A multiphasic approach to solve misidentification of Cutibacterium acnes as Atopobium vaginae during routine bacterial screening of platelet concentrates using the VITEK 2 system
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Skin flora bacteria, such as Cutibacterium acnes , are the predominant contaminants of blood products used for transfusion. Platelet concentrates (PCs), a therapeutic product used to treat patients with platelet deficiencies, are stored at ambient temperature under agitation, providing ideal conditions for bacterial proliferation. At Canadian Blood Services, PCs are screened for microbial contamination using the automated BACT/ALERT culture system. Positive cultures are processed and contaminating organisms are identified using the VITEK 2 system. Over a period of approximately 2 years, several PC isolates were identified as Atopobium vaginae to a high level of confidence. However, since A. vaginae is associated with bacterial vaginosis and is not a common PC contaminant, a retrospective investigation revealed that in all cases C. acnes was misidentified as A. vaginae . Our investigation demonstrated that the media type used to grow PC bacterial isolates can have a significant impact on the results obtained on the VITEK 2 system. Furthermore, other identification methods such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALD-TOF MS) and PCR amplification of the 16S RNA gene were only partially successful in the identification of C. acnes . Therefore, our findings support a multiphasic approach when PC isolates are identified as A. vaginae by the VITEK 2 system for proper identification of C. acnes using macroscopic, microscopic and other biochemical analyses.
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This study would be a valuable contribution to the existing literature.
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The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Both reviewers have agreed that in principle your study would be of interest to the community and other researchers. However, both reviewers have raised some concerns in the methods used and the clarity of results presented. In particular, I invite you to carefully consider and address the concerns raised by reviewer 2 before resubmission of a revised version of the manuscript. Both reviewers have agreed to review an edited version of the manuscript.
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Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data: The authors need to include more details regarding how they set-up the VITEK ANC card, specifically how was the density of the cell suspension performed and did they follow the vendor's instructions for use (IFU). The IFU incudes precautions and limitations that the authors do not address in their paper such as using only polystyrene tubes and vendor recommended media to name just two. It is also important to include what MALDI-TOF system including database was used to determine identification. The authors reference table 2 (line 149) however it is not included in the manuscript. Table 1 should include all of the available data to allow readers to easily interpret the study, this includes defining the media that was used …
Comments to Author
1. Methodological rigour, reproducibility and availability of underlying data: The authors need to include more details regarding how they set-up the VITEK ANC card, specifically how was the density of the cell suspension performed and did they follow the vendor's instructions for use (IFU). The IFU incudes precautions and limitations that the authors do not address in their paper such as using only polystyrene tubes and vendor recommended media to name just two. It is also important to include what MALDI-TOF system including database was used to determine identification. The authors reference table 2 (line 149) however it is not included in the manuscript. Table 1 should include all of the available data to allow readers to easily interpret the study, this includes defining the media that was used in the initial ID, MALDI-TOF, PCR of C acnes 16S rRNA gene, and API 20A strip ID results. See more detailed comments under 5. 2. Presentation of results: Table 1 contains only the VITEK 2 results however the authors compare the strains using other identification methods such as MALDI-TOF, PCR, and API 20A. All of the data should be presented in table 1 to allow the user to better understand the data and the level of misidentification and need for additional testing. See more detailed comments under 5. 3. How the style and organization of the paper communicates and represents key findings. The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media (this needs to be verified as the authors do not confirm that Columbia agar with 5% sheep blood was used). They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach'. See more detailed comments under 5. 4. Literature analysis or discussion. The authors should reference the vendor's instruction for use and verify that the correct vendor recommended media (Columbia agar with 5% sheep blood (CBA)) was used. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification. The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae. See more detailed comments under 5. 