Protective Factors and Risk Factors in Patients with COVID-19 Pneumonia and/or Infection

This study examined 50 COVID-19 patients who have been cured in Anhui Province, China. The protective factors and risk factors for these patients were investigated. By comparing CT-negative and CT-positive patients, we found protective factors in blood: lymphocytes, eosinophils number and %, basophils%, reticulocyte%, high uorescence reticulocyte ratio, and reticulocyte absolute value. Comparing patients with underlying disease and without underlying disease, we found protective factors in blood: lymphocytes%, basophils%, large platelets, and low-uorescent reticulocyte ratio. Regarding the biochemistry indicators, albumin/globulin, apolipoprotein and prealbumin can be considered as protective factors for patients without lung symptoms. Urea, glucose, total bile aicd, creatinine and hypersensitivity CRP can be considered as risk factors for patients with underlying diseases. For patients with repeatedly negative and positive results in nucleic acid tests, they were at a medium level in terms of both protective and risk factors, explaining the mild symptoms and repeatedly results in nucleic acid tests.


Cases
We enrolled 50 patients (26 pneumonia patients) who initially presented to the Second People's Hospital of Hefei and the First A liated Hospital of Anhui Medical University with fever, and/or respiratory symptoms, and/or pulmonary in ltrates on chest radiographs. The patients with con rmed diagnosis were implemented and reported according to 'Technical Guidelines for Laboratory Testing of Pneumonia of New Coronary Disease Infection (Seventh Edition)' of the National Health and Health Commission (National Health Commission of China).
All patients had mild to moderate symptoms without signs of life dying. We recorded and analyzed the history, physical ndings, haematological, biochemical, radiological, and microbiological investigation results. Brie y, nasopharyngeal and throat swabs and stool samples were taken and put into viral transport media. This study was approved by the Institutional Review Board of the University of Anhui Medical University. We obtained written consent from the patients.

Lab Tests
The three major routines were blood routine, urine routine, and feces routine. The mainly checked items in blood tests included: red blood cells, hemoglobin, white blood cells, platelet counts, absolute lymphocyte values, absolute intermediate cells, absolute neutrophils, lymphocyte percentage, intermediate cell percentage, neutral percentage of granulocytes, hematocrit, average red blood cell volume, average red blood cell hemoglobin, average hemoglobin concentration, red blood cell distribution width, average platelet volume, platelet distribution width, platelet hematology, etc. Secondly, the main items of urine routine were: urine color, urine pH value, urine speci c gravity, and protein qualitative and microscopic examination. Thirdly, the main items of feces routine were: stool color, hardness and mucus. Microscopic examination of the stool for helminthiasis (hookworm eggs, roundworm eggs, whipworm eggs) and other parasites.

CT of Lungs
Using Siemens somatom de nition 64-row spiral CT scan, patient took the supine position, with the head in advance, patient was told to hold the breath during the scan. The scanning range was from the top to the bottom of two lungs and cross-sectional. the scanning parameters were: tube voltage 120 kV, tube current 320 mA, matrix 512*512, layer thickness and layer moment 5 mm.
Generally, in patients with COVID-19 pneumonia, the texture of two lungs was typically enhanced, blurred, and disordered. Multiple patchy ground-glass density lesions and consolidation lesions were seen on both lungs, with the bronchial vascular bundles and subpleural distribution predominantly. The shadow of blood vessels was thickened, and the shadow of bronchial was partially visible. The bronchial opening of each leaf was usually not obstructed. No obvious enlarged lymph nodes were seen in the mediastinum. The rib cages were symmetrical, there was no effusion in the chest cavity.

Treatment
All patients were actively treated after admission, with daily droplet isolation, air isolation, contact isolation, routine care for Class A infectious diseases, lopinavir/ritonavir tablets 500 mg x 2 per day, 2 ml saline nebulized inhalation, recombinant human interferon alpha 2b injection 5 million unit nebulized inhalation, Chinese medicine decoction, vitamin C tablet 0.2 g, abidol tablet 0.2 g, thymosin enteric-coated tablet 15 mg, chloroquine hydrogen sulfate tablet 300 mg. Patients all improved after 4-34 days treatment and were discharged from hospital. Three of them were re-examined after discharged and were hospitalized again because of positive results in the follow-up nucleic acid tests.

