Patients with COVID-19 Interstitial Pneumonia Exhibit Pancreatic Hyperenzymemia and Not Acute Pancreatitis

Raffaele Pezzilli Department of Gastroenterology, San Carlo Hospital Stefano Centanni Respiratory Unit, ASST Santi Paolo e Carlo, San Paolo Hospital, Department of Health Sciences, Università degli Studi di Milano Michele Mondoni Respiratory Unit, ASST Santi Paolo e Carlo, San Paolo Hospital Rocco F. Rinaldo Respiratory Unit, ASST Santi Paolo e Carlo, San Paolo Hospital, Department of Health Sciences, Università degli Studi di Milano Matteo Davì Respiratory Unit, ASST Santi Paolo e Carlo, San Paolo Hospital Rossana Stefanelli Laboratory of Clinical Chemistry, ASST Santi Paolo e Carlo, San Paolo Hospital GianVico Melzi d’Eril Università degli Studi di Milano Alessandra Barassi (  alessandra.barassi@unimi.it ) Laboratory of Clinical Chemistry, ASST Santi Paolo e Carlo, San Paolo Hospital, Department of Health Sciences, Università degli Studi di Milano


Introduction Introduction
Novel coronavirus (SARS-CoV-2)-infected pneumonia  was originally reported to be associated with exposure to the seafood market in Wuhan; it then spread to more than 100 countries and led to tens of thousands of cases within a few months [1]. On March 11 th 2020, the World Health Organization (WHO) o cially declared the outbreak of COVID-19 to be a pandemic [2].
Gastrointestinal manifestations of COVID-19 have been well established [3]; COVID-19 also involves the liver [4]. Acute pancreatic involvement in patients with COVID-19 has recently been reported; acute pancreatitis was de ned on the basis of an elevation of serum pancreatic enzymes [5]. Con rmed cases of acute pancreatitis in COVID-19 patients have been anecdotally reported in two of three family members with the remaining one having only hyperamylasemia [6]. The present study was undertaken to assess the frequency of acute pancreatitis in consecutive patients affected by COVID-19.

Methods
The study was carried out in the Respiratory Unit, ASST Santi Paolo e Carlo, San Paolo Hospital, Milan, Italy and it was approved by local Ethical Committee Milano Area 1 (n. 2020/ST/057) with a priori patient or appropriate proxy consent obtained prior to the participants' entry into the study which was then carried out in accordance with the Helsinki Declaration of the World Medical Association.
Acute pancreatitis was de ned as the presence of prolonged typical pancreatic pain associated with the ndings of pancreatic abnormalities at imaging techniques and a three-fold increase in serum amylase and lipase activity [7].
Our patients did not required orotracheal intubation and sedation as those in Intensive Care Unit that are severely ill [8] because we had previously demonstrated that serum pancreatic enzymes could be elevated in those patients [9].
The inclusion criteria were age equal to or greater than 18 years and novel coronavirus infection con rmed by real-time polymerase chain reaction (PCR) and having been diagnosed as having COVID-19 according to the World Health Organization (WHO) interim guidelines [10] plus chest computed tomography demonstrating lung involvement. Both genders were included.
Serum samples were obtained from all subjects at their initial observation; they were kept frozen at -20°C until analysis.

Patients
From April 1 st 2020 to April 30 th 2020, 110 consecutive patients (69 males, 41 females; mean age 63.0 years, range 24-93 years) met these criteria and were enrolled in the study.
The average time from the onset of respiratory symptoms to the blood samples was 22.2 days (range 0-47).
Novel Coronavirus (SARS-CoV-2) antibodies (Immunoglobulin [Ig] M/IgG) were also evaluated using the commercially available kit BioMedomics IgM-IgG Combined Antibody Rapid Test (Morrisville NC, USA). It is one of the world's rst rapid point-of-care lateral ow immunoassays for the diagnosis of coronavirus. The test has been used widely by the Chinese Center for Disease Control to combat COVID-19 infections and is now being made available globally. This newly developed test kit, the IgG-IgM combined antibody test kit, has a sensitivity of 88.66% and a speci city of 90.63% [11].
All patients were treated according to current therapeutic modalities [12]; regarding ventilation support, seven patients (6.4%) did not receive any ventilator support, 42 (38.2%) received oxygen via nasal cannula, oxygen mask or an oxygen mask with a reservoir, 41 (37.3%) were on a continuous positive airway pressure device (CPAP) and 20 (18.2%) on noninvasive mechanical ventilation (NIMV).
All authors had access to the study data and reviewed and approved the nal manuscript.

Serum assays
Serum pancreatic amylase and serum lipase were assayed using commercially available kits. Pancreatic amylase was assayed using pancreatic isoamylase (Sentinel Ch. S.p.A., Milan, Italy). The linearity of the method was 4.0 to 2000 U/L; within-run coe cient of variation (CV) was 0.3 to 0.7% and total imprecision CV was 3.0 to 5.7%. The upper reference limit (URL) of pancreatic amylase was 53 U/L. Lipase was assayed using VITROS Chemistry Products LIPA Slides (Ortho-Clinical Diagnostics, High Wycombe, United Kingdom). The URL of lipase was 300 U/L. The linearity of the method was 10 to 2000 U/L; within-run CV was 1.1 to 6.1% and total imprecision CV was 1.8 to 12.2%.
Total bilirubin (upper reference value equal to 1.30 mg/dL), direct bilirubin (upper reference value equal to 0.3 mg/dL), alanine aminotrasferase (ALT) (upper reference value equal to 35 U/L), aspartate aminotransferase (AST) (upper reference value equal to 36 U/L), gamma-GT (GGT) (upper reference value equal to 58 U/L) and C-reactive protein (CRP) (upper reference value equal to 10 mg/L) were assayed using commercially available kits (Ortho-Clinical Diagnostics, High Wycombe, United Kingdom).

