Diagnostic, Prognostic, and Therapeutic Roles of Gut Microbiota in COVID-19: A Comprehensive Systematic Review

Introduction The Coronavirus Disease 2019 (COVID-19) pandemic caused by Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) emerged in late December 2019. Considering the important role of gut microbiota in maturation, regulation, and induction of the immune system and subsequent inflammatory processes, it seems that evaluating the composition of gut microbiota in COVID-19 patients compared with healthy individuals may have potential value as a diagnostic and/or prognostic biomarker for the disease. Also, therapeutic interventions affecting gut microbial flora may open new horizons in the treatment of COVID-19 patients and accelerating their recovery. Methods A systematic search was conducted for relevant studies published from December 2019 to December 2021 using Pubmed/Medline, Embase, and Scopus. Articles containing the following keywords in titles or abstracts were selected: “SARS-CoV-2” or “COVID-19” or “Coronavirus Disease 19” and “gastrointestinal microbes” or “dysbiosis” or “gut microbiota” or “gut bacteria” or “gut microbes” or “gastrointestinal microbiota”. Results Out of 1,668 studies, 22 articles fulfilled the inclusion criteria and a total of 1,255 confirmed COVID-19 patients were examined. All included studies showed a significant association between COVID-19 and gut microbiota dysbiosis. The most alteration in bacterial composition of COVID-19 patients was depletion in genera Ruminococcus, Alistipes, Eubacterium, Bifidobacterium, Faecalibacterium, Roseburia, Fusicathenibacter, and Blautia and enrichment of Eggerthella, Bacteroides, Actinomyces, Clostridium, Streptococcus, Rothia, and Collinsella. Also, some gut microbiome alterations were associated with COVID-19 severity and poor prognosis including the increment of Bacteroides, Parabacteroides, Clostridium, Bifidobacterium, Ruminococcus, Campylobacter, Rothia, Corynebacterium, Megasphaera, Enterococcus, and Aspergillus spp. and the decrement of Roseburia, Eubacterium, Lachnospira, Faecalibacterium, and the Firmicutes/Bacteroidetes ratio. Conclusion Our study showed a significant change of gut microbiome composition in COVID-19 patients compared with healthy individuals. This great extent of impact has proposed the gut microbiota as a potential diagnostic, prognostic, and therapeutic strategy for COVID-19. There is much evidence about this issue, and it is expected to be increased in near future.


INTRODUCTION
A pandemic caused by Severe Acute Respiratory Coronavirus 2 (SARS-CoV-2) emerged in late December 2019 . The World Health Organization (WHO) named the consequent disease as Coronavirus Disease 2019  and declared it as a global emergency due to the serious public health effects (Jamshidi et al., 2021). According to the report of the WHO, until February 1, 2022, there have been about 376 million confirmed cases and about 5.6 million deaths due to COVID-19 around the world.
The angiotensin-converting enzyme 2 (ACE2) receptor is a known SARS-CoV-2 receptor for entering host cells (Li et al., 2003;Zhou et al., 2020). This receptor is detected in various cells of the body such as the respiratory, digestive, renal, and skin epithelium, suggesting that each of these organs could be a potential target for the virus (Jamshidi et al., 2021;Xue et al., 2021). Moreover, virus RNA and viral particles have been identified in the fecal sample of COVID-19 patients, which may indicate the possibility of virus replication and activity in the human intestine (Gu et al., 2020a;Lamers et al., 2020;Xu et al., 2020).
Gut microbiota plays a well-known role in regulating immune system responses (Donaldson et al., 2016;;Schirmer et al., 2016). Recent studies indicate the role of gut dysbiosis in the pathogenesis of various diseases such as inflammatory bowel disease, type 1 and type 2 diabetes, and celiac disease, as well as chronic respiratory diseases such as asthma, COPD, and cystic fibrosis (Jamshidi et al., 2019;Enaud et al., 2020).
Bacteria in the human intestinal flora appear to affect the respiratory system and lungs (especially the lung microbiota) by producing metabolites, endotoxins, cytokines, and intestinal hormones reaching the bloodstream, which is called the gutlung axis (Budden et al., 2017;Dang and Marsland, 2019;Zhang et al., 2020).
On the other hand, there is evidence of the role of gut dysbiosis in the severity and prognosis of bacterial (e.g., Streptococcus pneumonia, Klebsiella pneumonia, Pseudomonas aeruginosa, Mycobacterium tuberculosis) and viral (e.g., H1N1 influenza) respiratory infectious diseases in animal models (Ichinohe et al., 2011;Fagundes et al., 2012;Fox et al., 2012;Brown et al., 2017). The use of broad-spectrum antibiotics that target the gut microbiota has led to a poor prognosis in mouse models with infectious lung diseases (Enaud et al., 2020).
Considering the important role of gut microbiota in maturation, regulation, and induction of the immune system and subsequent inflammatory processes, it seems that evaluating the composition of gut microbiota in COVID-19 patients compared with healthy individuals may have potential value as a diagnostic and/or prognostic biomarker of the disease. Also, therapeutic interventions affecting gut microbial flora may open new horizons in the treatment of COVID-19 patients and accelerating their recovery.

