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Abstract and Figures

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad range of human tissues by using the host receptor angiotensin-converting enzyme 2 (ACE2). Individuals with comorbidities associated with severe COVID-19 display higher levels of ACE2 in the lungs compared to those without comorbidities, and conditions such as cell stress, elevated glucose levels and hypoxia may also increase the expression of ACE2. Here we showed that patients with Barrett's esophagus (BE) have a higher expression of ACE2 in BE tissues compared to normal squamous esophagus, and that the lower pH associated with BE may drive this increase in expression. Human primary monocytes cultured in reduced pH displayed increased ACE2 expression and viral load upon SARS-CoV-2 infection. We also showed in two independent cohorts of COVID-19 patients that previous use of proton pump inhibitors is associated with 2-to 3-fold higher risk of death compared to those not using the drugs. Our work suggests that pH has a great influence on SARS-CoV-2 Infection and COVID-19 severity.
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The influence of pH on SARS-CoV-2 infection and COVID-19 severity
Authors:
Leandro Jimenez1,2, Ana Campos Codo3, Vanderson de Souza Sampaio4,5,6,7, Antonio E.R.
Oliveira1, Lucas Kaoru Kobo Ferreira1, Gustavo Gastão Davanzo3, Lauar de Brito Monteiro3,
João Victor Virgilio-da-Silva3, Mayla Gabriela Silva Borba4, Gabriela Fabiano de Souza3,
Nathalia Zini8, Flora de Andrade Gandolfi8, Stéfanie Primon Murano3, José Luiz Proença-
Modena3, Fernando Almeida Val4,5,7, Gisely Cardoso Melo4,5, Wuelton Marcelo Monteiro4,5,
Maurício Lacerda Nogueira8, Marcus Vinícius Guimarães Lacerda4,5,9, Pedro M. Moraes-
Vieira3,10,11, Helder I Nakaya1,2,*
Affiliations:
1 Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences,
University of São Paulo, São Paulo, Brazil.
2 Scientific Platform Pasteur-University of São Paulo, São Paulo, Brazil.
3 Department of Genetics, Evolution, Microbiology and Immunology, Institute of Biology,
University of Campinas, SP, Brazil.
4 Fundação de Medicina Tropical Dr Heitor Vieira Dourado, Manaus, Brazil
5 Universidade do Estado do Amazonas, Manaus, Brazil
6 Fundação de Vigilância em Saúde do Amazonas, Manaus, Brazil
7 Faculdade de Medicina da Universidade Federal do Amazonas, Manaus, Brazil, UFAM
Amazonas, Manaus, Brazil
8 Faculdade de Medicina de São José do Rio Preto, São Paulo, Brazil
9 Faculdade de Medicina da Universidade Federal do Amazonas, Manaus, Brazil, UFAM
Amazonas, Manaus, Brazil
10 Obesity and Comorbidities Research Center (OCRC), University of Campinas, SP, Brazil.
11 Experimental Medicine Research Cluster (EMRC), University of Campinas, SP, Brazil.
*Correspondence to: hnakaya@usp.br
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Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect a broad
range of human tissues by using the host receptor angiotensin-converting enzyme 2 (ACE2).
Individuals with comorbidities associated with severe COVID-19 display higher levels of
ACE2 in the lungs compared to those without comorbidities, and conditions such as cell
stress, elevated glucose levels and hypoxia may also increase the expression of ACE2. Here
we showed that patients with Barrett’s esophagus (BE) have a higher expression of ACE2 in
BE tissues compared to normal squamous esophagus, and that the lower pH associated with
BE may drive this increase in expression. Human primary monocytes cultured in reduced pH
displayed increased ACE2 expression and viral load upon SARS-CoV-2 infection. We also
showed in two independent cohorts of COVID-19 patients that previous use of proton pump
inhibitors is associated with 2- to 3-fold higher risk of death compared to those not using the
drugs. Our work suggests that pH has a great influence on SARS-CoV-2 Infection and
COVID-19 severity.
Keywords
COVID-19; pH; SARS-CoV-2; proton pump inhibitors; Barrett’s esophagus.
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Introduction
As of August 2020, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-
2) infected over 20 million people worldwide (World Health Organization). The new
coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is characterized by a broad
range of symptoms, from respiratory to neurological and digestive problems (1, 2). Although
a small fraction of patients develop highly lethal pneumonia, at least 20% of COVID-19
patients may display one or more gastrointestinal (GI) symptoms (1), such as diarrhea,
vomiting, and abdominal pain (2, 3).
SARS-CoV-2 tissue tropism can be directly linked to the diverse clinical manifestations
of COVID-19. The receptor used by the virus to enter the cells is the angiotensin-converting
enzyme 2 (ACE2), which is found in several tissues, including the GI epithelial cells and liver
cells (4, 5). SARS-CoV-2 was detected in biopsies of several tissues, including esophagus,
stomach, duodenum and rectum, and endoscopy of hospitalized patients revealed
esophageal bleeding with erosions and ulcers (2, 6).
