Alterations in Fecal Fungal Microbiome of Patients With COVID-19 During Time of Hospitalization until Discharge

Tao Zuo, Hui Zhan, Fen Zhang, Qin Liu, Eugene Y K Tso, Grace C Y Lui, Nan Chen, Amy Li, Wenqi Lu, Francis K L Chan, Paul K S Chan, Siew C Ng, Tao Zuo, Hui Zhan, Fen Zhang, Qin Liu, Eugene Y K Tso, Grace C Y Lui, Nan Chen, Amy Li, Wenqi Lu, Francis K L Chan, Paul K S Chan, Siew C Ng

Abstract

Background & aims: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects intestinal cells, and might affect the intestinal microbiota. We investigated changes in the fecal fungal microbiomes (mycobiome) of patients with SARS-CoV-2 infection during hospitalization and on recovery.

Methods: We performed deep shotgun metagenomic sequencing analysis of fecal samples from 30 patients with coronavirus disease 2019 (COVID-19) in Hong Kong, from February 5 through May 12, 2020. Fecal samples were collected 2 to 3 times per week from time of hospitalization until discharge. We compared fecal mycobiome compositions of patients with COVID-19 with those from 9 subjects with community-acquired pneumonia and 30 healthy individuals (controls). We assessed fecal mycobiome profiles throughout time of hospitalization until clearance of SARS-CoV-2 from nasopharyngeal samples.

Results: Patients with COVID-19 had significant alterations in their fecal mycobiomes compared with controls, characterized by enrichment of Candia albicans and a highly heterogeneous mycobiome configuration, at time of hospitalization. Although fecal mycobiomes of 22 patients with COVID-19 did not differ significantly from those of controls during times of hospitalization, 8 of 30 patients with COVID-19 had continued significant differences in fecal mycobiome composition, through the last sample collected. The diversity of the fecal mycobiome of the last sample collected from patients with COVID-19 was 2.5-fold higher than that of controls (P < .05). Samples collected at all timepoints from patients with COVID-19 had increased proportions of opportunistic fungal pathogens, Candida albicans, Candida auris, and Aspergillus flavus compared with controls. Two respiratory-associated fungal pathogens, A. flavus and Aspergillus niger, were detected in fecal samples from a subset of patients with COVID-19, even after clearance of SARS-CoV-2 from nasopharyngeal samples and resolution of respiratory symptoms.

Conclusions: In a pilot study, we found heterogeneous configurations of the fecal mycobiome, with enrichment of fungal pathogens from the genera Candida and Aspergillus, during hospitalization of 30 patients with COVID-19 compared with controls. Unstable gut mycobiomes and prolonged dysbiosis persisted in a subset of patients with COVID-19 up to 12 days after nasopharyngeal clearance of SARS-CoV-2. Studies are needed to determine whether alterations in intestinal fungi contribute to or result from SARS-CoV-2 infection, and the effects of these changes in disease progression.

