Efficient Recovery of Candida auris and Five Other Medically Important Candida Species from Blood Cultures Containing Clinically Relevant Concentrations of Antifungal Agents

ABSTRACT Candida auris and other Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei) are important causes of bloodstream infection. Early or prolonged treatment with antifungal agents is often required. The inhibitory effect of antifungal agents in the patients’ bloodstream may compromise the sensitivity of blood culture (BC) to diagnose and/or monitor patients with candidemia. Using a clinical BC simulation model, we compared antimicrobial drug-neutralizing BC media in BacT/Alert FA PLUS (FAP) or Bactec Plus Aerobic/F (PAF) bottles with non-neutralizing BC media in Bactec Mycosis IC/F (MICF) bottles to allow Candida growth in the presence of 100%, 50%, or 25% peak serum level (PSL) antifungal concentrations. In total, 117 organism/antifungal combinations were studied, and Candida growth was detected after incubating bottles into BacT/Alert VIRTUO or Bactec FX BC systems. Compared to control (without antifungal) bottles, both FAP and PAF bottles with 100% PSL antifungal concentrations allowed 100% recovery for C. auris, C. glabrata, and C. parapsilosis, whereas recovery was below 100% for C. albicans, C. krusei, and C. tropicalis. MICF bottles were less efficient at 100%, 50%, or 25% PSL antifungal concentrations, for all Candida species, except for C. auris. While azoles and amphotericin B did not hinder Candida growth in FAP or PAF bottles, echinocandins allowed C. auris, C. glabrata, and C. parapsilosis to grow in FAP, PAF, or MICF bottles. Overall, the maximum time to detection was 4.6 days. Taken together, our findings emphasize the reliability of BCs in patients undergoing antifungal treatment for candidemia. IMPORTANCE While echinocandins remain the preferred antifungal therapy for candidemia, bloodstream infections caused by C. auris, C. glabrata, or, at a lesser extent, C. parapsilosis may be difficult to treat with these antifungal agents. This is in view of the high propensity of the above-mentioned species to develop antifungal resistance or tolerance during treatment. Azoles and amphotericin B are possible alternatives. Thus, optimizing the recovery of Candida from BCs is important to exclude the likelihood of negative BCs for Candida species, owing to the inhibitory effect of antifungal agents present in the blood sample with which BCs are inoculated. Consistently, our results about the recovery of medically important Candida species (including C. auris) from simulated BCs in BacT/Alert FAP, Bactec PAF, or Bactec MICF bottles containing clinically relevant antifungal concentrations add support to this research topic, as well as to the use of BCs for monitoring the clinical and therapeutic course of candidemia.

are useful for daily medical practice, given the high number of patients receiving antifungal drugs and requiring blood culturing per their clinical manifestations. Some comments and inquiries: 1. Lines 112-116. The main conclusion of the study is that the neutralizing resins used may not be adequate for echinocandins but appear to neutralize the effect of azoles and amphotericin B in general. May the authors comment on this "further", considering the chemical structure of echinocandins? 2. Similary, regarding the varying effects on different species of Candida --What are the possible factors that may have influence on this? (growth rate, etc...) Adding an additional short discussion may be helpful. 3. Line 158. "Slightly", "moderately", or "highly delayed": Please insert exact definitions and relevant references at this point. 4. Lines 277-278. CDC tentative C. auris breakpoints should also be referenced and acknowledged at this point.

Reviewer #3 (Comments for the Author):
This is an interesting and well-executed study which provides valuable information on the reliability of blood culture in the presence of various antifungals. The authors are to be congratulated on the thoroughness of their methods.
I have a few small suggestions that I feel would be beneficial for the manuscript.
Candida glabrata and Candida krusei have relatively recently had name changes which are increasingly being used. Whilst I understand for familiarity the authors may choose to continue using their older names, I feel it would be helpful to also mention their newer names: Nakaseomyces glabrata and Pichia kudriavzevii respectively. P5 -L87-91. It would make the reading of the manuscript slightly easier if the authors included the range of antifungal concentrations used as 100%, 50% and 25% PSL in the main body of the text as well as in the supplementary information.
Staff Comments:

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Editor (Comment for the Author):
Both reviewers found the study well executed and useful for our community. They both pointed out that bioMérieux was involved in reviewing the manuscript. The authors should precise if the results were amended or modified after these reviewing or if it was only minor modifications. Please explain in detail. Answer: I have added a specification of how bioMérieux reviewed the manuscript via a short sentence in the Acknowledgments section. See page 15, lines 342 to 344 of the revised manuscript.

Reviewer #2 (Comments for the Author):
The authors have investigated the efficiency of different blood culture media of the automated blood culture systems containing clinically relevant concentrations of antifungal drugs in isolation of six Candida species, including Candida auris. A clinical blood culture simulation model was used. Ultimately, the effect of the neutralizing resins on the existing antifungal drug was explored and differential data for different classes of antifungal drugs and varying Candida species were obtained. The presented data are useful for daily medical practice, given the high number of patients receiving antifungal drugs and requiring blood culturing per their clinical manifestations. Some comments and inquiries: Answer: While I am very grateful to the reviewer for appreciating the manuscript, I have tried to substantiate my responses to the comments/questions raised by the reviewer. This implied the addition of two references (nos. 22 and 23). 1. Lines 112-116. The main conclusion of the study is that the neutralizing resins used may not be adequate for echinocandins but appear to neutralize the effect of azoles and amphotericin B in general. May the authors comment on this "further", considering the chemical structure of echinocandins? Answer: As suggested by the reviewer, I have added a few sentences to "further" comment on the main conclusion of the study, discussing how the chemical structure of the echinocandins may have affected the main findings of the study. See page 10, lines 213 to 220, with enclosed reference no. 22, of the revised manuscript. 2. Similarly, regarding the varying effects on different species of Candida --What are the possible factors that may have influence on this? (Growth rate, etc...) Adding an additional short discussion may be helpful. Answer: As in the previous comment, I elaborated on factors that may have affected the apparent species-specificity of the study findings. See page 10, lines 220 to 223, with enclosed reference no. 23, of the revised manuscript. 3. Line 158. "Slightly", "moderately", or "highly delayed": Please insert exact definitions and relevant references at this point.

Reviewer #3 (Comments for the Author):
This is an interesting and well-executed study which provides valuable information on the reliability of blood culture in the presence of various antifungals. The authors are to be congratulated on the thoroughness of their methods. I have a few small suggestions that I feel would be beneficial for the manuscript. Answer: While I am very grateful to the reviewer for appreciating the manuscript, I have tried to substantiate my responses to the suggestions raised by the reviewer. Candida glabrata and Candida krusei have relatively recently had name changes which are increasingly being used. Whilst I understand for familiarity the authors may choose to continue using their older names, I feel it would be helpful to also mention their newer names: Nakaseomyces glabrata and Pichia kudriavzevii respectively. Answer: As suggested by the reviewer, I have mentioned the newer names of Candida glabrata and Candida krusei. See page 4, lines 55 to 56 of the revised manuscript. P5 -L87-91. It would make the reading of the manuscript slightly easier if the authors included the range of antifungal concentrations used as 100%, 50% and 25% PSL in the main body of the text as well as in the supplementary information. Answer: As suggested by the reviewer, I have added in the main text (as already reported in Table S1) the antifungal concentrations used as 100%, 50%, and 25% PSL, respectively. See page 5, lines 86 to 89 of the revised manuscript.