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The study by Lee et al1 showed that the short-term current use of proton pump inhibitors (PPIs) for less than 1 month was associated with severe clinical outcomes for patients with COVID-19. The authors speculated that individuals taking PPIs had increased gastric pH, leading to higher SARS-CoV-2 viral loads associated with a severe course of COVID-19. Many studies suggested that a proportion of patients with COVID-19 experiencing GI symptoms such as diarrhoea, nausea and vomiting had overall more severe disease.2 However, it is not clearly understood how SARS-CoV-2 could survive the passage through the harsh gastric acidity and persist through the intestinal contents to infect the intestinal epithelia. It is known that the gastric pH varies greatly, depending on whether the individual is in a fasting or feeding state (between 1.23 and 6.7, respectively).3 Similarly, bile concentrations in the small intestine can fluctuate from as low as 2.6 mM in fasted state to over 15 mM in the fed state.4 Whether changes in gastric pH and bile allow the virus to escape gastric and intestinal inactivation to infect the intestine is not well understood. Therefore, to understand the effect of stomach acidity, digestive components and meals on the infectivity of swallowed SARS-CoV-2, the virus ~6 log 50% tissue infective dose (TCID50)/mL was incubated at 37°C for 60 min in simulated gastric fluid of different pH (1.5–6.0), pepsin (0–8 mg/mL), pancreatin (0–5 mg/mL) or bile (0–15 mM) solutions and for up to 120 min in simulated biorelevant gastric and intestinal fluids supplemented with digestive enzymes that represent either fasting or feeding states (see online supplemental materials for details). SARS-CoV-2 was highly inactivated by gastric pH of ≤2.5 (~5.8 log reduction), showed a~3.2 log reduction at pH 3 and was less affected by pH between 3.5 and 6.0 (~1 log reduction) (figure 1A). Pepsin had no significant effect on SARS-CoV-2 infectivity (figure 1B). The virus infectivity was significantly reduced by pancreatin in a dose-dependent manner and by bile in an inversely proportional manner (figure 1C,D). Furthermore, under fasting, gastric followed by intestinal fluids highly reduced SARS-CoV-2 infectivity by ~4 log TCID50/mL within 10 min (figure 2B). In contrast, under feeding state, there was only ~1 log TCID50/mL reduction in SARS-CoV-2 infectivity (figure 2D). To our knowledge, this is the first report that examines the effect of individual digestive enzymes on SARS-CoV-2 infectivity and shows that infectious SARS-CoV-2 can escape the stomach and intestinal inactivation during feeding. During meal consumption, the pH of the stomach rises to ~6, and the meal’s effect on gastric pH may still be apparent for over 3 hours, after which the pH decreases to ~2.5.3 The feeding gastric fluid used in our study represented the acidity (pH 3) of the stomach fluids when the stomach is 75% empty within 3–6 hours of consuming a high-fat Food and Drug Administration (FDA) FDA meal, suggesting that at a higher pH during early hours of digestion, there would be even lesser inactivation effect on SARS-CoV-2. The PPIs are known to raise the pH of the stomach, allowing microbes to escape the gastric pH barrier, which leads to increased acute gastroenteritis and community-acquired pneumonia.5 6 In fact, Middle East respiratory syndrome coronavirus(MERS-CoV) injected intragastrically in mice showed worse outcomes when the mice were pretreated with antiacid drugs.7 Previous studies that assessed the effect of pH on SARS-CoV-2 infectivity are unreliable as they did not simulate the temperature or physiological fluids of the stomach as well as studies using biorelevant fluids because the authors did not supplement the fluids with digestive enzymes nor did they test the consecutive effect of the fluids8 9 (see online supplemental material). The inverse proportional effect of bile on SARS-CoV-2 might be explained by the fact that bile salts form primary micelles at lower concentrations (representing fasting state) which can be better at solubilising the lipid bilayer in the SARS-CoV-2’s envelope than stabilised micelles formed at higher bile concentrations (representing fed state).10 Taken together, higher stomach pH and higher bile concentrations allow ingested SARS-CoV-2 to escape the GI inactivation, which would give the virus a higher chance to infect the intestine, supporting Lee et al’s speculations. Further studies using dynamic in vitro digestion models, animal models and human biopsies from patients with COVID-19 are needed to understand the various factors affecting the infectivity of SARS-CoV-2 as it passes through the GI tract.
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The author thanks Amy Mann for propagating Vero E6 cells and preparing tissue culture plates, Revati Narwankar for making various buffers and determining the volume of NaOH needed for neutralisation, Dr Issmat Kassem for editing the manuscript and Robert Hogan for providing the SARS-CoV-2 and infectivity assay protocol.
Contributors MAE designed and conducted all the experiments, analysed the data and wrote the manuscript.
Funding Faculty startup provided by the University of Georgia (17ESSTART).
Disclaimer The views expressed in the submitted article are the author’s own and not an official position of the institution or funder.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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