METHODS
This review conforms to the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) statement (Moher et al., 2009).

Search Strategy and Selection Criteria
To investigate the diagnostic, prognostic, and therapeutic role of the gut microbiota composition in COVID-19, a systematic search was conducted for relevant studies published from December 2019 to December 2021 using Pubmed/Medline, Embase, and Scopus.
Articles containing the following keywords in titles or abstracts were selected: "SARS-CoV-2" or "COVID-19" or "Coronavirus Disease 19" and "gastrointestinal microbes" or "dysbiosis" or "gut microbiota" or "gut bacteria" or "gut microbes" or "gastrointestinal microbiota". Only studies included if they contained data about the gut microbiota composition in COVID-19 patients. There were no language restrictions. Review articles, duplicate publications, letters, commentary, animal studies, and articles with no relevant data were excluded from the analysis. Two authors (MA and FV) independently screened the articles by title and abstract. Full-text screening was conducted by two other authors independently (AT and YF). In each step, contrarieties were discussed with a third reviewer (PJ).

Data Extraction
A data extraction form designed by two authors (PJ and MJN) and, finally, selected data were extracted from the full text of eligible publications by PJ, YF, AT, MA, and FV. The following data were extracted for further analysis: first author's name, year of publication, country where the study was executed, type of study, study population, mean age, gender, COVID-19 severity of the cases, comorbidity(ies), microbiota analysis technique, intestinal microbiota alterations, biochemical and immunological alterations, and studied value of gut microbiota alterations in COVID-19. The data were jointly reconciled, and disagreements were discussed and resolved by review authors (PJ, MJN).

Quality Assessment
The critical appraisal checklist for case reports provided by the Joanna Briggs Institute (JBI) was used to perform a quality assessment of the studies (Institute, 2021). Figure 1, the primary search resulted in 1,668 relevant articles, of which 47 articles were selected after title and abstract screening. Following the full-text screening, 22 articles fulfilled the inclusion criteria. Most of the studies were casecontrol (n = 9) followed by cohort (n = 9), clinical trial (n = 2), and cross-sectional (n = 2) studies. Fifteen of the studies were executed in China, 2 in Italy and 1 in UK, Portugal, India, Egypt, and Korea (Table 1).
Ten studies assessed gut microbiota composition alteration by fecal samples; one study used plasma samples, and it was not mentioned by the rest. The most commonly used techniques in these studies for detection and assessment of gut microbiota were 16s rRNA sequencing and shotgun metagenomic sequencing analysis ( Table 1).
Three articles surveyed the gut mycobiota alterations, and different results have been reported for different species of the same genus. About the Candida spp., an increase in Candida albicans and a decrease in Candida glabrata and Candida parapsilosis were mentioned. In regard to Aspergillus spp., enrichment of Aspergillus flavus and Aspergillus niger and a depletion of Aspergillus rugulosus, Aspergillus tritici, and Aspergillus penicillioides were reported. Also, one study indicated a reduction in seven unclassified species belonging to order Helotiales, Pleosporales, and Sordariales, family Exidiaceae, and genera Microscypha and Emericellopsis in COVID-19 patients ( Table 4).
According to all gut microbiota changes that were mentioned in the reviewed articles, a decrease in phyla Firmicutes and Bacteroidetes and an increase in phylum Actinobacteria among COVID-19 patients were inferred.
The alteration of the gut virome composition in severe COVID-19 cases was mentioned in two studies. In severe cases, fourteen Microviridae phages, one Inoviridae phage, one Podoviridae phage, and one unclassified virus were increased and  Table 4.
According to one study, a positive correlation between Bifidobacterium spp. and prothrombin time (PT) and lactate dehydrogenase (LDH) was shown. Also, a negative correlation was reported between Atopobium spp. and D-dimer, Bacteroides spp. and LDH and creatine kinase (CK) level, Clostridium b u t y r i c u m , a n d C R P a n d n e u t r o p h i l c o u n t , a n d Faecalibacterium prausnitzii and CRP in critical COVID-19 patients. One study showed a specific relation between some genus of gut microbiota and immunological and biochemical modifications in critical and severe COVID-19 patients. In severe patients, Faecalibacterium prausnitzii and Clostridium leptum had a positive correlation with neutrophil count as well as Eubacterium rectale with IL-6 and Enterobacteriaceae with AST.
Another study indicated the specific relation of some species of gut microbiome and immune cells as the following: Bacteroides ovatus, Lachnospiraceae bacterium, and Eubacterium ventriosum had a positive correlation with CD4 and CD8 lymphocytes and other T-cells, in contrast to Bifidobacterium animalis and Escherichia spp. On the other hand, Faecalibacterium prausnitzii had a positive correlation with NK cells and Coprobacillus spp., Clostridium ramosum, and Clostridium symbiosum had a negative correlation with them.