5. Any other relevant comments Lines 2-3: The title is misleading as the authors are stating a deficiency of an identification system to correctly identify an organism. The authors show marked improvement in correct identification when using the vendor's recommended media. They also present that there is no one perfect identification system, specifically they show a failure to properly identify C. acnes using MALDI-TOF and that PCR amplification of the C acnes 16S rRNA gene failed to provide an identification. Given these facts, it is highly recommended that the title be changed to reflect the full study. A more suitable title would be 'Identification of Cutibacterium acnes during routine bacterial screening of platelet concentrates requires a multiphasic approach Line 22: add multiphasic, MALDI-TOF, 16S RNA to keywords Lines 38-40. The authors should state that MALDI-TOF and PCR amplification of the C acnes 16S rRNA gene were also not able to properly identify C. acnes. The findings from their studies indicate the need for a multiphasic approach for proper identification of this common PC contaminant including using the recommended growth media and supplemental biochemical assays. Line 48. Bold statement. Instead recommend wording as 'to our knowledge we report an issue not reported in the literature concerning misidentification ….' Line 55. Alternative manual identification systems such as MALDI-TOF and PCR amplification of C acnes16S rRNA , to confirm the VITEK results. Lines 58-60. Table 1 needs to include both MALDI-TOF and molecular results. Authors may reference outside supporting laboratories in the table. The table should also reference what media was used to generate the initial ID. Line 67. Define the type of anaerobic agar. Lines 69-71. Include the VITEK software version and define the media that is used by CBS to perform the VITEK 2 system identification. The vendor includes performance characteristics including percent misidentification and no identification that can be observed for each software version. Line 99. Please state if the Columbia agar is with or without blood and if it is with blood please include details, for example with 5% sheep blood and reference it as Columbia blood agar or CBA. Line 99. Columbia agar with 5% sheep blood is the vendor recommended media. Please clarify here and throughout the entire manuscript what type of Columbia agar was used. If 5% sheep blood is not included the Columbia agar referenced by the authors is not the vendor recommended media. Line 106. Add descriptor for Columbia agar. Was the agar with or without blood? See comment for page 5, line 99. Line 106. All of the isolates were grown on Columbia agar with 5% sheep blood as recommended by the vendor, while 5 of the isolates were randomly chosen to be grown on all three media types. Note: If Columbia agar did not contain sheep blood it is not the recommended media. Line 109. The authors should confirm if they correctly achieved the vendor recommended density prior to loading the ANC card. How was the density performed? If the authors followed the exact vendor recommended instructions they can reference the vendor instructions for use. The vendor states the importance of cell density, media type, and requirement for using polystyrene tubes in their instructions for use. Line 111. MALDI-TOF is not a molecular method therefore this section should be renamed as Additional identification methods. Line 122. Since the authors also relied on API test strips they should include information about how they were used in this section that should be renamed to additional identification methods. Lines 125-126. Include a description on how the catalase test was performed. Catalase testing should not be performed from media containing blood as it could lead to false positive results. Line 133, It is important to state what media was used to initially identify the organisms listed in Table 1 under initial ID column. The authors were not using the vendor recommended media for initial identification. Line 134. Columbia agar with or without blood (see previous comments). Please clarify. Lines 136-137. Columbia agar with or without blood (see previous comments). If using the recommended Columbia agar with 5% sheep blood the authors should further clarify by stating '… grown on the vendor recommended CBA'. Line 142. The media study clearly shows the importance of using the vendor recommended culture media for identification using the VITEK 2 system. This result confirms the precaution listed in the vendor instructions for use that 'use of culture media other than the recommended types must be validated by the customer laboratory for acceptable performance'. Line 143. This subtitle is misleading. All of the isolates were not identified as C acnes by just one method. Full identification required a multiphasic approach. The authors used MALDI-TOF, PCR amplification of a section of the C acnes 16S RNA gene as well as using API Rapid ID 32A strip testing to confirm proper identification. This section should be renamed as 'Multiple alternative identification tests were required to identify all isolates initially identified as A vaginae as C acnes.' Lines 145-147. Include the MALDI-TOF and 16S rRNA results in Table 1. Line 149. Table 2 is missing from the manuscript. Recommend adding API results to Table 1. Table 1 should provide all of the data together so readers are able to easily analyze the data in order to better illustrate the importance of using vendor recommended media and the need for additional testing to provide proper identification. Lines 170. ….for quality control and routine testing of the ANC cards. Reference Table 2 in the vendor's instruction for use. Table 2 in the instructions for use provides a list of the media that were used in the identification product database development and that will give the optimal performance. Lines 166-170. The results of the study do show that the VITEK 2 system may misidentify C acnes as A vaginae however the authors initial results show a higher percentage of misidentification due to using media that is not recommended by the vendor. Using the vendor recommended CBA markedly improves identification. The results also show that alternative identification systems such as MALDI-TOF and PCR amplification of 16S RNA gene from C acnes does not provide 100% identification and that further confirmation was needed by using API Rapid ID test strips. The authors should conclude that it is important to perform a multiphasic approach for identification of C acnes especially when initial identification using VITEK 2 identifies an anaerobic gram positive organism as A. vaginae. Line 205. Recommend referencing the vendor instructions for use for the VITEK 2 system.