Statistics
We divided all patients into two groups, lung CT-positive and lung CT-negative. After age and gender matching, we performed independent sample t-tests on blood routine indicators and biochemical indicators. Since patients were measured with blood routine and biochemical exams several times during the hospital stay, we choose the exam results measured when the symptoms were the most serious.
Some of patients had underlying diseases, and the patients with underlying diseases were more prone to infect COVID-19 (Huang et al. 2020;Barraquer et al. 1988) we divided the patients into two groups according to whether they have underlying diseases. After age and gender matching, we performed independent sample t-tests on blood routine indicators and biochemical indicators.
After above comparisons, we identi ed some indicators that were more prominent than others, we performed z-transformation on these indicators rst and then added them into two indexes: I and J. Since there were three patients in our sample, who showed repeatedly negative and positive results in the nucleic acid tests, we extracted these three patients and placed them in the whole distribution of the sample for observation.

Results:
1. Lung CT-negative vs. Lung CT-positive: We have 26 patients who were diagnosed with COVID-19 pneumonia, and 24 patients were diagnosed with COVID-19 infection without respiratory symptoms. Age range 12-87 years, with mean 48.20 and SD 16.06, 27 males. After gender and age matching, we had 22 in CT-positive group and 24 in CT-negative group.
By comparing two groups, we found that patients with CT-negative have the number of lymphocytes, number of eosinophils, percentage of eosinophils, number of basophils, the absolute value of reticulocytes, the percentage of reticulocytes and the ratio of high uorescent reticulocytes greater than those of patients with CT-positive. ps < 0.05.
In patients without underlying diseases, lower number of neutrophils, lower number of monocytes, higher percentage of lymphocytes, higher percentage of basophils, greater number of large platelets, lower immature reticulocyte ratio, higher low-uorescent reticulocyte ratio and lower medium-uorescent reticulocyte ratio were found.
In addition, higher chloride, lower urea, lower creatinine, lower glucose, lower total bile acid, lower cystatin C, lower apolipoprotein B, lower hypersensitive CRP in blood were found in patients without underlying disease. For results, see Table 2a,b and Fig. 2a,b. 3. Three patients with repeatedly positive and negative nucleic acid tests There were three patients with repeatedly positive and negative results in nucleic acid tests. Patient A, male, age 79, lung CT negative, with a history of chronic bronchitis with emphysema, cervical spondylosis, femoral head necrosis and cerebral infarction. In addition, he was in a family cluster case, with three of his family members diagnosed with COVID-19 pneumonia and four of his family members diagnosed with COVID-19 infection, including Patient B. He was staying in hospital for total 59 days, he has been tested with nucleic acid tests, including pharyngeal swabs and anal swabs, for 19 times, with 6 negative and 13 positive. Patient B, female, age 34, lung CT negative, without an underlying disease. She was in the family cluster case with Patient A (her father-in-law). She was in hospital for total 63 days, and was tested with nucleic acid tests, including pharyngeal swabs and anal swabs, for 9 times, with 6 negative and 3 positive. Patient C, male, age 44, lung CT positive, without an underlying disease. He was in hospital for total 35 days. He was tested with nucleic acid tests, including pharyngeal swabs and anal swabs, for 17 times, with 7 negative and 10 positive.
For the indicators most prominent in the tests 1 and 2 above, we performed z-transformation and added them into two indexes: I and J. Index I included albumin/globulin, apolipoprotein, and prealbumin. Index J included urea, glucose, total bile aicd, creatinine, and hypersensitivity CRP. We draw the distributions for I and J separately, and placed these three patients into the distributions for observation, as shown in Fig. 3a, b.