Studied Value of Gut Microbiome in COVID-19
All of the included studies showed a correlation between intestinal microbiota and COVID-19, and they studied the correlation in different aspects as in the following.
Four studies suggested that microbiota could have therapeutic properties with reducing gastrointestinal (GI) symptoms. Streptococcus, Lactobacillus, and Bifidobacterium were the most common bacterial genera interventions used so far. Nine articles demonstrated intestinal microbiota modifications in infected cases with COVID-19 in which two of them confirmed the value of specified gut microbiota as a diagnostic tool and one of them studied gut microbiota changes during recovery time. Lachnospiraceae are a large family including Fusicathenibacter, Eubacterium hallii group, and Roseburia, and the Ruminococcaceae family including Faecalibacterium prausnitzii and Ruminococcus as well as Clostridium spp., Bacteroides spp., Lactobacillus spp., Rothia spp., Actinomyces spp., Lactobacillus spp., and Streptococcus spp. were the most common bacteria with diagnostic value. Thirteen studies demonstrated a relationship between gut microbiota changes and the intensity and prognosis of COVID-19. Eubacterium, Faecalibacterium, Ruminococcus, Bacteroides, Clostridium, Lactobacillus, Bifidobacterium, and Roseburia were the most notable bacterial genera with prognostic values. Details are shown in Table 3.