Keywords: Coronovirus; Intestine; Microbe; Yeast.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
Schematic diagram of stool specimen collection and duration of hospitalization in patients with COVID-19 (n = 30). “CoV” denotes patient with COVID-19. Stool specimens were serially collected for shotgun metagenomics sequencing. “D0” denotes baseline date when the first stool was collected after hospitalization; the following time points starting with “D” represent days since baseline stool collection. “-ve nasopharyngeal/throat swab”: the first negative result for SARS-CoV-2 virus in 2 consecutive negative nasopharyngeal/throat/pooled swab tests, on which patient was then discharged.
Figure 2
Figure 2
Gut mycobiome (fungal community) alterations in patients with COVID-19. (A) Fecal mycobiome alterations in COVID-19, viewed by NMDS (nonmetric multidimensional scaling) plot based on Bray-Curtis dissimilarities. The fecal mycobiome was compared among healthy controls (n = 30), COVID-19 (n = 30), and pneumonia patient controls (n = 9). (B) Interindividual dissimilarities between fecal mycobiomes within each group. The mycobiome dissimilarity was calculated as Bray-Curtis dissimilarity. Between-group comparison was conducted by t test. (C) The relative abundance of Candida albicans in the fecal mycobiome. Between-group comparison was conducted by Wilcoxon rank sum test.
Figure 3
Figure 3
Gut mycobiome (fungal community) alterations in patients with COVID-19 and longitudinal changes during time of hospitalization. Temporal compositional changes in fecal mycobiome with respect to each COVID-19 case were viewed by NMDS (nonmetric multidimensional scaling) plot based upon Bray-Curtis dissimilarities. The aqua cluster denotes the fecal mycobiome cluster of healthy controls. “CoV” denotes patient with COVID-19. “Day0” denotes baseline date when the first stool was collected after hospitalization; the following time points starting with “Day” represents days since baseline stool collection.
Figure 4
Figure 4
Bloom of gut fungi in patients with COVID-19 during time of hospitalization. (A) The diversity of fecal mycobiome (fungi) in patients with COVID-19 over time of hospitalization (plotted as the baseline time point and the last follow-up time point after hospitalization), compared with healthy controls (n = 30) and pneumonia patient controls (n = 9). Between-group comparison was conducted by t test, last follow-up versus baseline comparison for the hospitalized patients with COVID-19 were conducted by paired t test. (B) The richness of fecal mycobiome (fungi) in patients with COVID-19 over time of hospitalization (plotted as the baseline time point and the last follow-up time point after hospitalization), compared with healthy controls (n = 30) and pneumonia patient controls (n = 9). Between-group comparison was conducted by t test, last follow-up versus baseline comparison for the hospitalized patients with COVID-19 were conducted by paired t test. (C) Overrepresented fungal species in feces in patients with COVID-19 during time of hospitalization, compared with healthy controls. LefSE analysis was conducted to identify differential species, only species with LDA effect size > 2 and false discovery rate P < .1 were plotted.
Figure 5
Figure 5
The presence of Aspergillus flavus (A) and Aspergillus niger (B) in patients with COVID-19 over time of hospitalization. “CoV” denotes patient with COVID-19. “Controls” denotes healthy controls (n = 30). “Pneumonia” denotes patient controls with pneumonia (n = 9). “Day0” denotes baseline date when the first stool was collected after hospitalization; the following time points starting with “Day” represent days since baseline stool collection. “throat swab -ve” indicates the first negative result for SARS-CoV-2 virus in 2 consecutive negative nasopharyngeal/throat/pooled swab tests, on which patient was then discharged.
Supplementary Figure 1
Supplementary Figure 1
Temporal changes in the diversity of gut mycobiome (fungal) in COVID-19 patients over time of hospitalization.
Supplementary Figure 2
Supplementary Figure 2
Temporal changes in the relative abundance of Candida albicans in the gut mycobiome of COVID-19 patients over time of hospitalization. (A) Longitudinal changes of C. albicans in each COVID-19 case. (B) Alteration in the relative abundance of C. albicans compared between baseline and the last follow-up; statistical comparison was conducted by paired Wilcoxon rank sum test. (C) The relative abundance of C. albicans across all time points in all patients with COVID-19 during hospitalization, as compared with healthy controls. Statistical comparison was conducted by paired Wilcoxon rank sum test.
Supplementary Figure 3
Supplementary Figure 3
Temporal changes in the relative abundance of Candida auris in the gut mycobiome of COVID-19 patients over time of hospitalization. (A) Longitudinal changes of C. auris in each COVID-19 case. (B) Alteration in the relative abundance of C. auris compared between baseline and the last follow-up; statistical comparison was conducted by paired Wilcoxon rank sum test. (C) The relative abundance of C. auris across all time points in all patients with COVID-19 during hospitalization, as compared with healthy controls. Statistical comparison was conducted by paired Wilcoxon rank sum test.

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