Diagnostic Role of Laboratory Markers in Egyptian Patients with Mild Coronavirus Disease-2019: A Case-Control Study

Methods: This case-control study included patients with reverse transcriptionpolymerase chain reaction (RT-PCR)-confirmed COVID-19 and healthy participants. Patients were recruited from the National Hepatology and tropical research institute. Routine laboratory markers, Ferritin, D-Dimer, Pro-calcitonin, C-reactive proteins (CRP), Tumor necrosis factor-alpha (TNF-Alpha), interleukin-6 (IL-6) levels were assessed. Regression analysis was performed to detect the independent markers associated with COVID-19 diagnosis and receiver operating characteristic (ROC) curves were used.


Introduction
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing coronavirus disease 2019 (COVID- 19), emerged as a multifaceted problem in December 2019. The first case infected with the newly discovered SARSCoV-2 in Africa was reported in Egypt on February 14, 2020 [1]. Egypt reached the first peak of SARS-CoV-2 infections in June-July 2020 [2]. Diagnosis of SARS-CoV-2 infection is based on immunological and molecular tests. Immunological tests include the detection of viral-specific antibodies in serum (diagnose previous infection, infection of at least 3-4 weeks' duration, asymptomatic infections in close contacts) or SARS-CoV-2 antigens in samples from respiratory secretions (diagnose current infection) [3][4][5]. Molecular testing is the nucleic acid amplification test (NAAT) including real-time polymerase chain reaction (RT-PCR) to diagnose current infection.
The performance of RT-PCR depends on the type and quality of the test and the respiratory secretions specimen in addition to the duration of COVID-19 disease. False-negative results ranged from <5 to 40% [3][4][5]. The favored initial diagnostic test is RT-PCR. In some settings, antigen detection may be used initially but with lower sensitivity. A negative antigen detection testing should be confirmed with NAAT [6]. Molecular tests require adequate laboratory infrastructure [5].
If possible, all symptomatic patients with suspected SARA-CoV-2 infection should be tested. However, in some poor areas, limited capacity could preclude testing all suspected individuals.
Local health authorities may have specific criteria for testing. Different non-specific laboratory biomarkers were used to support the diagnosis, prognosis, and monitoring of patients infected with COVID-19. Although nonspecific, some biomarkers were reported to be linked to the infectious process of SARS-CoV-2.
Previous studies and meta-analyses found that the most common laboratory abnormalities reported with COVID-19 have decreased serum albumin, platelet count, lymphopenia, elevated CRP, lactate dehydrogenase, transaminases, and ESR [7,8] as well as increased D-dimer and low hemoglobin [9].
Other abnormalities reported were increases in neutropenia, total bilirubin, creatinine, cardiac troponin, PT, and PCT [10,11]. In some countries, biomarkers such as CRP, PCT, lymphopenia, and IL-6 and IL-10 are already being used to aid diagnosis or to provide early evidence of more severe disease progression [12]. cytokine storms with high levels of IL-2R, IL-6, IL-10, and TNF-α and a reduction in the absolute numbers of CD4+ and CD8+ T lymphocytes have been related to severe COVID-19, with progression to cardiovascular collapse, multiple organ failure, and rapid deaths [5]. This work aimed to study simple laboratory markers that could be involved in the diagnosis of early mild COVID-19 disease within a cohort of Egyptian patients.

Materials and Methods
This case-control study included consecutive patients with and without COVID-19 disease presented at the National Hepatology and Tropical medicine Research Institute (NHTMRI) who agreed to participate in the study and provide blood samples.