DISCUSSION
The interaction between gut microbiota and viral respiratory diseases such as COVID-19 is a complex, bilateral, and dynamic association. The current study emphasizes the role of the gutlung axis (GLA) in the pathogenesis of COVID-19. One of the important aspects of GLA is the impact of gut microbiota on the supply and maintenance of the lung immune system, and its correlation with respiratory diseases and infections (He et al., 2017;Dang and Marsland, 2019;Ahmadi Badi et al., 2021;Allali et al., 2021). The gut and lung microbiome are closely related in health or disease conditions (Dickson and Huffnagle, 2015;Enaud et al., 2020;Ahmadi Badi et al., 2021). SARS-CoV-2 may cause a dysbiosis in the lung microbiota to increase the population of inflammatory bacteria such as Klebsiella oxytoca and Rothia mucilaginosa which is associated with acute respiratory distress syndrome (Aktaşand Aslim, 2020; Han et al., 2020;van der Lelie and Taghavi, 2020;Battaglini et al., 2021;Chattopadhyay and Shankar, 2021). The high levels of inflammatory cytokine productions during SARS-CoV-2 invasion interfere with gut mucosal integrity and increasing risk of bacterial disposition to the bloodstream (Chattopadhyay and Shankar, 2021;Prasad et al., 2021).
Based on our findings, gut microbiota in patients with SARS-CoV-2 is significantly affected, possibly due to systemic inflammatory response. Although the underlying mechanism for the observed dysbiosis is unclear, it might happen via downregulation of ACE2 expression that alleviates the intestinal absorption of tryptophan leading to decreased secretion of antimicrobial peptides and changes the composition of gut microbiota . In a healthy individual, intact bacteria and their fragments or metabolites such as des-aminotyrosine and short-chain fatty acids (SCFAs) pass across the intestinal barrier via the mesenteric lymphatic system, reach the lung, and activate the innate immune system by the production of cytokines like type 1 interferon (IFN1) (Antunes et al., 2019). In the current study, Faecalibacterium prausnitzii and Clostridium leptum have a positive correlation with neutrophil counts in  COVID-19 patients and a negative correlation with Clostridium butyricum. In addition to the innate immunity, gut microbiota improves the function of CD8+ T-cell effectors (Trompette et al., 2018). There is also evidence that few bacterial species such as Bacteroides ovatus, Lachnospiraceae bacterium 5_1_63FAA, and Eubacterium ventriosum have an anti-inflammatory property in CD4 + and CD8 + T cells (Cao et al., 2021). Natural killer (NK) cells and B cells are also affected by gut microbiota; Coprobacillus spp., Clostridium ramosum, and Clostridium symbiosum are negatively associated with NK cell activity and Bacteroides uniformis, Faecalibacterium prausnitzii, and Subdoligranulum are positively correlated with B cells (Cao et al., 2021). We found that the dysbiosis of gut microbiota may be a determinant factor in the clinical severity of COVID-19. Increase of the dominant Enterococcus and reduction of Ruminococcaceae and Lachnospiraceae are reported in severe cases of COVID-19 who were admitted to the medical intensive unit (MICU) (Gaibani et al., 2021).
Diversity of gut microbiota reduces in patients with COVID-19, which is associated with pro-inflammatory reaction and increased risk of opportunistic infections. GI symptoms of COVID-19 are reported to be strongly affected by gut microbial composition (Din et al., 2021;Katz-Agranov and Zandman-Goddard, 2021). The expression of the ACE-2 receptor on the surface of small intestine epithelial cells has been significantly associated with the extent of GI symptoms and fecal viral shedding during the course of disease (D'Amico et al., 2020;Patel et al., 2020;Wu et al., 2020;Katz-Agranov and Zandman-Goddard, 2021;Suaŕez-Fariñas et al., 2021). An increased expression of ACE-2 receptor in COVID-19 may occur due to a dominance of Coprobacillus in gut microbiota (Geva-Zatorsky et al., 2017;Enaud et al., 2020;Zuo et al., 2020a;Massip Copiz, 2021;Rajput et al., 2021;Walton et al., 2021). It is also noteworthy to imply that the expression of ACE-2 on luminal cells may be a determinant factor in microbial composition as the ACE-2 receptor plays some role in amino-acid absorption (Harmer et al., 2002;Dang and Marsland, 2019).
Eggerthella is another genus of bacteria which significantly increases in patients with COVID-19 (d'Ettorre et al., 2020;Cao et al., 2021). Eggerthella may induce colitis via abnormal activation of Th17 in patients with inflammatory diseases. It can interfere with gut integrity and make the patient more susceptible to pathogen invasion including SARS-CoV-2 (Alexander et al., 2019;Katz-Agranov and Zandman-Goddard, 2021).
There is a dominancy of genus Clostridium in patients with COVID-19 (Gu et al., 2020b;Zuo et al., 2020a;Cao et al., 2021;Yamamoto et al., 2021). The increase in Clostridium ramosum and Clostridium hathewayi is associated with the disease severity which may be a risk factor of acute portal vein thrombosis (Zuo et al., 2020;Rokkam et al., 2021). Clostridium difficile may complicate COVID-19 and worsen the GI symptoms (Ferreira et al., 2020;Oba et al., 2020;Sandhu et al., 2020;Khanna and Kraft, 2021). Two butyrate-producing members of this genus, Clostridium butyricum and Clostridium leptum, are decreased in patients with COVID-19 . Bacteroides spp. was decreased in all groups, but the decrease was within the lower normal limits. There was no significant difference between the groups. c Study1: comparison between COVID-19 patients and healthy controls, Study2: comparison between COVID-19 patients and non-COVID-19 ICU admitted control.
The genus Collinsella is an opportunistic pathogenic genus which is widely found in the gut of patients with COVID-19 especially in severe cases and higher infectivity status (Chhibber-Goel et al., 2021;Liu et al., 2021;Massip Copiz, 2021;Rajput et al., 2021;Zuo et al., 2021a). Collinsella aerofaciens abundance altered with the gut mucosal integrity and production of inflammatory mediators such as IL-17, CXCL1, and CXCL5 from the luminal cells (Kalinkovich and Livshits, 2019).
Genus Roseburia is another commensal gut microbiota which decreases in patients with COVID-19 and other viral diseases such as influenza Gu et al., 2020b;Cao et al., 2021;Lv et al., 2021a). SCFAs produced by Roseburia maintain mucosal integrity in healthy adults via modulation of inflammatory mediators especially IL-10 (Koh et al., 2016;Zheng et al., 2017;Haak et al., 2018;Gautier et al., 2021). It has been shown previously that butyrate may preserve lung integrity from cytokine-induced injuries in influenza (Chakraborty et al., 2017;Dang and Marsland, 2019). If we assume that it is true in COVID-19, lower levels of Roseburia result in lower levels of butyrate and consequently more extensive lung injuries due to inflammatory processes.
Other members of the gut microbiota are viruses and fungi. Although pathogenic gut viruses are known for more than a century, the term "gut virome" is recently introduced (Reyes et al., 2012). Most of the gut virome consists of bacteriophages that can explain the fact that the virome structure is related to gut bacterial composition in both healthy and COVID-19 people (Minot et al., 2011;Cao et al., 2021). There is a bidirectional relationship between gut virome and infectious diseases; bacteriophages have a significant role in protecting against bacterial infections (Wilks and Golovkina, 2012). Gut virome composition might be affected during COVID-19 (Cao et al., 2021). There are limited data on the alterations of commensal viral and fungal populations in the gut during COVID-19 infection.
Lv et al. showed a strong correlation between altered fungal gut microbiome and inflammatory blood biomarkers (Lv et al., 2021b). Further studies focusing on viral and fungal alterations during the COVID-19 are desired.
The gut microbiota alteration in COVID-19 patients should be considered as a dynamic process (d' Ettorre et al., 2020;Liu et al., 2021;Hussain et al., 2021;Zuo et al., 2020a). To the date of revising this manuscript (January 2022), several registered clinical trials are in progress and the results are not provided yet; however, growing evidence supports the effectiveness of microbiota modulatory actions on fastening the recovery of patients with COVID-19 (Chen and Vitetta, 2021;Hussain et al., 2021;Wang et al., 2021a).

