A total of 25 patients diagnosed as COVID-19 and 13 suspected cases were enrolled between Jan 23 and March 16, 2020. The mean age of COVID-19 group was 45.52 ± 16.99 (11 females) with comparison to 35.69 ± 10.89 (6 females) in suspect COVID-19 group (P=0.0433). More than half of the COVID-19 patients (14, 56%) came from or had been to Wuhan recently (first generation). COVID-19 patients were classified into 4 groups based on the severity of the disease in strict accordance with the Diagnosis and Treatment Guidelines for 2019 Novel Coronavirus Pneumonia (Trial Version 7): 4 (16%) mild, 17 (68%) moderate, 3 (12%) severe and 1 (4%) critical. One patient (80-year-old, female, 4%) died due to respiratory failure at the 6th day after admission. A majority of the COVID-19 patients (19, 76%) had underlying diseases: 7 (28%) hypertension, 2 (8%) cardiovascular disease, 2 (8%) diabetes, 2 (8%) chronic obstructive pulmonary disease (COPD), 5 (20%) chronic liver disease and 1 (4%) hepatitis B virus (HBV) carrier. Diverse clinical presentations could be observed among COVID-19 patients at onset and on admission, albeit respiratory system symptom being the most widely. During hospitalization, digestive (7, 28%), neuromuscular (6, 24%) and cardiovascular (4, 16%) symptoms have also emerged and occupied a certain proportion. These data and other basic characteristics were presented in Supplemental Table 1.
Patients were divided into 4 groups according to symptom: 3 (12%) in digestive system symptom group, 14 (56%) in respiratory system symptom group, 4 (16%) in combined group and 4 (16%) in asymptomatic group. Baseline and clinical characteristics were summarized respectively. Digestive system symptom included diarrhea, nausea and vomiting. All 4 patients in combined group reported respiratory system symptom first, and then digestive system symptom emerged within 48 hours prior to admission. For each patient, their symptom persisted on admission. Nearly all baseline characteristics did not differ in the 4 groups, neither did clinical characteristics except for length of stay (IOS), where the digestive system symptom group reported the longest (Supplemental Table 2).
LABORATORY EXAMINATIONS AND CT SCANS
Clinical examinations on admission to hospital (Table 1 and Figure 1) were analyzed. For blood routine examination, lymphocyte (LYM) count (x109/L, P=0.0101) and percentage (%, P=0.0033) typically remained near the normal range in digestive system symptom group, in comparison with significant reductions in other groups, which indicated unaffected systemic immunity in this group. Not surprisingly, digestive system symptom group suffered the most from alimentary injury, as 3 liver function indexes (alanine aminotransferase [ALT, U/L, P＜0.0001], alkaline phosphatase [ALP, U/L, P=0.0167] and γ-Glutamyl transpeptidase [GGT, U/L, P=0.0018]) increased significantly. Two infection related biomarkers, erythrocyte sedimentation rate (ESR, mm/h, P＜0.0001) and serum ferritin (ng/ml, P＜0.0001) had the highest level in this group. ESR generally points to bacterial infection. We believed that intestinal flora being interrupted by SARS-COV-2 could not be excluded, as could be observed in other reports(22, 23). Considering the prevalent liver damage in digestive system symptom group, elevation in serum ferritin could also be interpreted as affected liver function. Other inflammatory factors were not notably high in this group.
Interleukin-6 (IL-6, pg/ml, P=0.0046) and C-reaction protein (CRP, mg/L, P=0.0004) were remarkably elevated in combined group, implying a different infective pattern in combined group from other groups. We emphasized on subsets of LYM cell count and percentage, and reported that CD3+ cell count (106/L, P=0.0011), CD3+ cell percentage (%, P=0.0014) and CD8+ cell count (106/L, P=0.0021) were in the normal range in digestive system symptom group, as well as a steady CD8+ cell count (%, P=0.0013) in combined group, with contrast to significant reductions in respiratory system symptom group. CD4+/CD8+ did not change much in digestive system symptom group compared to other groups (P=0.0206). Nearly all laboratory examinations were similar in asymptomatic group to those in respiratory system symptom group, including immune indices (LYM and its subsets), with a few indicators being less severe in asymptomatic group. All these data did not support obvious immune dysregulation in digestive system symptom group, while it seemed the most depressed in the respiratory system symptom and asymptomatic group, and moderately disturbed in combined group. What needs to be noted, however, is that the 4 patients in the combined group could first be categorized as the respiratory system symptom group. Then, digestive system symptom emerged within 48 hours prior to admission and they were grouped into combined symptom. The subsequent digestive system symptom in combined group, which signaled an emerging local inflammation(14), as evidenced by elevated IL-6 and CRP in our study, probably limited immunodepression caused by pneumonia.