Higher levels of ACE2 in the tissues may explain in part some of the comorbidities
associated with severe COVID-19. Recently, we showed that ACE2 was highly expressed in
the lungs of people with pulmonary arterial hypertension and chronic obstructive diseases
(7). Since the expression of ACE2 changes under conditions of cell stress, elevated glucose
levels and hypoxia (8, 9), other comorbidities related to the GI tract can be associated with
different forms of COVID-19.
Here we suggest that gastroesophageal reflux disease (GERD) and Barrett’s
esophagus (BE) may represent novel comorbidities associated with COVID-19. In the United
States, it has been estimated that 5.6% of adults have BE, a disease where GERD damages
the esophageal squamous mucosa (10). Here we demonstrated that ACE2 is highly
expressed in the esophagus of patients with BE and that the acid pH associated with this
condition is a key inducer of ACE2 expression. Human primary monocytes cultured in
reduced pH display increased expression of ACE2 and increased viral load upon SARS-CoV-
2 infection. We also show that patients using proton pump inhibitors, which are recommended
for GERD treatment, have a higher risk of developing severe COVID-19, observed by an
increased risk of ICU admittance and death.
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Methods
Acidosis and Barrett’s esophagus meta-analysis
We manually curated the Gene Expression Omnibus (GEO) repository
(https://www.ncbi.nlm.nih.gov/geo/) to find esophagus transcriptome datasets related to
“Barrett’s esophagus” and cell line transcriptome datasets related to “acidosis” and “pH
reduction”. Author-normalized expression values and metadata from these datasets were
downloaded using the GEOquery package (11). We performed differential expression
analyses using the limma package (12). The GEO study ID and the groups of samples
compared are listed in Supplementary Table 1. The MetaVolcanoR package (13) was used
to combine the P values using the Fisher’s method. To adjust for multiple comparisons, we
calculated the false discovery rate (FDR) using the Benjamini-Hochberg procedure. For
enrichment analyses, we utilized the EnrichR tool (14) and fgsea R package (15) with gene
sets from the Gene Ontology Biological Process database. We then selected pathways with
a P value adjusted for multiple comparisons lower than 0.10.
Single cell transcriptomic analysis of Barrett’s esophagus
The single cell RNA-seq (scRNA-seq) data from esophagus, Barrett’s esophagus, gastric and
duodenum cells from patients with BE were acquired from Owen et al. 2018 (16). Cells with
less than 1,000 genes were excluded from analysis using Seurat v3 (17). Raw UMI counts
were log transformed and variable genes called on each dataset independently based on the
VST method. The AddModuleScore function was used to remove batch effects between
samples and based on C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5,
SNRPD3, VCP, VPS29 genes. We assigned scores for S and G2/M cell cycle phases based
on previously defined gene sets using the CellCycleScoring function. Scaled z-scores for
each gene were calculated using the ScaleData function and regressed against the number
of UMIs per cell, mitochondrial RNA content, S phase score, G2/M phase score, and
housekeeping score. Scaled data was used as an input into PCA based on variable genes.
These PCA components were used to generate the UMAP reduction visualization. To identify
the number of clusters, UMI log counts were used as input to SC3 (18). Technical variation
was tested using BEARscc (19), which models technical noise from ERCC spike-in
measurements. The clusters were then annotated based on genes previously characterized
(16).
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Peripheral blood mononuclear cells (PBMC) isolation
Buffy coats provided by the Hematology and Hemotherapy Center of the University of
Campinas (SP-Campinas, Brazil) were used for PBMC isolation as described (9). The study
was approved by the Brazilian Committee for Ethics in Human Studies (CAAE:
31622420.0.0000.5404). Briefly, buffy coats were mixed and then diluted in Phosphate Buffer
Saline (PBS) (1:1) and carefully to 50 mL tube containing Ficoll (Sigma-Aldrich) and
centrifuged. PBMCs were cultured in RPMI 1640 for 2-3h to allow cell adhesion. Next, cells
were washed twice with PBS and adherent cells, enriched in monocytes, were further
incubated until infection in RPMI 1640 containing 10% fetal bovine serum (FBS) and 1%
Penicillin-Streptomycin (Pen-Strep) at 37ºC with 5% CO2. Monocytes were maintained in
different pH levels (6, 6.5, and 7.4) during 24h and subsequently infected with SARS-CoV-2,
as described below.
Viruses and infection
HIAE-02 SARS-CoV-2/SP02/human/2020/BRA (GenBank MT126808.1) virus was isolated
as described (9). Stocks of Sars-CoV-2 were prepared in the Vero cell line. The supernatant
was harvested at 23 dpi. Viral titers were obtained by plaque assays on Vero cells.