Limitations and Suggestions
A few studies have documented the comorbidities of subjects. However, almost all the studies have missed the impact of comorbidities on gut microbiota alterations in COVID-19 patients compared with the healthy control. It has been shown that gut microbiota may change in patients with hypertension, cardiovascular diseases, diabetes mellitus, hyperlipidemia, and thrombotic events (Huynh, 2020;Avery et al., 2021;Kyriakidou et al., 2021;Mineshita et al., 2021). We strongly propose to investigate the effects of underlying comorbidities in gut microbial composition in patients with COVID-19.
Due to the variations in data analysis techniques such as 16S rRNA sequencing, qPCR, and metagenome sequencing, there was a challenge to compare the bacterial taxa across studies. Since there was a fair diversity in geographical distribution of the current studies (most of them are from China), we cannot ignore the effect of diet and genetic predisposing factors like HLA in gut microbiome compositions. Future studies in different countries are required in this regard.
It is important to mention that different levels of p-value significance were reported in reviewed articles; however, in this study we used only statistically significant findings from the included studies.
Further studies with a larger study population, including the range of patients from mild to severe symptoms, involving the patients who are managed out patiently, focusing on the effectiveness of gut microbiota-targeted therapies for prevention and improvement of COVID-19 patients' symptoms are desired to light up this topic.

CONCLUSION
Our study showed a significant alteration of gut microbiome composition in patients with COVID-19 compared to healthy individuals. This great extent of impact has proposed the gut microbiota as a potential diagnostic, prognostic, and potentially therapeutic strategy for COVID-19.

AUTHOR CONTRIBUTIONS
MN and PJ designed the study. MN, YF, AT, PJ, MA, and FV performed the search and data extraction and wrote the first draft of the manuscript. LS, AHSB, and MM reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
This study was supported by Shahid Beheshti University of Medical Sciences, Tehran, Iran.