Clinical and Laboratory Findings of COVID-19 in High-Altitude Inhabitants of Saudi Arabia

Background: SARS-CoV-2, the causative agent of COVID-19, continues to cause a worldwide pandemic, with more than 147 million being affected globally as of this writing. People's responses to COVID-19 range from asymptomatic to severe, and the disease is sometimes fatal. Its severity is affected by different factors and comorbidities of the infected patients. Living at a high altitude could be another factor that affects the severity of the disease in infected patients. Methods: In the present study, we have analyzed the clinical, laboratory, and radiological findings of COVID-19-infected patients in Taif, a high-altitude region of Saudi Arabia. In addition, we compared matched diseased subjects to those living at sea level. We hypothesized that people living in high-altitude locations are prone to develop a more severe form of COVID-19 than those living at sea level. Results: Age and a high Charlson comorbidity score were associated with increased numbers of intensive care unit (ICU) admissions and mortality among COVID-19 patients. These ICU admissions and fatalities were found mainly in patients with comorbidities. Rates of leukocytosis, neutrophilia, higher D-dimer, ferritin, and highly sensitive C-reactive protein (CRP) were significantly higher in ICU patients. CRP was the most independent of the laboratory biomarkers found to be potential predictors of death. COVID-19 patients who live at higher altitude developed a less severe form of the disease and had a lower mortality rate, in comparison to matched subjects living at sea level. Conclusion: CRP and Charlson comorbidity scores can be considered predictive of disease severity. People living at higher altitudes developed less severe forms of COVID-19 disease than those living at sea level, due to a not-yet-known mechanism.


INTRODUCTION
Humans worldwide have been besieged by the relentless spread of the SARS-CoV-2 pandemic. As of this writing, SARS-CoV-2 laboratory-confirmed cases have exceeded 147 million globally. Patients infected with this newly discovered coronavirus can be affected by either asymptomatic or symptomatic forms of the disease. The latter includes fever, sore throat, cough, shortness of breath, and a loss of smell and taste sensation. Some patients develop severe pneumonia and acute respiratory distress syndrome with fatal consequences (1). Older people and those with other health problems such as diabetes, cancer, lung diseases, and heart disease are the most vulnerable groups afflicted by the severe form of COVID-19. Early diagnosis and treatment of infected patients are important to avoid a complicated form of the disease and to prevent further spread of the virus to uninfected subjects (2). Lymphopenia, elevated inflammatory markers such as high C-reactive protein (CRP), ferritin, and elevated D-dimer, in association with abnormal radiological findings in both chest X-rays and CT scans, are suggestive of COVID-19 (3). The COVID-19 Reporting and Data System (CO-RADS) is used as an indicator of the pulmonary engagement of COVID-19 (4). However, nucleic acid testing on respiratory tract specimens is the only laboratory confirmation of SARS-CoV-2. The angiotensin-converting enzyme 2 (ACE2) receptor has been recognized as the binding site of SARS-CoV-2 in the target cells (5). ACE2 plays a major role in patients with hypertension, CVD, obesity, and type 2 diabetes by opposing the harmful effects of the renin-angiotensin-aldosterone system (RAAS); therefore, the ACE/ACE2 ratio could be a good indicator of SARS-CoV-2 disease progression. There is a direct proportional relationship between COVID-19 infectivity and virulence that depends on the level of ACE2 expression on target cells (6,7). High-altitude-linked hypoxia has been reported to cause systemic inflammation associated with hypercoagulability and promotes venous thromboembolism development (8)(9)(10). Exposure to high altitudes has been reported to predispose patients to a series of thromboembolic events, due to increased blood hypercoagulability (11,12). Platelet counts have been observed to rise within 1 week of exposure to high altitudes (13). Moreover, exposure to high altitudes for 1 year has been shown to increase plasma fibrinogen concentration by as much as 61% (14). In addition, a recent study showed that the mean platelet count is higher among high-altitude dwellers compared to low-altitude dwellers in Saudi Arabia, predisposing them for hypercoagulability (15).
The development of coagulation test abnormalities seen in COVID-19-infected patients is most likely a result of the profound inflammatory response caused by the virus and its associated coagulopathy (16). Based on these data, the combined causes of hypoxia and increased platelet counts in high-altitude dwellers may leave these people prone to more severe attacks of disseminated intravascular coagulation if they develop a COVID-19 infection. However, a recent study has reported less severe manifestations of the disease in people living in high-altitude regions (17).
The current study aimed to analyze the clinical characteristics, laboratory findings, risk factors, associated comorbidities, radiological findings, and outcomes in patients admitted to the hospital with a confirmed COVID-19 infection in Taif, Saudi Arabia. In addition, we also aimed to investigate the effect of high altitude on the severity of the disease, based on the clinical outcomes and disease severity in the hospitalized COVID-19 patients from one high-altitude city, Taif, and a sea-level city, Jeddah.

