name in parentheses after first mentioned C. glabrata (Line 53).

Reviewer Point P2.4

- Table 1 - spelling error on "guilliermondii"

Reply: Thank you for finding the error. The spelling error was corrected.

Reviewer Point P2.5

- Why were clinical isolates included only for C.glabrata, not the other species

Reply: As you commented, more valuable results could have been obtained if we had experimented with other Candida species. However, the number of bottles available during the experiment was limited (especially old bottles), and as we mentioned (Line 63-65), the manufacturer's claim mentioned that the change in bottle formulation shortened the detection time of Candida glabrata as the main finding. In this study, we wanted to know the results of clinically derived isolates of Candida glabrata rather than other species, so clinical isolates of other species were not included in the study.

Reviewer Point P2.6

Retrospective data analysis: were there any diagnostic or management differences between the 2 time periods which could have affected the TTD of the 2 bottles. Were the 2 periods collecting specimens from the same patient population?

Reply: Although the specimen collections from both periods were obtained from patients within the same institution, they cannot be considered the same patient population. We did not intentionally designate or exclude specific patient groups, and samples were included in this study as soon as they were received, so we believe there is no bias, but the results may have been affected. Therefore, the time period-related issue was additionally mentioned in the discussion as a limitation (Line 261-263):

“Last, in retrospective study, the substantial patient population might be different between two time periods. Although the specimen collections from both periods were obtained from within same institution, patient characteristics may differ between two periods.”

Reviewer Point P2.7

Results:

- Line 150: how many of each species was included in each period? Overall were there a similar number of isolates in both periods?

Reply: A summary of the species of specimens included in each period has been added as a supplementary table, S1 table. Since the number and distribution of isolates included in the analysis in each period were different, these differences were added to the discussion (Line 264-266):

“Furthermore, the number and distribution of isolates included in the analysis in each period were different, therefore, it is possible that the differences may have affected the results.”

Table R 1 S1 Table A summary of the Candida specimen included in retrospective analysis

Candida species Old FA Plus

Number of blood specimen

(Peripheral blood specimen) New FA Plus

Number of blood specimen

(Peripheral blood specimen)

Candida albicans 8(2) 19(10)

Candida glabrata 8(3) 11(7)

Candida parapsilosis 16(6) 13(8)

Candida tropicalis 17(11) 7(3)

Total 49(22) 50(28)

Reviewer Point P2.8

- greater variability in TTD as per figure 1. These findings are inconsistent with the conclusion of the paper which suggests that the newer bottle performs better. It only performed better for 1 species.

Line 155: "For the other 3 species...there was no significant difference" This statement is incorrect. For C.parapsilosis, TTD was much longer with the new bottle (p= 0.05)...which is bordering on a significant difference.

Reply: As you commented, this paper claims that Candida glabrata showed better performance, but there was no clear performance improvement in other species. Reviewing Figure 1 (revised using Mann-Whitney U test, not t-test), the TTD of C. tropicalis increased significantly (p=0.0361) in the new bottle, and similarly, the TTD of C. parapsilosis was longer (p=0.02) in the new bottle when only peripheral blood specimens were included (Figure 1B). Therefore, the following was added to the results (Line 179-183) and discussion(Line 216-224) in the main text:

“In particular, when analyzing all blood samples for C. tropicalis (Old FA Plus 9.58 versus New FA Plus 20.28 hours; P =0.0361) and only peripheral blood samples for C. parapsilosis (Old FA Plus 21.33 versus New FA Plus 27.17 hours; P =0.02), New FA Plus showed a statistically significant increase of TTD compared to Old FA Plus.”

“Retrospective data analysis results showed that some Candida species had an extended TTD due to aerobic culture bottle changes, which can also be related to the simulated study data. In the simulated study, the median TTD values for C. parapsilosis and C. tropicalis were approximately 1 hour longer in the New FA Plus bottle, but the difference was not statistically significant. In the retrospective analysis, it is difficult to generalize because the TTD prolongation was not significantly observed in all sample groups. However, although the existing blood culture bottle formulation change was known to be substantially equivalent except for the detection of C. glabrata, this study suggests the possibility of causing some detection delay for C. parapsilosis and C. tropicalis.”

Reviewer Point P2.9

Discussion:

- line 181: spelling error again on C.guilliermondii

- Line 188 - Suggest reword to "Although C.auris is resistant to multiple antifungal agents"

- line 189 - blood infections should be bloodstream infections

Reply: We apologize for repeated spelling errors, and the errors been corrected.

The sentence for which a reword was requested was modified as follows (Line 225-226): “Although C. auris is often known to have multidrug-resistance”.

“Blood infections” was replaced to “Bloodstream infections”. Thank you for reviewing the shortcomings of the overall paper.

Reviewer Point P2.10

Additional Limitations:

- only including ATCC strains for the majority of species and including clinical isolates only for C.glabrata

- comparing retrospective clinical data over 2 separate time periods

Reply: We agree with the additional limitations of this study you raised and have added them to the discussion (Line 251-252).:

“Third, the simulation study only included ATCC strains for the majority of species and clinical isolates were included only for C. glabrata.”

To. Reviewer #3

Comment from reviewer:

Dear Authors,

In your study, in which you compared the reproduction times of Candida species in your study, it does not include any information or discussion about the blood culture system, except for the time period. In order for your study to have a scientific result, you should reveal the differences in the old and new blood culture bottles, and the issues that can be developed technically or that are recommended to be developed should be expressed. The scientific contribution of the study in this state is very limited. The introduction, material, method and discussion parts of the study contain insufficient or inappropriate information. The work in its current form is unacceptable to me.