Please rate the manuscript for methodological rigour
Satisfactory
Please rate the quality of the presentation and structure of the manuscript
Satisfactory
To what extent are the conclusions supported by the data?
Partially support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
No
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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Comments to Author
This work demonstrates that bacterial misidentification of Cutibacterium acnes as Atopobium vaginae using the VITEK 2 system is most likely caused by the growth medium used for sub-culturing of isolates. It also describes alternative identification strategies that leads to a better differentiation of the two species. Although the paper is short, it highlights the importance of a sound assement of doubtful results generated by automated systems like the VITEK 2. I do have the following remarks (minor comments): 2. Abstract line 36 - please abbreviate the genus Cutibacterium (full genus name already in line 28) line 36 - delete "a common PC contaminant" as it is already mentioned in line 28 5. Introduction line 74 - please choose a different reference for description of C. acnes line 76-79 - it is …
Comments to Author
This work demonstrates that bacterial misidentification of Cutibacterium acnes as Atopobium vaginae using the VITEK 2 system is most likely caused by the growth medium used for sub-culturing of isolates. It also describes alternative identification strategies that leads to a better differentiation of the two species. Although the paper is short, it highlights the importance of a sound assement of doubtful results generated by automated systems like the VITEK 2. I do have the following remarks (minor comments): 2. Abstract line 36 - please abbreviate the genus Cutibacterium (full genus name already in line 28) line 36 - delete "a common PC contaminant" as it is already mentioned in line 28 5. Introduction line 74 - please choose a different reference for description of C. acnes line 76-79 - it is stated that the VITEK system was adopted for identification of blood product contaminants in 2019 which the led to the described misidentification. How was the situtation before 2019 regarding the identification of C. acnes? 6. Methods line 91 - "...initially misidentified as..." might be better even though the description with "catalase positive" indicates the the PC isolates were wrongly identified line 99 - delete the first "card" line 106 - based on table 1, all isolates were grown on Columbia agar. It is stated that 5 of the isolates were chosen to be grown on the three media types. 5 isolates of what? If these isolates belong to the initially identified A. vaginae isolates, there are only 4 isolates listed in table 1 that were grown on TSAb. This is a bit confusing, please modify this paragraph for a better clarification. line 108 - isolates were grown for up to 72 hours indicating that some isolates were cultivated also for a shorter period dependent on the growth that was obtained. However, as test results of mature cultures might influence the VITEK 2 results, the incubation time is important. Do the authors have any information on the cultivation period of the samples that were used for identification on the VITEK 2 system? line 113 - how is the MALDI measurement performed? any reference to a standard procedure/protocol? 7. Results and Discussion line 126-128 - The colony morphology is described briefly. An illustration of the colony morphology in Figure 1 (close-up or enlarged colony) would be preferable. line 149 - There was no Table 2 in section 8. Tables and Figures. Please add the table. 8. Tables and Figures Table 2 is missing. 9. Conclusion line 166-167 - Do the authors have any information on which of the 36 colorimetric reactions of the VITEK 2 system is responsible for the misidentification? line 172 - The authors hypothesise that due to a faster growth on OxyPRAS Plus, cultures are more matured leading to a distorted enzymatic profile. An experimentally proof that a shorter incubation time of C. acnes on OxyPRAS Plus results in a better identification would be advisable to support this idea. 1. Methodological rigour Based on the manuscript, the identification for each isolate was only done once per medium and assay. Can the authors comment on the reproducibility of the results e.g. when an isolate is pre-cultured on one specific media type and tested serveral times on the VITEK 2 system? 2. Presentation of results No comment. 3. Literature analysis or discussion It might be worth to highlight the critical assessment of results generated by automated test system in general at the end of the discussion. Operators in the clinical setting use these systems more and more frequently, however, they need to keep in mind not to trust blindly the results.
Please rate the manuscript for methodological rigour
Good
Please rate the quality of the presentation and structure of the manuscript
Good
To what extent are the conclusions supported by the data?
Strongly support
Do you have any concerns of possible image manipulation, plagiarism or any other unethical practices?
No
Is there a potential financial or other conflict of interest between yourself and the author(s)?
Yes: The corresponding author is head of an international expert group of which I am also a member. Due to our activities within the group (e.g. international collaborative studies), we are co-authors in studies published in the past.
If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?
Yes
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