Monocytes were infected with SARS-CoV-2 at MOI 0.1 under continuous agitation at 15 rpm
for 1 h. Next, monocytes were washed twice and incubated in RPMI with 10% FBS and 1%
Pen-Strep for 24h at 37°C with 5% CO2 for 24 hours.
Viral load and gene expression analyses
Total RNA extraction was performed using TRIzol Reagent (Sigma-Aldrich). RNA
concentration was measured with NanoDrop 2000 spectrophotometer (Thermo Scientific).
RNA was reverse-transcribed using GoScript™ Reverse Transcriptase cDNA synthesis kit
following manufacturer’s instructions. SARS-CoV-2 viral load was determined with primers
targeting the N1 region and a standard curve was generated as described (20). Viral load
and gene expression were made using SYBR Green Supermix in BIO-RAD CFX394 Touch
Real-Time PCR Detection System. Fold change was calculated as 2^-ΔΔCt. Primer
sequences used: 18S (Forward: 5’-CCCAACTTCTTAGAGGGACAAG-3’; Reverse: 5’-
CATCTAAGGGCATCACAGACC-3’); ACE2 (Forward: 5’-GGACCCAGGAAATGTTCAGA-3’;
Reverse: 5’-GGCTGCAGAAAGTGACATGA-3’); SARS-CoV-2_IBS_N1 (Forward: 5’-
CAATGCTGCAATCGTGCTAC-3’; Reverse: 5’-GTTGCGACTACGTGATGAGG-3’).
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Clinical data analysis
We retrieved clinical data from two independent cohorts of 551 and 806 RT-qPCR
confirmed COVID-19 patients aged 18 years or older that went to reference hospitals for
COVID-19 in Manaus, Amazonas, Brazil (North region cohort) and in São José do Rio Preto
city, São Paulo, Brazil (Southeast region cohort), respectively. They were followed for at least
28 days (North region cohort) or 120 days (Southeast region cohort) after recruitment.
Information about the previous history of proton pump inhibitors use (e.g. omeprazole and
pantoprazole), a surrogate evidence of low gastric pH-related diseases, time of
hospitalization, ICU admittance, and time to death, as well as demographics, previous use of
other drugs, clinical, laboratory, and outcome variables were collected. The protocol was
approved by the Brazilian Committee of Ethics in Human Research (CAAE:
30152620.1.0000.0005 and 30615920.2.0000.0005 for North region cohort, and
31588920.0.0000.5415 for Southeast region cohort). Data were collected and managed
using REDCap (v. 10.2.1) electronic data capture tools hosted at Fundação de Medicina
Tropical Dr. Heitor Vieira Dourado.
Adjusted hazard ratios and risk ratios with respective 95% confidence intervals (CI)
were estimated for time to death and ICU admittance, respectively by Cox regression and
log-binomial generalized linear model models. To adjust for confounders, ages higher than
60 years old and obesity, defined by both BMI and fat percentage, were used as covariables
in the multivariable analyses. Wilcoxon Rank-Sum analysis was used to test differences in
the days of hospitalization. A 2-tailed P< 0.05 was considered significant. The statistical
analyses were carried out using Stata v. 13.0 (StataCorp LP, College Station, TX).
Results
To evaluate whether people with BE may have higher chances of being infected with
SARS-CoV-2 when compared to people without the disease, we performed a meta-analysis
of 8 transcriptomic studies of BE (Figure 1A, Table S1). A total of 304 and 256 genes
displayed, respectively higher and lower expression BE compared to normal esophagus
tissue in at least 7 of these studies (Figure 1B). ACE2 was among the genes consistently up-
regulated in the BE compared to normal esophagus (Figure 1C). While pathways related to
keratinocyte differentiation and epidermis development were enriched with down-regulated
genes, we found that bicarbonate transport and regulation of intracellular pH pathways were
enriched with up-regulated genes (Figure 1D), suggesting that pH may influence ACE2
expression. In fact, when human coronary artery endothelial cells were treated with proton
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pump inhibitors omeprazole or lansoprazole the expression of ACE2 decreased in
comparison to untreated cells (Figure 1E). Gene set enrichment analysis (GSEA) confirmed
that Barrett’s esophagus tissues have higher expression of genes related to pH alterations
(Figure 1F).
Figure 1. Meta-analysis of gastroesophageal junction transcriptomes of patients with
Barrett’s esophagus. A. Meta-analysis of 8 studies of Barrett's esophagus transcriptomes.