Patients and Time Frame
An observational retrospective study was conducted on a total of 760 COVID-19 laboratory-confirmed cases between April and September 2020 in Taif, Saudi Arabia. All patients were at least 18 years old and had confirmed positive polymerase chain reaction (PCR) results for COVID-19. Data from subjects were collected from the Alameen and King Faisal Hospitals, in Taif. An additional 208 subjects were recruited from Jeddah (which is at sea level), from patients at King Abdulaziz Hospital. The Jeddah data (n = 208) were compared to matched patients from Taif (n = 272/760). Matching patients' data from Taif were selected from the original 760 subjects based on patients' ages and comorbidities.

Clinical and Laboratory Investigations
Initial investigations included complete blood count, highly sensitive CRP, and chest X-rays. CT chest scans were used as a routine investigation for most of the Taif patients during the early months of the epidemic. X-rays were performed on all patients, while the CT chest scans were only applied to 722/760 (95%) of the patients. Other investigations, such as D-dimer, ferritin, alanine aminotransferase, aspartate aminotransferase (AST), creatinine, international normalized ratio, prothrombin time, and partial thromboplastin time were also conducted. Supplemental oxygen therapy was used if patients' oxygen levels were <90%, to keep these levels between 92 and 96%. Patients who were admitted to an intensive care unit (ICU) were closely monitored for adequate hemodynamic support and fluid therapy, depending on both dynamic and static parameters. Patients who required supplemental oxygen were also given dexamethasone (6 mg daily), either orally or via intravenous injection.

The Charlson Comorbidity Index
The relationships among the severity of COVID-19, ICU admission, disease outcome, and associated comorbidities were assessed in patients from Taif. Twenty-nine points were assigned to different comorbidities: a history of myocardial infarction (one point), congestive heart failure (one point), peripheral vascular disease (one point), dementia (one point), chronic pulmonary disease (one point), rheumatologic disease (one point), peptic ulcer disease (one point), liver disease (three points), diabetes (two points), hemiplegia or paraplegia (two points), renal disease (two points), malignancy (six points), and HIV/AIDS (six points). The Charlson comorbidity index was used as previously described (18)(19)(20).

CO-RADS Classification
CO-RADS classification was used to classify patients from Taif according to their CT results (4). Those in the CO-RADS 1 group had a normal CT or showed non-infectious radiological changes, while those in CO-RADS 2 had a CT that showed changes relevant to non-COVID- 19   Age is a major factor in the severity of COVID-19, as greater ages were associated with increased ICU admission and mortality rates, with p < 0.0001. We found no significant differences between the patient subgroups for gender and nationality. Patients who were admitted to the ICU and/or who died showed significant histories of hypertension, diabetes, chronic kidney disease, peripheral vascular disease, and coronary artery disease, with a p value of 0.0001 for all. We observed no significant differences in most of the initial clinical presentations among the patient subgroups, though patients with a history of ICU admission had a longer duration of hospital length of stay, with a p value of <0.0001. Charlson's comorbidity scores were significantly higher in patients admitted to the ICU and/or who died, with a p < 0.0001 ( Table 1).  Patients' mean oxygen saturation percentage at the time of admission was 94.6%, and 45.1% of our patients showed absolute lymphocytopenia. The mean level of high-sensitivity CRP was 23.6 (3.8) mg/L, the mean ferritin level was 540 (47.7) ng/ml, and the mean D-dimer level was 0.7 (0.1) mcg/ml. Patients with a history of ICU admission and/or death showed significant hypoxia, with a p < 0.001. Leukocytosis and neutrophilia were found to be significant among patients admitted to the ICU and/or who died, in addition to significantly higher D-dimer, ferritin, and highly sensitive CRP levels, with a p < 0.0001 for all patients ( Table 2).