Some additional manifestations could be noticed. More frequent proteinuria (P=0.0003) could be observed in digestive system symptom group, being mildly positive. Glucose (mmol/L, P=0.0152) did not seem to be affected in digestive system symptom group, as it only remained in the normal range in digestive system symptom and asymptomatic group. Troponin T (ng/L, P=0.0015) differed but remained normal among all groups (Supplemental Figure 1).
High-resolution computed tomography (HRCT) of the patients were read by the radiologists who recorded CT findings for each pulmonary lobe and then gave the total CT scores. Scores were not significantly different among the 4 groups. All patients showed signs of pneumonia on CT scans, although only respiratory system symptom and combined groups reported respiratory system symptom. Different evaluation scores were used for severity of illness, and no difference was found among MuLBSTA, CURB-65, PSI grading and PII (Table 1).
In order to explore the reason why alimentary system was affected in COVID-19, especially in view of immune response being hardly dysregulated in digestive system symptom group than that in other groups, we attempted to investigate whether this phenomenon could be a result of direct infiltration of SARS-COV-2 through a combination of bioinformatic and clinical analysis. As is known that SARS-COV-2 could be detected in various tissues, we obtained bioinformatic data from the “TISSUE” units of “THE HUMAN PROTEIN ATLAS” (http://www.proteinatlas.org/) to analyze mRNA and protein expression profiles in various human tissues(20). Figure 2 summarized tissue-specific expressions of binding and endocytosis proteins of SARS-COV-2. The main binding protein of SARS-COV-2, ACE2, displayed high expressions of protein in kidney, small intestine and testis, and mild expression in adrenal gland and colon. It’s worth noting that the mRNA expression in small intestine was also the highest. Another protein, CD147, which was found to be a binding protein of SARS-COV-2 recently, showed ≥ moderate expressions in the digestive tract (colon, stomach and esophagus and small intestine).
Several proteins assist the binding and endocytic process of SARS-COV-2. Moderate expression of TMPRSS2 existed in pancreas, salivary gland and small intestine. One of endocytosis proteins of SARS-COV-2, Cathepsin L (CTSL), exhibited moderate expression in liver, and mild expressions in colon, pancreas and salivary gland. We also found 3 endocytosis proteins (PIKfyve, TPC2 and CTSB) and 1 binding-related protein (Furin) that were widely expressed in different tissues (Figure 3). The results revealed ACE2, TMPRSS2 and Furin were co-expressed in small intestine, which strongly suggested small intestine being a major target of SARS-COV-2. Other binding and endocytosis proteins were widely expressed in alimentary system, small intestine included.
We believed that digestive system symptoms were more caused by direct infiltration of SARS-COV-2 into alimentary tract than subject to hyperinflammation or immune dysregulation brought by pneumonia based on findings above: (a) All necessary virus entry and endocytosis proteins could be found in alimentary system through bioinformatic analysis; (b) Re-elevation of IL-6 and CRP in combined group might signal an acute “reinfection” in alimentary tract by the virus; (c) Appearance of digestive system symptom seemed to combat immune dysregulation, rather than be caused by it.
CORRELATION BETWEEN SYMPTOM AND VIRUS LOCATION
We continued to determine whether the location of SARS-COV-2 positive was associated with symptom (Supplemental Table 3). Nasal and throat swab were sensitive enough that the virus could be detected positive regardless of time and symptom. SARS-COV-2 RNA was hardly found in urine, even with proteinuria being found in digestive system symptom group and all SARS-COV-2 entry proteins being expressed in the kidney (Figure 2 and 3). Detection of SARS-COV-2 for cerebrospinal fluid (CSF) was conducted only once and turned out negative. Other locations (sputum, bronchoalveolar lavage fluid, stool and blood) of SARS-COV-2 were not significantly correlated with symptom (P=0.5281, 0.3865, 0.8454 and 0.5488 respectively).