B. Number of differentially expressed genes in Barrett's esophagus compared with non-
Barrett’s esophagus. The lines show the number of genes (y-axis) considered up-regulated
(red lines) or down-regulated (blue lines) in Barrett's esophagus (P value < 0.05; log2 fold-
change > 1; combined FDR < 0.01) in one or more datasets (x-axis). The numbers of up-
regulated and down-regulated genes in at least 7 studies are indicated. C. ACE2 is
upregulated in patients with Barrett’s esophagus. Each bar represents the log2 expression
fold-change between patients and control individuals. The error bars indicate the 95%
confidence interval. Bars in red represent a P value < 0.05 and in grey a non-significant P
value. D. Pathway enrichment analysis using the up-regulated and down-regulated genes in
at least 7 studies. The bars represent the combined score (x axis) calculated by Enrichr tool
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for selected Gene Ontology gene sets (y axis). E. ACE2 expression in cells treated with
proton pump inhibitors. Each boxplot represents the log2 expression of untreated (CTRL)
cells and cells treated with either omeprazole (OPZ) or lansoprazole (LPZ). F. Gene Set
Enrichment Analysis (GSEA) of the 8 studies of Barrett's esophagus transcriptomes using
pH-related gene sets. The size and color of the circles are proportional to the normalized
enrichment score (NES) of the gene sets (columns) on each study (rows). The Gene Ontology
IDs are indicated at the top.
We also investigated ACE2 expression in Barrett’s esophagus at single-cell level. Our
analysis showed that single cells from Barrett’s esophagus patients are distinct than normal
esophagus cells, as well as cells from duodenum and gastric tissues (Figure 2A). While a
large fraction of duodenum cells expresses ACE2 (21), only 11% of the single cells from
Barrett’s samples have ACE2 expression above 0 (Figure 2B). However, among the cells
expressing ACE2, higher levels of the gene were found in gastric, Barrett’s, and duodenum
cells when compared to esophagus cells (Figure 2C). Using GSEA, we found that genes
associated with regulation of cellular pH were enriched among the up-regulated genes in
gastric, Barrett’s and duodenum cells when compared to esophagus cells (Figure 2D).
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Figure 2. Single cell transcriptomics of Barrett’s esophagus. A. Dimension reduction of
single cells using Uniform Manifold Approximation and Projection (UMAP). Cells from 4
patients with Barrett’s esophagus (n = 1,168) are shown. The colors represent the tissue
types. B. ACE2 expression by tissue type. The pie charts show the number of single cells
with (black) or without (grey) ACE2 expression (expression values > 0). The fractions of
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ACE2-expressing cells are indicated. C. Distribution of ACE2 expression by cells from
different tissue types. The colors of histograms represent the tissue types. The dashed
vertical line shows the median values of each tissue type. Student’s t-test P-value between
tissue types versus esophagus is indicated. D. Gene Set Enrichment Analysis (GSEA) of the
3 tissue types compared to esophagus using the regulation of cellular pH gene set. The
normalized enrichment score (NES) are shown in the x-axis for each one of the tissue types.
The adjusted P-value of the enrichment is displayed right next to the corresponding bar.
To further evaluate whether pH may influence the expression of ACE2, we analyzed
publicly available transcriptomic studies of cells under experimentally-induced acidosis. Cells
cultured at lower pH displayed higher expression levels of ACE2 when compared to those
cultured under higher pH (Figure 3A and B). We validated this finding with human primary
monocytes cultured at pH 7.4, 6.5 and 6.0 under normoxia. ACE2 expression was
significantly increased at pH 6.5 and 6.0 compared to pH 7.4 (Figure 3C). The reduction of
pH alone also significantly increased SARS-CoV-2 infection of human monocytes (Figure
3D), indicating that pH plays a role in ACE2-mediated SARS-CoV-2 infection.
Figure 3. Acidosis increases ACE2 expression and SARS-CoV-2 infection. A. Human
cells exposed to acidosis. Each boxplot represents the log2 expression of samples untreated
(grey) or treated with lactic acidosis (brown) for two microarray studies (GSE9649 and
GSE70051). Student’s t-test P-values are indicated. B. MCF7 cells exposed to pH reduction
increases ACE2 expression. Grey and brown lines represent, respectively cells treated with
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control media or with 25mM lactic acid for 1, 4, and 12 hours (x-axis). Each point represents
the mean log2 expression and the error bars the standard deviation of biological replicates.
C. Acid pH increases ACE2 expression in monocytes. Human peripheral blood monocytes
were incubated in medium at 3 different pH (6, 6.5, 7.4) for 24h. Each boxplot represents the
fold change ACE2 expression. D. Acid pH increases SARS-CoV-2 viral load. Human
peripheral blood monocytes were incubated in medium at 3 different pH (6, 6.5, 7.4) for 24h.
The cells were infected with CoV-2 (MOI 0.1) for 1h under continuous agitation. The RNA
viral load was measured by qPCR.
Proton pump inhibitors (PPI) decrease the amount of acid produced in the stomach
and are often utilized to treat subjects with GERD symptoms or with certain stomach and
esophagus problems (22). The use of PPIs prior to COVID-19 may serve as a proxy for
identifying subjects with tissue irritation and inflammation caused by stomach acid. In two
independent cohorts of 551 and 806 RT-qPCR confirmed COVID-19 patients from North and
Southeast regions of Brazil, respectively, we investigated the effects of gastrointestinal
discomfort and COVID-19 severity. Survival curve analysis showed that people using PPIs
had a 2- to 3-fold higher risk of death compared to those not using the drug (Figure 4A).
