Long-Term Survival of Three Fungal Species in Lyophilized Human Blood Plasma

The low prevalence of some diseases, such as invasive fungal diseases, limits the availability of clinical specimens needed for developing and testing novel diagnostics for such diseases. Thus, the development and testing of such diagnostics usually require the use of banked specimens (1). However, the use of such specimens mandates the evaluation of the impact of the storage conditions on the stored specimens (1). Lyophilization allows the long-term storage of biological samples at room temperature and lowers the cost of storage and shipping, which makes it a valuable method for biobanking (2, 3). Moreover, lyophilization improves the performance of some spectroscopic methods such as infrared spectroscopy (4). In a previous study, plasma samples spiked with Aspergillus and Penicillium species were lyophilized and then analyzed by infrared spectroscopy to study the potential of infrared spectroscopy as a bloodbased diagnostic tool for invasive aspergillosis (4). As the survival of microorganisms in samples stored in a biobank can affect the assay being investigated as a novel diagnostic, this study reports the survival of these fungal species in these lyophilized plasma samples after 4 years of storage at room temperature. Samples used in this study were prepared in a previous study, as follows (4). The mycelia of clinical isolates of Aspergillus flavus, Aspergillus niger, and Penicillium chrysogenum were used to aseptically spike fresh-frozen plasma in blood tubes (1ml), and the samples were then lyophilized overnight at 250°C to be used in the study. These isolates were identified to the species level by Sanger sequencing and matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) identification in addition to their macroscopic and microscopic morphology; the isolates were subcultured on Sabouraud dextrose agar (SDA) at 30°C for 7days, which resulted in adequate mycelial development to be used for spiking the samples (4). The remaining lyophilized plasma samples in addition to a nonspiked lyophilized plasma sample, as a negative control, were stored at room temperature in the dark until the time of conducting this study. After 4 years, Sabouraud dextrose broths (SDBs) were aseptically spiked by a few milligrams of the stored lyophilized samples (directly, without prior reconstitution) and then incubated at 30°C for 2 to 3 days. Once the mycelia were visible, part of the mycelia was transferred onto SDA and then incubated at 30°C and examined daily for typical colonies for 7 days. Colonies from the plate and mycelia from growth in SDB were also examined under a microscope for characteristic features of the fungi. Microscopic examination included examining the colonies on the plate itself with a low-power lens. To examine the fungi by high-power and oil immersion lenses, an agar plug from the SDA with the corner of the colony and/or mycelial growth from SDB was transferred to a flame-sterilized slide. Citation Elkadi OA, Elanany M, Ramadan MA. 2021. Long-term survival of three fungal species in lyophilized human blood plasma. Microbiol Spectr 9:e00222-21. https://doi.org/ 10.1128/Spectrum.00222-21. Editor N. Esther Babady, Memorial Sloan Kettering Cancer Center Copyright © 2021 Elkadi et al. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Omar Anwar Elkadi, Omar.elkadi@live.com.


Reviewer comments:
Reviewer #1 (Comments for the Author): Elkadi et al present a nice report about reviving fungi from lyophilized state in a plasma matrix. The findings have value for a number of applications. I have only some minor comments to further refine this well-written report: Did the authors prepare the fungi in a special way prior to lyophilization? For instance, did they assure sporulation was happening, or a certain level of sporulation? Even if this was described in cited work, please share again here briefly for completeness.
Line 35: were the samples reconstituted first, or was the lyophilized powder used as the inocoulum itself?
Reviewer #2 (Comments for the Author): While this an important step in assessing if lyophilization is a useful way to preserve this type of specimen this study is very limited in its evaluation (only one strain each of three fungal species with no replicates) and does not measure performance against any other method of preservation.
The authors demonstrated the limited number of fungi were still viable after 4 years of storage however it is unclear how this compared (or if there is a benefit)to other storage methods. The authors state this method offers potentially "improved stability" (line 15) but no comparator evaluation or data are provided. If the goal of the study was to identify a way to preserve actual clinical specimens (in this case plasma, as well as the pathogen) for later use in diagnostic assay development/performance evaluation (line 12) there should also be some method of evaluation to determine if reconstituted plasma has similar properties as fresh specimen.
This study is very limited in scope in its current form.

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While this an important step in assessing if lyophilization is a useful way to preserve this type of specimen this study is very limited in its evaluation (only one strain each of three fungal species with no replicates) and does not measure performance against any other method of preservation.
The authors demonstrated the limited number of fungi were still viable after 4 years of storage however it is unclear how this compared (or if there is a benefit)to other storage methods. The authors state this method offers potentially "improved stability" (line 15) but no comparator evaluation or data are provided. If the goal of the study was to identify a way to preserve actual clinical specimens (in this case plasma, as well as the pathogen) for later use in diagnostic assay development/performance evaluation (line 12) there should also be some method of evaluation to determine if reconstituted plasma has similar properties as fresh specimen.
This study is very limited in scope in its current form.

Dear Dr. Babady,
We are grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to address most of the reviewers' comments, which are highlighted in the "Marked Up Manuscript" pdf in blue for those corresponding to the comments of reviewer #1 and red for those of reviewer#2. A point-by-point response to the reviewers' comments and concerns is listed below.

Comments from Reviewer#1:
1. Did the authors prepare the fungi in a special way prior to lyophilization? For instance, did they assure sporulation was happening, or a certain level of sporulation? Even if this was described in cited work, please share again here briefly for completeness.
The details of subculturing the fungi have been briefly described at lines 20-22.

Line 35: were the samples reconstituted first, or was the lyophilized powder used as the inocoulum itself?
We have further elaborated that it was used "directly, without prior reconstitution" at line 26.

The authors demonstrated the limited number of fungi were still viable after 4 years of storage however it is unclear how this compared (or if there is a benefit) to other storage methods.
This study was not intended to be a comparative study nor creating a new storage method, we were just reporting the survival of the fungi in lyophilized human blood plasma in samples prepared in one of our previous studies four years ago, and we were just elaborating the cases where such samples can be encountered/created. For that purpose, we only submitted the manuscript for publication as an observation (and has been modified as a new data letter as requested by the editor) as we have noticed that this has not been reported before. We have modified the statement about the purpose of the study in line 13 to emphasize that we are only reporting this observation.

The authors state this method offers potentially "improved stability" (line 15) but no comparator evaluation or data are provided.
In this sentence (now in line 6 after the amendments), we were just mentioning the advantages of lyophilization that was reported in previous studies (see references 2 and 3) to elaborate why plasma samples can be encountered in lyophilized forms. We have rephrased the statement to make it clearer.
3. If the goal of the study was to identify a way to preserve actual clinical specimens (in this case plasma, as well as the pathogen) for later use in diagnostic assay development/performance evaluation (line 12) there should also be some method of evaluation to determine if reconstituted plasma has similar properties as fresh specimen.
We agree with the reviewer that other aspects should also be considered in diagnostic assay development/performance evaluation, but as mentioned in the first comment, we were just reporting the survival of the fungi in lyophilized human blood plasma in samples prepared in one of our previous studies four years ago, which is one of the aspects that also can have an impact on diagnostic assay development/evaluation. "identifying a way to preserve clinical specimens" is not in the goal of the study, we were just mentioning when such samples can be encountered.

Other Changes:
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