With the growth of the rehabilitation group, the long-term follow-up and management of COVID-19 patients has become the focus of social attention. Large cohort follow-up studies have shown that numerous recovery patients with COVID-19 still have Long COVID-19 symptoms such as cough, palpitation, insomnia and fatigue. In fact, the oral and intestinal flora of COVID-19 patients are obviously disordered and exist for a long time after nucleic acid turns negative, and Long COVID-19 is also closely related to the flora disorder. However, most relevant studies have been conducted for 3 months or 6 months, and no longer studies have been reported.
We collected oral, fecal, and serum samples of 35 COVID-19 patients at discharge and one year after follow-up for microbiome and metabolomics analysis to explore the alterations of flora and metabolism in the process of disease recovery. The results indicated that butyric acid-producing microbes and bifdobacterium gradually increased, while lipopolysaccharide-producing microbes gradually decreased. Sphingosin-1-phosphate, which is closely associated with the COVID-19 inflammatory factor storm, increased significantly during recovery 1.
Although fungi occupy a relatively small proportion in human microecosystem, they can coordinate and restrict each other with bacteria, regulate host homeostasis and pathophysiological processes, and play an important role in the diversity and function of microflora. There are also many ways of interaction between flora and immunity, which can directly or indirectly participate in host immunity and metabolism through secretion of metabolites. Therefore, based on the previous findings, we further perform detection and analysis of the fungal flora and cytokines in these samples through internal transcribed spacer (ITS) sequencing and Luminex liquid chip technology. Subsequently, the key fungal microorganisms and cytokines in the recovery process were identified based on the random forest model and five-fold cross-validation, and the noninvasive prediction model of neutralizing antibodies was established, which confirmed the strong predictive value of fungal microorganisms in the recovery of COVID-19.
According to the diagnostic criteria and treatment plan for common patients in the fifth and sixth editions of the Guidelines for the Diagnosis and Treatment of Novel Coronavirus Pneumonia issued by the National Health Commission of China, the patients infected with the original strain were strictly screened. In order to make the detection more accurate, fresh tail stool samples and tongue coating samples after rinsing with normal saline were collected from 6 a.m. to 10 a.m., and immediately separated into a -80 ℃ refrigerator, and samples left at room temperature for more than 2 hours were discarded. Single-end sequencing analysis was performed on the Illumina MiSeq platform (Shanghai Mobio Biomedical Technology, China). Subsequently, the original sequencing data were qualitatively filtered, clustered and identified, and the OTUs were classified and annotated after the chimeric sequences were removed. SARS-CoV-2 Surrogate Virus Neutralization Test Kit (Nanjing GenScript Biological Co., Ltd., China) was used to detect neutralizing antibodies against COVID-19. Serum Anti-SARS-CoV-2 IgG and IgM were detected by direct chemiluminescence microparticle technology (YHLO Biotech Co., Ltd., Shenzhen, China), and serum cytokines were detected by Luminex liquid chip (Chengdu Milian Biotechnology Co., Ltd., China).
The results showed that α diversity of fungal microbiome in oral cavity and feces of COVID-19 patients increased to varying degrees during disease recovery, but it did not completely return to normal level after 1 year, and the recovery time of intestinal fungal microorganisms may be longer, which is consistent with the results of previous bacterial studies. A fraction of fungal microbes showed significant changes in abundance compared with healthy controls, but oral and fecal dominant phyla did not change, with total abundances of Ascomycota, Basidiomycota, and Mucoromycota consistently accounting for more than 80% of disease recovery. In addition, we selected patients with complete clinical information to build models to predict the neutralizing antibody level after 1 year, and finally identified 3 oral fungal biomarkers and 18 fecal fungal biomarkers, with the ability to predict the neutralizing antibody level 1 year later up to 80.95% and 96.67%. We also found that most cytokines, such as IL-1b, MIP-3a, IL-5, and IL-10, declined during disease recovery, while IL-8 concentrations gradually increased.
Microecology and immunity are vital and inseparable throughout COVID-19 infection. Hence, we conducted Spearman correlation analysis to further understand the potential role of the microbial-immune axis in disease recovery. The results indicated that neutralizing antibody and IgG were positively correlated with albumin and negatively correlated with monocytes and total bile acid. They were also negatively correlated with OTU3924 (Saccharomyces_cerevisiae) in fecal fungal microorganisms, and OTU13 (Cryptococcus_longus) and OTU30 (Aspergillus_subflavus) in oral fungal microorganisms. In addition, OTU4 (Zanclospora_jonesii), OTU12 (Acrodictys_fluminicola), OTU19 (Candida_solani) and OTU69 (Apiotrichum_domesticum) in fecal fungal microorganisms showed negative correlation with most of the key bacterial microorganisms, demonstrating that the bacteria in the gastrointestinal tract may restrict fungal colonization and invasion.
This study is the first to report alterations of oral and gut fungal microfloras and serum cytokines levels in patients with COVID-19 1 year after discharge. The prediction models of neutralizing antibodies based on oral and gut fungal microbiota achieved excellent prediction effect, and contributed to the long-term research on COVID-19 related microecological alterations. Spearman correlation analysis also indicated the relationship between fungi, bacteria and immunity, suggesting that targeted regulation of flora is of great significance in improving the prognosis of COVID-19, promoting immune recovery, and preventing reinfection. However, this study has a small sample size and is an observational study, so it is necessary to design a large sample experiment to verify the research results. Therefore, we support the disclosure of relevant data, welcome cooperation and exchange, and hope that it can be seen and used by more experts in the field, so as to contribute to the prevention and treatment of COVID-19 and the improvement of prognosis.
1 Cui, G. Y. et al. Characterization of oral and gut microbiome and plasma metabolomics in COVID-19 patients after 1-year follow-up. Military Medical Research 9, 32, doi:10.1186/s40779-022-00387-y (2022).