When controlling for potential confounders (i.e. age above 60 years old, diabetes, and
hypertension), the adjusted hazard ratio was 2.183 (95CI: 1.635 - 2.914; P<0.0001) for the
North region cohort and 2.332 (95CI: 1.661 - 3.274; P<0.0001) for the Southeast cohort
(Figure 4B). These clinical findings indicate that the reduction of physiological pH (caused by
stomach acid) may play a significant role in SARS-CoV-2 infection and COVID-19 severity.
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Figure 4. Increase risk of death in individuals with COVID-19 using proton pump
inhibitors prior infection. A. Time to death. Kaplan-Meier survival curves showing a higher
risk of death for the group of patients that used PPIs (brown) prior to admittance when
compared to those not using them (grey). The North region cohort result is shown at the top
and Southeast region cohort result is shown at the bottom. B. Risk of death. The forest plot
presents the hazard ratios and respective 95CI for the main explanatory variable (brown), as
well as the potential confounders (black) used in the multivariate model. The North region
cohort result is shown at the top and Southeast region cohort result is shown at the bottom.
Discussion
Our findings suggest that acid pH increases SARS-CoV-2 infection by up-regulating
the ACE2 receptor, and this may have clinical implications for patients with GERD or Barrett’s
esophagus. No clear mechanism exists linking alterations in pH and ACE2 expression.
Although evidence indicates that hypoxic conditions can increase the expression of ACE2 (8,
9), the expression of neither SIRT1 nor HIF1A seem to be associated with Barrett’s
esophagus (Table S2). We found that known regulators of ACE2 HNF1B (23) and FOXA2
(24) were up-regulated in 6 out of 8 Barrett’s esophagus transcriptomic studies (Table S2),
suggesting that they may be involved with the pH-induced ACE2 expression in Barrett’s
esophagus.
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Pulmonary damage, one of the main features of severe COVID-19, may lead to acute
hypoxia and further respiratory acidosis. It is possible that the acidosis in the blood of some
patients with severe COVID-19 (25) worsen the disease by increasing the levels of ACE2
and facilitating the entry of SARS-CoV-2 into human cells. Hypoxia itself may contribute to
the regulation of ACE2 (9, 26). In addition, elevated levels of the enzyme lactate
dehydrogenase (which converts lactate from pyruvate) has been associated with worse
outcomes in patients with COVID-19 (27). The excess of lactate may directly alter the
extracellular and intracellular pH which in turn can impact ACE2 expression. The extent to
which acute systemic acidosis contributes to COVID-19 severity is poorly known and
deserves further research.
The drug famotidine suppresses gastric acid production by blocking the histamine 2
receptor in the stomach. Recently, Freedberg et al (28) have shown that early treatment of
patients tested positive for SARS-CoV-2 significantly improved clinical outcomes among the
hospitalized patients. Although the authors hypothesized that famotidine may have antiviral
effects, it is possible that pH itself can play an important role in regulating ACE2 expression
and limiting SARS-CoV-2 infection in patients.
We showed here that the previous use of PPIs is associated with unfavorable
outcomes, such as the time of hospitalization, ICU admittance, and death. To the best of our
knowledge, none of these associations were previously reported. Almario et al. (29) recently
described that individuals using PPIs had higher chances for testing positive for COVID-19
when compared to those not using PPIs. Their hypothesis is that PPIs might increase the risk
for COVID-19 by undermining the gastric barrier to SARS-CoV-2 and reducing the microbial
diversity in the gut (29). Rather, we believe that PPIs are important markers of hidden
comorbidities that involve the damage caused by the excess stomach acid in GI tissues.
By going from disease (Barrett’s esophagus) to molecule (ACE2) to cells (in vitro
experiments) and back to clinical findings (COVID-19 patients), we showed that pH may have
a great influence on SARS-CoV-2 infection and COVID-19 severity. Additional studies should
be performed to not only confirm the clinical findings on a larger scale but also to assess the
molecular mechanism related to pH-induced ACE2 expression.
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. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 11, 2020. .https://doi.org/10.1101/2020.09.10.20179135doi: medRxiv preprint
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Funding:
This work was supported by Brazilian National Council for Scientific and Technological
Development (grant number 313662/2017-7); the São Paulo Research Foundation (grant
numbers 2018/14933-2; 2018/219345; 2017/27131-9; 2013/08216-2; 2020/04836-0); and
CAPES.
Author declaration
Authors declare no competing interests.