Discussion:
Most patients with COVID-19 pneumonia had typical imaging features, including ground glass opacity (GGO) (86%) or mixed GGO and merge (65%), dilated blood vessels at the lesion (72%) and traction bronchiectasis (53%). Disease changes usually have a surrounding distribution (88%), bilateral involvement (83%), and predominantly in the lower lobes (55%) and multi-focus (55%) (Zhao et al., 2020; Yang et al., 2020). CT in the middle-stage of the disease showed an increase in the number and size of GGO. CT found the highest severity visible around 10 days after the onset of symptoms. Acute respiratory distress syndrome was the most common indication for transferring COVID-19 patients to the ICU and was the major cause of death in these patients (Li et al. 2020). CT model corresponding to clinical improvement usually occurred after the second week of the disease, including the gradual resolution of comorbidities, the reduced number of turbidity, lesions and affected leaves (Salehi et al., 2020; Pan et al., 2020).
Consistent with the literature, our patients had the same features in lung CT scans. As our patients had mild to moderate symptoms, there were no case of death or ICU cases. The literature generally described CT ndings in patients with COVID-19 pneumonia, and these CT features changed with the disease development, from mild to severe to death, and there were also CT features in the recovery period (Wang et al. 2020). However, no study has investigated the differences between patients with positive CT scans and negative CT scans in lungs.
We found that patients with CT-negative ndings (infected patients) had the number of lymphocytes, number of eosinophils, percentage of eosinophils, number of basophils, the absolute value of reticulocytes, the percentage of reticulocytes, and the ratio of high uorescent reticulocytes increased, compared to patients with CT-positive ndings (pneumonia patients). Generally, elevated number of lymphocytes means a healthy immunity and resistance to viruses (Guan et al. 2020;Jiang et al. 2020). Therefore, the infected patients without pneumonia might have better immunity and hematopoietic function than patients with pneumonia. The chance for them to be infected in lungs became smaller and they were less threatened by death due to ARDS.
In addition, the infected patients had higher calcium, lower creatinine, higher urea creatinine ratio, lower globulin, higher albumin/globulin ratio, lower creatine creatinine, lower lactate dehydrogenase, higher HDL/CHOL, higher prealbumin, higher apolipoprotein a1, and lower SAA in blood than the pneumonia patients. In the infected but not pneumonia patients, calcium, urea/creatinine ratio, albumin/globulin ratio, HDL/CHOL ratio, prealbumin and apolipoprotein a1 became greater, indicating that these indicators may play a potentially protective role in ghting COVID-19 viruses.
In patients without underlying disease, higher percentage of lymphocytes, higher percentage of basophils, greater number of large platelets, higher low-uorescent reticulocyte ratio were found. These indicators might be protective factors, similar to CT-negative patients, they had better hematopoietic function and immunity (Adams et al. 2020). In addition, higher chloride, lower urea, lower creatinine, lower glucose, lower total bile acid, lower cystatin C, lower apolipoprotein B, lower hypersensitive CRP in blood were found in this patient group. Patients with hypertension, hyperlipidemia and diabetes account for 15/23 in patients with underlying diseases, this might be an explanation for the changes in the biochemical exams. We suggested that the biochemical differences between two groups are mainly from the underlying diseases.
There were three patients with repeatedly positive and negative results in nucleic acid tests in our sample. Their hospital stay was relatively long. In order to study their characteristics, we extracted the most prominent indicators in group comparisons and synthesized them into two index values. Index I can be seen as a sum of factors that were advantage for patients, and index J can be seen as a sum of factors that were disadvantage for patients. We then put these three patients in the sample distributions, and found that these three in the middle of the distributions of index I and J. We then concluded that patients with repeated positive and negative results in nucleic acid tests have both protective factors and risk factors. Therefore, their morbidity and symptoms were in a medium level, neither too serious nor easy to remove the virus.
In conclusion, CT-negative patients and patients without underlying diseases had mild symptoms, which were closely related to the number of lymphocytes and hematopoietic function. Blood routine testing was an good indicator of immunity. By comparing CT-negative and CT-positive patients, we found protective factors in blood: lymphocytes, eosinophils number and %, basophils%, reticulocyte%, high uorescence reticulocyte ratio, and reticulocyte absolute value. By comparing patients with underlying disease and without underlying disease, we found protective factors in blood: lymphocytes%, basophils%, large platelets, and lowuorescent reticulocyte ratio. Regarding the biochemistry indicators, albumin/globulin, apolipoprotein and prealbumin can be considered as protective factors for patients without lung symptoms. Urea, glucose, total bile aicd, creatinine and hypersensitivity CRP can be considered as risk factors for patients with underlying diseases. However, for these patients, their biochemical changes were mainly from the underlying diseases. For patients with repeatedly negative and positive results in nucleic acid tests, they were at a medium level in terms of both protective and risk factors, which explained the mild symptoms and repeatedly negative and positive results in nucleic acid tests.