Demographic Data of Patients From Taif
A logistic regression analysis for patients admitted to an ICU showed that the level of highly sensitive CRP was the most independent predictor for death, with a p value of 0.003 ( Table 3). Radiological investigations of our confirmed cases showed that 74.5% had positive results in a chest x-ray. By contrast, 95% of our patients had CT chest affections and were classified according to the CO-RADS classification system (Figure 1); 18.2% were CO-RADS 1, 3.8% were CO-RADS 2, 1.6% were CO-RADS 3, 2.9% were CO-RADS 4, and 68.6% were CO-RADS 5. There was a significant difference among patient subgroups regarding the chest CT and chest X-ray results, with p-values of 0.03 and 0.0001, respectively ( Table 4).
Pearson's correlation was conducted between the different parameters with disease severity and radiological findings. Items that only showed significant correlation was presented in Table 5. Age, AST, D-dimer, ferritin, and CRP showed highly significant correlation (P > 0.01) with both disease severity and radiological findings.

Comparison of Matched Patients in High Altitude and Sea Level
Upon comparing the clinical data of matched patients from Taif and Jeddah, we noted that SARS-CoV-2 infection rates were found to be significantly higher in males than in females, and among subjects between the ages of 21-40 years and 41-60 years in both the high-altitude (Taif) and sea-level (Jeddah) regions ( Table 6). Interestingly, we detected a significantly higher percentage of recovery in the high-altitude region, in comparison with those living at sea level. Importantly, the death rate in the sea level region was nearly threefold that reported in the high-altitude region ( Table 7).
Contrary to the above results, significantly higher rates of deep vein thrombosis, PE, MI, and acute large vessel occlusion were reported in the high-altitude region, compared to the sealevel region. However, the incidence of fever, cough, shortness of breath, and acute respiratory distress syndrome (ARDS) during the course of the disease were higher in Jeddah than in Taif. Importantly, the incidence of comorbidities such as diabetes mellitus, hypertension, asthma, ischemic stroke, and coronary disease showed no significant differences between the highaltitude and sea-level regions ( Table 7).