Ethics Statement
All procedures contributing to this work comply with the ethical

Subjects
This study included adults above 18 years, male and female

Results
This case-control study included 135 patients with PCRconfirmed non-severe COVID-19 disease and 137 healthy controls.
We recruited the participants during the period from1st of May to the 15 th of July, 2020. There was no difference among the 2 groups regarding their age, gender distribution, comorbidities, or BMI. All Patients with COVID-19 disease had mild to moderate disease and all recovered and were discharged from the hospital (Table 1). Patients with COVID-19 disease showed significantly higher white blood cell count, neutrophils, lymphocytes, and monocytes percentages. Inflammatory markers of Serum ferritin, Pro-calcitonin, LDH, and CRP were significantly higher in patients with COVID-19 disease. D-dimer, Tumor necrosis factor-alpha, and Interleukin-6 serum levels were significantly higher in patients with COVID-19 disease (Table 1) [14,15]. Another situation that raises the importance of auxiliary laboratory studies is negative nucleic acid amplification testing in suspicious symptomatic patients [16,17].
In such a situation, the clinical diagnosis of COVID-19 could be supported by distinctive laboratory or imaging results which can be a reason to establish infection control measures [18]. In addition to their diagnostic role, initial cytokines assessments are predictable of disease outcome and, accordingly, arouse the importance of utilizing serum cytokine levels for treatment decisions [19].
This study aimed to use routine laboratory and inflammatory markers to help in the diagnosis of mild COVID-19 cases that can be managed by home isolation preserving molecular diagnosis in low-income countries for severe cases to optimize and prioritize hospitalization. We chose these markers because they were proved to be elevated during the disease course of COVID-19.
They also were proved to affect disease severity and mortality. and lactate dehydrogenase were significantly lower in patients with mild disease than in severe cases while procalcitonin did not show any increase in mild cases [15].
In our study, serum ferritin was within the normal range but significantly higher in COVID-19 patients. Ferritin is a protein that is iron storing. It increases during viral infection and reflects active viral replication [20]. Patients with mild COVID-19 disease usually present with normal ferritin levels on hospital admission (30-400 μg/L) [14]. Elevated serum ferritin levels (>400 μg/L) were usually observed in patients with severe disease on admission [21]. A meta-analysis by Cheng and colleagues, 2020 that involved 10 614 COVID-19-confirmed cases, reported that ferritin level is significantly higher in patients with severe COVID-19, nonsurvivors, patients with one or more chronic diseases [20].
TNF-alpha starts to rise early in COVID-19 infection and continues to rise during the disease [21]. Chen and colleagues, 2020 [10] found that IL-2R, IL-6, and TNF-α increase with the increasing severity of COVID-19. Although IL-6 levels were significantly higher in univariate analysis in patients with COVID-19 of our study, it was not proved by multivariate analysis to help in the diagnosis of mild COVID-19 cases. IL-6 was proved to be a trustworthy indicator of disease severity and outcome of mechanical ventilation [22]. A study by Del Valle et al. 2020 [19] that included confirmed COVID-19 cases of different severities (from mild to severe cases with organ damage) found that IL-6 followed by TNF-alpha were the highly independent factors associated with severity and prediction of outcome. They used a cut-off above 70 pg ml −1 for IL-6 while in our study the mean value of IL-6 in COVID-19 patients was 28.5±9.8 pg ml −1 . They used a cut-off above 35 pg ml −1 for TNF-α which was near our mean value of TNFα of 32.5±12.8 pg ml −1 . They found that IL-6 levels were the most dynamic, but they did not compare their levels among patients with different COVID-19 severities.
Disturbed coagulation profile and overt disseminated intravascular coagulation is an early indicator of high mortality in patients with COVID-19. D-dimer appears in the blood after coagulation occurs. D-dimer was reported to be a strong independent factor of mortality in COVID-19 [14]. During the early stages of COVID-19 disease, D-dimer level is usually normal or slightly increased [23] like our study. Limitations of the current study include the small sample size, and only a few markers are assessed and other cytokines which could have an important role were not measured.

Conclusion
This work showed that ferritin, D-Dimer, CRP, TNF-Alpha, were the independent laboratory variables associated with the diagnosis of Egyptian patients with mild COVID-19 who did not progress to specific-PCR is available.

Funding Statement
This research received no specific grant from any funding agency, commercial or not-for-profit sectors. The data that support the findings of this study are available on request from the corresponding author, [Shousha HI].

Disclosure of Interest
The authors report no conflicts of interest.