Statistical analysis
No statistical sample size calculation was carried out a priori, and the sample size was equal to the number of patients treated during the study period. Data were reported as mean values ± standard deviations for the continuous variables and as absolute number and percentage for the categorical variables. The Kolmogorov-Smirnov test was used to evaluate the normal distribution of the blood parameters. Statistical analyses were carried out using the Mann-Whitney U-test, the Spearman rank correlation and the chi squared test The statistical analyses were carried out by running the SPSS/PC+ statistical package (SPSS Inc., ver. 23.0 Chicago, IL, USA) on a personal computer. Two-tailed P values less than 0.05 were considered to be statistically signi cant.

Results
There were no differences in age between male (62.1±15. Regarding the oxygen support, a statistical difference was found in amylase serum activities among the various types of oxygen support used (amylase P = 0.047) whereas this difference was not found for lipase (P = 0.065) ( Table 3).

Discussion
The gastrointestinal manifestation of COVID-19 patients is well known; this study found that 12.7% of patients had diarrhea and 2.7 % had nausea/vomiting; these gures are similar to those reported by Wang et al. [13]. Nausea, vomiting and abdominal discomfort may also appear during the course of the disease [14]. The reason why (SARS-CoV-2)infected pneumonia may involve the gastrointestinal tract is probably due to the fact that the virus has been found more commonly in he saliva [15] but has also been found in the feces in 29% of patients [16]. However, more recently, it has been reported that SARS-CoV-2 infection may also cause acute pancreatic damage [17]. The Authors found, in a retrospective study, that 17% of patients experienced a pancreatic injury. This nding was supported only by the elevation of serum amylase and lipase. In addition, con rmed cases of acute pancreatitis in COVID-19 patients have been anecdotally reported in two of three family members with the other one having only hyperamylasemia [6]. The present study was undertaken for this reason. Acute pancreatitis was de ned according to accepted International criteria [7] and the Authors found that none of their patients had an episode of acute pancreatitis as de ned by the presence of persistent abdominal pain associated with a 5-fold increase in serum pancreatic enzymes, and imaging showing acute alterations of the pancreatic gland. On the contrary, the Authors found that COVID-19 patients could have an increase in serum pancreatic enzymes, such as amylase (24.5% of the cases) and lipase (16.4% of the cases), and only one had a three-fold increase in this enzyme without pain and alteration of the pancreatic gland at imaging. The elevations of the serum pancreatic enzymes were not related to either the presence of diarrhea or nausea/vomiting, or to the IgM and IgG status. Why this happened requires additional studies. similarly to COVID-19, the presence of pancreatic hyperenzymemia in other viral diseases has been reported; the Authors found that, in 78 patients with chronic liver diseases due to hepatitis C virus (HCV) or hepatitis b virus (HBV) infection, the serum amylase levels were abnormally elevated in 27 patients (35%; 22 liver cirrhosis, 5 chronic active hepatitis) whereas the serum lipase levels were elevated in only 16 patients (21%; 15 liver cirrhosis, 1 chronic active hepatitis) [18]; this also happens in mumps and HIV [19,20].
Pancreatic hyperenzymemia in these patients could result from various causes; pancreatic cells highly express angiotensin converting enzyme 2, the transmembrane protein required for SARS-CoV-2 entry [21], and the pancreatic renin-angiotensin system plays important endocrine and exocrine roles in hormone secretion [22] which could be the effect of drugs used for antiviral therapy. Even if no studies regarding the pancreas of COVID-19 patients have been found, a recent pathological report from China found that, even if the damage was located predominantly in the lungs, there were slight alterations in the pancreas, mainly represented by degeneration of some islet cells [23]. The data in the present study regarding liver function tests con rmed those previously reported [24] and the Authors suggest that, in patients with elevated liver function tests who have suspected or known COVID-19, it is necessary to also consider alternative etiologies. In hospitalized patients, it is also useful to obtain these tests at the time of admission and throughout the hospitalization, particularly in the context of COVID-19 drug treatment.
Finally, this study used a new test for evaluating the immunological response to novel coronavirus infection. It was found that this assay, considering only strong positivity for the two classes of immunoglobulins, had a sensitivity of 73.4% for IgM and 87.3% for IgG. These preliminary data are interesting but should be con rmed by studies involving a larger number of patients.
In conclusion, during COVID-19, serum amylase is more frequently elevated than serum lipase, but none of the patients with pancreatic hyperenzymemia showed acute clinical pancreatic injury. The presence of pancreatic hyperenzymemia in a patient with COVID-19 requires the management of these patients be guided by clinical evaluation and not merely by evaluation of the biochemical results.

Declarations
Funding: None Con icts of interest: the authors disclose no con icts Author Contributions: Tables   Table 1. Values of the various biochemical assays in the 110 patients studied. Results are reported as mean, standard deviation (SD) and range.