DISCUSSION
In our study, as reported in other studies, patient age was a critical risk factor for ICU admission, with elderly patients being the most frequently admitted. In addition, comorbidities such as hypertension, diabetes mellitus, chronic kidney disease, ischemic heart disease, peripheral vascular disease, and malignancies were found to correlate with an increased probability of ICU admission and fatal consequences. Our findings also agree with previously available studies in this respect (21)(22)(23)(24)(25). Patients who were admitted to the ICU and those who died showed significantly higher Charlson comorbidity scores than other patients did. These results were similar to previously published data from Denmark (26). Our results indicated that the Charlson comorbidity index is a valid indicator for COVID-19 patients with poor outcomes, in concordance with previous studies (27,28).
In this study, we found no significant relationship between the presence of fever, cough, shortness of breath, GIT symptoms, and being a healthcare worker or with ICU admission or mortality. Our results did not match other studies (29,30) that found a high fever to be associated with COVID-19 progression; we attribute this distance to differences in the studies' designs.
In contrast to the above, sore throat and a loss of smell and taste sensation were associated with a good prognosis among confirmed cases of COVID-19. Our results agree with a previous study that reported a correlation between smell loss and decreased hospitalization, intubation, and acute respiratory distress syndrome (31). In addition, another study also found that patients with a sore throat had better outcomes than others (32). However, there is no available explanation for these correlations.
Our study showed that low oxygen saturation at the time of admission was associated with poor outcomes. Hypoxia was found to be a powerful independent predictor of mortality in COVID-19 patients (33). Previous studies showed a strong relationship between hypoxia and both increasing the inflammatory response (34) and induction of viral replication  (35,36). It might also play a role in the pathogenesis of hypercoagulation (37).
In our study, we found that the presence of leukocytosis, neutrophilia, high levels of highly sensitive CRP, ferritin, D-dimer, and AST at the time of admission were associated with patients' deterioration, ICU admission, and death. Additionally, a significant correlation was found between such parameters and both the disease severity. These results were in line with previous studies that reported a significant association between disease progression and poor outcomes, with increased levels of inflammatory biomarkers and leukocytosis (38)(39)(40).
Logistic regression analysis showed that highly sensitive CRP was the most significant independent predictor of death among COVID-19 patients who were admitted to an ICU. This finding corresponds with previous reports that confirmed that CRP is the major determinant of disease severity in COVID-19 patients (41,42). It may also highlight the role of secondary infection in the deterioration of patients' health status. Interestingly, the use of steroids was reported to be associated with secondary infection (43,44).
In all, 18.2% of our patients showed normal chest CT appearance. Chest CTs were conducted early for most of our patients, and it is well-known that chest CTs continue to be normal in the first 4-5 days of infection (45). Meanwhile, among those who were admitted to the ICU and who later died, the chest CT results were significantly positive. At present, chest CT is not routinely ordered, and its use is limited to specific cases, especially in patients with false negative PCR (46,47). However, we found a significant correlation of the radiological findings and the severity of the disease. This finding was also in accordance with recent findings that found similar correlation (48,49).
We observed a lower mortality rate among people living at a high altitude, in comparison to those living at sea level. Another study has suggested a reduced severity of SARS-CoV-2 in high-altitude-dwelling patients (17); however, there are no current data explaining the mechanism involved in such an effect. The number of ACE2 receptors in target cells affects the susceptibility of such cells to SARS-CoV-2 infection. One speculation is the assumption that highaltitude inhabitants possess hypoxia-induced downregulation of ACE2. The RAAS is the expression of both ACE1 and ACE2 in negative feedback manner. During hypoxia, ACE2 is downregulated in the concomitant upregulation of ACE1 (50). Accordingly, decreased expressions of ACE2 in the pulmonary endothelial lining among high-altitude inhabitants is assumed to reduce the amplitude of virus replication (17). This speculation was argued strongly by Pun et al. (51), who presented conflicting results regarding ACE2. Their argument was augmented by many previous studies that reported the upregulation of ACE2 via hypoxia (52)(53)(54). Furthermore, deciphering hypoxia as a potential factor that alters the ACE2 expression is still obscure and needs further investigation. It is possible that some other, still-unknown mechanism renders high-altitude inhabitants more resistant to the severe consequences of COVID-19, compared to their sea-leveldwelling contemporaries.

CONCLUSION
Fever, cough, and shortness of breath are the main clinical presentations in COVID-19 patients. Age, multiple comorbidities, a high Charlson comorbidity score, leukocytosis, neutrophilia, higher levels of highly sensitive CRP, ferritin, D-dimer, and AST are associated with poor outcomes among COVID-19 patients. High-altitude inhabitants showed reduced disease severity, in comparison with sea-level-dwelling patients.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Research and Studies Department, Directorate of Health Affairs, Taif, Saudi Arabia (NO. 397 on 23/07/2020). The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
AF, AR, MR, RMMA, and AAA collected and analyzed the data from Alamin hospital. RMA, SAA, TA, and SMA collected and analyzed data from Jeddah. MA, ZI, DN, ANA, and DA analyzed the data and prepared the initial draft. DN, DA, ASA-M and AAA conceived the study protocol and critically revised the manuscript. All authors shared in data analysis and discussing and approved the final form of the manuscript.