Author contributions
L.J., A.E.R.O., L.K.K.F., H.I.N. performed the transcriptome analyses. A.C.C., G.G.D.,
L.B.M., J.V.V, G.F.S., S.P.M., J.L.P., P.M.M. performed the experimental work. V.S.S.,
M.G.S.B., N.Z., F.A.G., M.L.N., F.A.V., G.C.M., W.M.M., M.V.G.L. performed the clinical
analysis. H.I.N. coordinated the study. L.J. and H.I.N. wrote the manuscript with inputs from
all of the co-authors.
Supplementary Materials:
Tables S1 and S2
. CC-BY-NC-ND 4.0 International licenseIt is made available under a perpetuity.
is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint The copyright holder for thisthis version posted September 11, 2020. .https://doi.org/10.1101/2020.09.10.20179135doi: medRxiv preprint
... Association between PPI use and the risk of severe disease in patients with COVID-19: The results of 9 studies from 8 original articles (7,9,(16)(17)(18)(19)(20)(21) showed that PPI use was associated with an increased risk of severe disease, including admission to the intensive care unit, intubation, and death, in patients with COVID-19 (OR 1.67, 95% CI = 1.37-2.02, P < 0.00001; I 2 = 67%, P heterogeneity = 0.002) (Fig. 3). ...
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The findings of previous research on the association between proton pump inhibitor (PPI) use and the treatment and prevention of coronavirus disease 2019 (COVID-19) are inconsistent. Therefore, this meta-analysis was conducted to clarify the outcomes of patients taking PPIs. This analysis included 14 articles with more than 268,683 subjects. PPI use was not associated with increased or decreased risk of COVID-19 infection (odds ratio [OR] 1.64, 95% confidence interval [CI] = 0.54–5.00, P = 0.39) or mortality (OR = 1.91, 95% CI = 0.86–4.24, P = 0.11). However, PPI use increased the risks of severe disease (OR 1.67, 95% CI = 1.37–2.02, P < 0.00001) and secondary infection (OR 4.62, 95% CI = 2.55–8.39, P < 0.00001). In summary, PPI use was not associated with an increased risk of infection and mortality in COVID-19 but appeared to be associated with an increased risk of progression to severe disease and secondary infection. However, more original studies are urgently needed to further clarify the relationship between PPI use and COVID-19.
... Briefly, SARS-CoV-2 can induce anaerobic metabolism via the disruption of cell oxygenation and the induction of anaerobic glycolysis (95). As cell pH is controlled by Na + /H + and lactate/H + exchangers and symporters, respectively, high lactate serum levels in SARS-CoV-2 raise the activity of the lactate/H + symporter with subsequent cell acidosis (96). Dapagliflozin inhibits cell Na + /H + exchangers, thus reducing cell acidosis and SARS-CoV-2 activation at acidic pH. ...
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Coronavirus disease 2019 (COVID-19), triggered by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), may lead to extrapulmonary manifestations like diabetes mellitus (DM) and hyperglycemia, both predicting a poor prognosis and an increased risk of death. SARS-CoV-2 infects the pancreas through angiotensin-converting enzyme 2 (ACE2), where it is highly expressed compared to other organs, leading to pancreatic damage with subsequent impairment of insulin secretion and development of hyperglycemia even in non-DM patients. Thus, this review aims to provide an overview of the potential link between COVID-19 and hyperglycemia as a risk factor for DM development in relation to DM pharmacotherapy. For that, a systematic search was done in the database of MEDLINE through Scopus, Web of Science, PubMed, Embase, China National Knowledge Infrastructure (CNKI), China Biology Medicine (CBM), and Wanfang Data. Data obtained underline that SARS-CoV-2 infection in DM patients is more severe and associated with poor clinical outcomes due to preexistence of comorbidities and inflammation disorders. SARS-CoV-2 infection impairs glucose homeostasis and metabolism in DM and non-DM patients due to cytokine storm (CS) development, downregulation of ACE2, and direct injury of pancreatic β-cells. Therefore, the potent anti-inflammatory effect of diabetic pharmacotherapies such as metformin, pioglitazone, sodium-glucose co-transporter-2 inhibitors (SGLT2Is), and dipeptidyl peptidase-4 (DPP4) inhibitors may mitigate COVID-19 severity. In addition, some antidiabetic agents and also insulin may reduce SARS-CoV-2 infectivity and severity through the modulation of the ACE2 receptor expression. The findings presented here illustrate that insulin therapy might seem as more appropriate than other anti-DM pharmacotherapies in the management of COVID-19 patients with DM due to low risk of uncontrolled hyperglycemia and diabetic ketoacidosis (DKA). From these findings, we could not give the final conclusion about the efficacy of diabetic pharmacotherapy in COVID-19; thus, clinical trial and prospective studies are warranted to confirm this finding and concern.