Responses to comment

We accept your critical opinion that this study may have limited scientific contribution. As previously mentioned in Reviewer point P2.2, the details of the bottle composition changes were unknown due to proprietary issues and were not the focus of this study. Rather than exploring the scientific mechanism caused by changes in the composition of the bottle, this study reported and considered changes in detection time of several Candida species that may have clinical implications. In addition, this study focused on the research methodology of a simulation study using horse blood rather than human blood. In response to criticism that this study provided insufficient and inappropriate information, we tried to improve it through this revision, and hope that it has reached an acceptable level for publication.

To. Reviewer #4

Thank you for your thoughtful comments. I understand what you suggested "It is better to publish this manuscript after revision as a report or a short article.", but since this journal only publishes research articles, it seems impossible at this stage. Instead, we have tried to make overall improvements through revision, so we would appreciate it if you could take this into consideration.

Reviewer Point P4.1

- Please don’t use abbreviate words in the Title and Abstract.

Reply: As you commented, we checked all abbreviations of title and abstract and replaced them with full terminology.

Reviewer Point P4.2

- In this sentence” The overall and clinical samples of C. glabrata showed similar results. C. albicans (27.6 versus 25.1 hours; P < 0.001) and C. guilliermondii (28.8 versus 27.3 hours; P = 0.026) also showed significantly shorter TTDs between the Old and New FA Plus bottles”. The P value calculation is not correct because there is no significant difference between the presented numbers.

Reply: We agree with the questions you raised about statistical significance. The t-test was used for the statistics of the manuscript before revision, but as mentioned in Reviewer point P1.1 above, we judged that parametric analysis may not be appropriate because the number of observations is small and the data is not normally distributed. Instead, statistical analysis was performed again using the median value and the Mann-Whitney U test and modified as shown in table 2. Accordingly, the results and discussion were also modified (Line 139-144, 156-158, 209-210).

To reviewer #5

Reviewer Point P5.1

1. For the abstract, please ensure full meanings of words/expressions are written for the first time and then subsequently, abbreviations could be used.

Reply: As mentioned in reviewer point P4.1 above, we checked all abbreviations of title and abstract and replaced them with full terminology.

2. Line 53 "It has been reported that C. glabrata was more affected by the blood culture system and medium than C. albicans" this statement is vague, please clarify, affected in what way.

Reply: Since the mainly affected factor was the time until detection of Candida species according the cited literature, it was described more clearly (Line 57-58).

3. The methods/materials should start by stating clearly what study design this research employed and the study settings. This will help readers to understand the study concept from the start.

Reply: A summary of the overall study design has been added to the first paragraph of methods/materials (Line 74-79). We hope this will help readers understand the paper.

4. Without going deep into proprietary issues, the authors should at least describe in basic terms what difference there is among the culture media employed.

Reply: As you mentioned, excluding parts that cannot be confirmed due to proprietary issues, we have added the formulation change of the new medium and the composition of the existing medium to the introduction and method (Line 61-63, 96-101).

5. The conclusion does not bring out the full picture of this study. The absence of significant difference in the other Candida species between the new and old media should at least be acknowledged and then the major finding of interest (i.e C. glabrata) and its importance highlighted.

Reply: We have added information about differences in the other Candida species to the conclusion (Line 275-276).:

“In conclusion, most Candida species did not show remarkable TTD differences, but TTD of C. glabrata was markedly reduced in the New FA Plus bottle.”

6. Also, the authors should have explained in the methods or discussion what the new FA Plus bottle differentially has to make this conclusion "This study shows that the TTD of C. glabrata was markedly reduced in the New FA Plus bottle due to the optimized growth performance". Otherwise, "due to the optimized growth performance" could not be concluded from the findings in this study.

Reply: Considering your comment, we performed the yeast cell count study to provide evidence for optimized growth performance, but this alone appears to be insufficient to draw a conclusion. Therefore, we deleted the phrase about optimized growth performance in the conclusion (Line 276).

Again, thank you for giving us the opportunity to strengthen our manuscript with your valuable comments and queries. We have worked hard to incorporate your feedback and hope that these revisions persuade you to accept our submission.

We apologize for the extremely long delay.

Sincerely,

Mi-Kyung Lee, M.D., Ph.D.

102, Heukseok-Ro, Dongjak-Ku, Seoul 06973, South Korea

Tel: +82-2-6299-2719, Fax: +82-2-6298-8630

E-mail: cpworld@cau.ac.kr

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Decision Letter 1

Ali Amanati

10 Nov 2023

Comparison of New and Old BacT/ALERT Aerobic Bottles for Detection of Candida Species

PONE-D-23-19815R1

Dear Dr. Mi-Kyung Lee,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Ali Amanati

Academic Editor

PLOS ONE

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The current article is scientifically valid in its current form. So, based on my ‎opinion and the respected reviewers' comments could be published.‎

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Acceptance letter

Ali Amanati

17 Nov 2023

PONE-D-23-19815R1

Comparison of New and Old BacT/ALERT Aerobic Bottles for Detection of Candida Species

Dear Dr. Lee:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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on behalf of

Professor Ali Amanati

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. A summary of the Candida specimen included in retrospective analysis.

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