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Background: Previous researches on the association between proton pump inhibitors (PPIs) use and the treatment and prevention of COVID-19 have generated inconsistent findings. Therefore, this Meta-analysis was conducted to clarify the outcome in patients who take PPIs. Methods: We carried out a systematic search to identify potential studies until November 2020. Heterogeneity was assessed using the I-squared statistic. Odds ratios (ORs) with its 95% confidence intervals (CIs) were calculated by fixed-effects or random-effects models according to the heterogeneity. Sensitivity analyses and tests for publication bias were also performed. Results: Eight articles with more than 268,683 subjects were included. PPI use was not associated with increased or decreased risk of COVID-19 infection (OR:3.16, 95%CI = 0.74-13.43, P=0.12) or mortality risk of COVID-19 patients (OR=1.91, 95% CI=0.86-4.24, P=0.11). While it can add risk of severe disease (OR=1.54, 95% CI=1.20-1.99, P<0.001;) and secondary infection (OR=4.33, 95% CI=2.57-7.29). No publication bias was detected. Conclusions: PPI use is not associated with increased risk infection and may not change the mortality risk of COVID-19, but appeared to be associated with increased risk of progression to severe disease and secondary infection. However, more original studies to further clarify the relationship between PPI and COVID-19 are still urgently needed.
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Patients: who died from COVID-19 often had comorbidities, such as hypertension, diabetes, and chronic obstructive lung disease. Although angiotensin-converting enzyme 2 (ACE2) is crucial for SARS-CoV2 to bind and enter host cells, no study has systematically assessed the ACE2 expression in the lungs of patients with these diseases. Here, we analyzed over 700 lung transcriptome samples of patients with comorbidities associated with severe COVID-19 and found that ACE2 was highly expressed in these patients, compared to control individuals. This finding suggests that patients with such comorbidities may have higher chances of developing severe COVID-19. Correlation and network analyses revealed many potential regulators of ACE2 in the human lung, including genes related to histone modifications, such as HAT1, HDAC2, and KDM5B. Our systems biology approach offers a possible explanation for increase of COVID-19 severity in patients with certain comorbidities.
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Background: Since its discovery in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 2 180 000 people worldwide and has caused more than 150 000 deaths as of April 16, 2020. SARS-CoV-2, which is the virus causing coronavirus disease 2019 (COVID-19), uses the angiotensin-converting enzyme 2 (ACE2) as a cell receptor to invade human cells. Thus, ACE2 is the key to understanding the mechanism of SARS-CoV-2 infection. This study is to investigate the ACE2 expression in various human tissues in order to provide insights into the mechanism of SARS-CoV-2 infection. Methods: We compared ACE2 expression levels across 31 normal human tissues between males and females and between younger (ages ≤ 49 years) and older (ages > 49 years) persons using two-sided Student’s t test. We also investigated the correlations between ACE2 expression and immune signatures in various tissues using Pearson’s correlation test. Results: ACE2 expression levels were the highest in the small intestine, testis, kidneys, heart, thyroid, and adipose tissue, and were the lowest in the blood, spleen, bone marrow, brain, blood vessels, and muscle. ACE2 showed medium expression levels in the lungs, colon, liver, bladder, and adrenal gland. ACE2 was not differentially expressed between males and females or between younger and older persons in any tissue. In the skin, digestive system, brain, and blood vessels, ACE2 expression levels were positively associated with immune signatures in both males and females. In the thyroid and lungs, ACE2 expression levels were positively and negatively associated with immune signatures in males and females, respectively, and in the lungs they had a positive and a negative correlation in the older and younger groups, respectively. Conclusions: Our data indicate that SARS-CoV-2 may infect other tissues aside from the lungs and infect persons with different sexes, ages, and races equally. The different host immune responses to SARS-CoV-2 infection may partially explain why males and females, young and old persons infected with this virus have markedly distinct disease severity. This study provides new insights into the role of ACE2 in the SARS-CoV-2 pandemic.
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Introduction: Proton pump inhibitors (PPIs) increase the risk for enteric infections that is likely related to PPI-induced hypochlorhydria. Although the impact of acid suppression on severe acute respiratory syndrome coronavirus 2 is unknown thus far, previous data revealed that pH ≤3 impairs the infectivity of the similar severe acute respiratory syndrome coronavirus 1. Thus, we aimed to determine whether use of PPIs increases the odds for acquiring coronavirus disease 2019 (COVID-19) among community-dwelling Americans. Methods: From May 3 to June 24, 2020, we performed an online survey described to participating adults as a "national health survey." A multivariable logistic regression was performed on reporting a positive COVID-19 test to adjust for a wide range of confounding factors and to calculate adjusted odds ratios (aORs) and 95% confidence intervals (CIs). Results: Of 53,130 participants, 3,386 (6.4%) reported a positive COVID-19 test. In regression analysis, individuals using PPIs up to once daily (aOR 2.15; 95% CI, 1.90-2.44) or twice daily (aOR 3.67; 95% CI, 2.93-4.60) had significantly increased odds for reporting a positive COVID-19 test when compared with those not taking PPIs. Individuals taking histamine-2 receptor antagonists were not at elevated risk. Discussion: We found evidence of an independent, dose-response relationship between the use of antisecretory medications and COVID-19 positivity; individuals taking PPIs twice daily have higher odds for reporting a positive test when compared with those using lower-dose PPIs up to once daily, and those taking the less potent histamine-2 receptor antagonists are not at increased risk. These findings emphasize good clinical practice that PPIs should only be used when indicated at the lowest effective dose, such as the approved once-daily label dosage of over-the-counter and prescription PPIs. Further studies examining the association between PPIs and COVID-19 are needed.
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Coronavirus disease 2019 (COVID-19) infection has now reached a pandemic state, affecting more than a million patients worldwide. Predictors of disease outcomes in these patients need to be urgently assessed to decrease morbidity and societal burden. Lactate dehydrogenase (LDH) has been associated with worse outcomes in patients with viral infections. In this pooled analysis of 9 published studies (n = 1532 COVID-19 patients), we evaluated the association between elevated LDH levels measured at earliest time point in hospitalization and disease outcomes in patients with COVID-19. Elevated LDH levels were associated with a ~6-fold increase in odds of developing severe disease and a ~16-fold increase in odds of mortality in patients with COVID-19. Larger studies are needed to confirm these findings.
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Background & Aims Infection with SARS-CoV-2 causes COVID-19, which has been characterized by fever, respiratory, and gastrointestinal symptoms as well as shedding of virus RNA into feces. We performed a systematic review and meta-analysis of published gastrointestinal symptoms and detection of virus in stool, and also summarized data from a cohort of patients with COVID-19 in Hong Kong. Methods We collected data from the cohort of patients with COVID-19 in Hong Kong (n=59; diagnosis from February 2 through Feb 29, 2020), and searched PubMed, Embase, Cochrane and three Chinese databases through March 11, 2020 according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. We analyzed pooled data on the prevalence of overall and individual gastrointestinal symptoms (anorexia, nausea, vomiting, diarrhea, and abdominal pain or discomfort) using a random effects model. Results Among the 59 patients with COVID-19 in Hong Kong, 15 patients (25.4%) had gastrointestinal symptoms and 9 patients (15.3%) had stool that tested positive for virus RNA. Stool viral RNA was detected in 38.5% and 8.7% among those with and without diarrhea, respectively (P=.02). The median fecal viral load was 5.1 log10cpm in patients with diarrhea vs 3.9 log10cpm in patients without diarrhea (P=.06). In a meta-analysis of 60 studies, comprising 4243 patients, the pooled prevalence of all gastrointestinal symptoms was 17.6% (95% CI, 12.3%–24.5%); 11.8% of patients with non-severe COVID-19 had gastrointestinal symptoms (95% CI, 4.1%–29.1%) and 17.1% of patients with severe COVID-19 had gastrointestinal symptoms (95% CI, 6.9%–36.7%). In the meta-analysis, the pooled prevalence of stool samples that were positive for virus RNA was 48.1% (95% CI, 38.3%–57.9%); of these samples, 70.3% of those collected after loss of virus from respiratory specimens tested positive for the virus (95% CI, 49.6%–85.1%). Conclusions In an analysis of data from the Hong Kong cohort of patients with COVID-19 and a meta-analysis of findings from publications, we found that 17.6% of patients with COVID-19 had gastrointestinal symptoms. Virus RNA was detected in stool samples from 48.1% patients—even in stool collected after respiratory samples tested negative. Healthcare workers should therefore exercise caution in collecting fecal samples or performing endoscopic procedures in patients with COVID-19—even during patient recovery.
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Objective To study the GI symptoms in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. Design We analysed epidemiological, demographic, clinical and laboratory data of 95 cases with SARS-CoV-2 caused coronavirus disease 2019. Real-time reverse transcriptase PCR was used to detect the presence of SARS-CoV-2 in faeces and GI tissues. Results Among the 95 patients, 58 cases exhibited GI symptoms of which 11 (11.6%) occurred on admission and 47 (49.5%) developed during hospitalisation. Diarrhoea (24.2%), anorexia (17.9%) and nausea (17.9%) were the main symptoms with five (5.3%), five (5.3%) and three (3.2%) cases occurred on the illness onset, respectively. A substantial proportion of patients developed diarrhoea during hospitalisation, potentially aggravated by various drugs including antibiotics. Faecal samples of 65 hospitalised patients were tested for the presence of SARS-CoV-2, including 42 with and 23 without GI symptoms, of which 22 (52.4%) and 9 (39.1%) were positive, respectively. Six patients with GI symptoms were subjected to endoscopy, revealing oesophageal bleeding with erosions and ulcers in one severe patient. SARS-CoV-2 RNA was detected in oesophagus, stomach, duodenum and rectum specimens for both two severe patients. In contrast, only duodenum was positive in one of the four non-severe patients. Conclusions GI tract may be a potential transmission route and target organ of SARS-CoV-2.