Hypoalbuminemia, Hyper-a-1/–a-2-Globulinemia Associated with High CRP and IL-6 in COVID-19 Patients in Togo

Research Article

Austin J Infect Dis. 2023; 10(2): 1084.

Hypoalbuminemia, Hyper-a-1/–a-2-Globulinemia Associated with High CRP and IL-6 in COVID-19 Patients in Togo

Gnatoulma Katawa1*; Wemboo Halatoko2; Christèle Nguepou Tchopba1; Yawo Hozo Aloyi2; Adjoa Holali Ameyapoh1; Pélagie Edlom Tchadié1; Marthe Oukoe Amessoudji1; Simplice Damintoti Karou1; Manuel Ritter3; Ameyo M Dorkenoo4,5; Malewe Kolou4

1Unité de Recherche en Immunologie et Immunomodulation (UR2IM)/Laboratoire de Microbiologie et de Contrôle de Qualité des Denrées Alimentaire (LAMICODA), Ecole Supérieure des Techniques Biologiques et Alimentaires, Universite de Lomé, Lomé, Togo

2Institut National d’Hygiène, Lomé, Togo

3Institute for Medical Microbiology, Immunology and Parasitology (IMMIP), University Hospital Bonn (UKB), Bonn, Germany

4Faculté des Sciences de la Santé, Université de Lomé, Lomé, Togo

5Division des Laboratoires, Ministère de la Santé, de l’Hygiène Publique et de l’Accès Universel aux Soins, Lomé , Togo

*Corresponding author: Gnatoulma Katawa Unité de Recherche en Immunologie et Immunomodulation (UR2IM)/Laboratoire de Microbiologie et de Contrôle de Qualité des Denrées Alimentaire (LAMICODA), Ecole Supérieure des Techniques Biologiques et Alimentaires, Universite de Lomé, Lomé, Togo. Email: mahkatawa@yahoo.fr

Received: April 04, 2023 Accepted: May 16, 2023 Published: May 23, 2023

Abstract

Introduction: COVID-19, a viral infectious disease that has caused the global health crisis, is characterized by inflammatory responses. It remains necessary to provide other biomarkers for the pronostic of the disease.

Aim: To determine inflammatory biomarkers associated with COVID-19 disease and its severity in Togo.

Methods: Total proteins, C-reactive protein, protein fractions, TNF-a and IL-6 were measured in the serum of COVID-19 positive subjects by respectively Biuret method, immunoturbidimetry, serum protein electrophoresis by capillary method, and sandwich ELISA technique. Logistic regression analyses were performed using SPSS software.

Results: In total, 74 samples were included, among which COVID-19 symptomatic (n=26), asymptomatic (n=18) and COVID-19 negative (n=30). Symptomatic subjects were significantly more aged than other groups, with mean age of 46.31±15.60 years. Data showed elevated total protein in asymptomatic COVID-19; increased a-1 and a-2 globulin concentration and low albumin level in COVID-19 positive patients; high level of a-1 and a-2 globulin associated with a low level of albumin in symptomatic COVID-19 positive subjects; and symptomatic COVID-19 were characterized by elevated CRP level. Logistic regression analysis revealed that increase of IL-6 level was associated with COVID-19 infection.

Conclusion: This study showed that COVID-19 disease is characterized by an upregulation of CRP, IL-6, a-1 and a-2 globulins; and a low level of albumin. The increase in IL-6 level enhances the chance of having COVID-19. Thus these biomarkers may serve as biochemical and serological markers of probable COVID-19 disease.

Keywords: COVID-19; Hypoalbuminemia; Hyper-a-1-globulinemia; Hyper-a-2-globulinemia protein; CRP; TNF-a; IL-6

Introduction

COVID-19, caused by “severe acute respiratory syndrome coronavirus 2” (SARS-CoV-2) [1,2], remains a global health emergency about four years after description of the first case in December 2019 [3]. The virus spread worldwide, causing many deaths: as of 5 March 2023, over 759 million confirmed cases and over 6.8 million deaths reported globally [4]. In Africa, COVID-19 was not as severe as expected, with regards to the high mortality rate registered all over the world. Over the course of time, molecular biomarkers of SARS-CoV-2 [5,6] have been identified, allowing the early detection of the virus and also the development of vaccines [7,8]; and consequently, the reduction of the mortality rate [9]. As of the time of writing this article, no treatment for COVID-19 is available, SARS-CoV-2 obviously entered our lives and it might be useful to identify other biomarkers to be used for the presumptive diagnosis of the disease. Indeed, patients with COVID-19 present a large variety of symptoms, ranging from mild such as fever, dry cough, malaise, sore throat, fatigue, pain, loss of taste or smell; to moderate with dyspnea, diarrhea and pneumonia; and may progress to severe pneumonia and ultimately to Acute Respiratory Distress Syndrome (ARDS), septic shock and/or multi-organ failure [10,11]. Symptomatic forms of SARS-CoV-2 infection are accompanied by biological changes like increased neutrophil counts, decreased CD4 and CD8 T cells, and in rare cases, decreased hemoglobin and platelets [12,13]. In China, several authors found that an abnormal elevation of the C-reactive protein is observed [14] with immunological disturbances, dominated by considerably elevated serum levels of inflammatory cytokines and chemokines responsible of the so called “cytokine storm”. The latter includes elevation of IL-6, TNF-a, IL-1β, IFN-γ, IL-2, IL-8, IL-17, G-CSF, GM-CSF, IP10, MCP1 and MIP1a (also known as CCL3) [15-17]; and causes tissue damage in the heart, liver, and kidneys, as well as respiratory failure or multi-organ failure [18].

The first case of COVID-19 was confirmed in Togo on 6 March 2020 by WHO [19] and three years later, on 6 March 2023, 808,684 individuals have been tested of which 39,396 confirmed cases, 290 deaths registered and 25 known active cases [20]. Data on the inflammatory profile of COVID-19 patients in Togo remains unknowned and the reference method for the certainty diagnostic in the country is RT-PCR. Unfortunately, RT-PCR is not available in most of the hospital diagnosis platforms. Therefore, it appears necessary to find other common biomarkers appropriate and affordable for the pronostic of COVID-19.

In this study, we demonstrated the implication of biomarkers such as total proteins, C-Reactive Protein (CRP), protein fractions (albumin, a-1-, a-2-, β-1-, β-2- and γ-globulins) and pro-inflammatory cytokines (IL-6 and TNF-a) in COVID-19 disease and its severity in patients infected in Togo.

Material and Methods

Study Population and Samples

This was a cross-sectional study conducted at the Togo’s national laboratory of public health called “Institut National d’Hygiène” (INH) and the immunology and immunomodulation research unit « Unité de Recherche en Immunologie et Immunomodulation (UR2IM) of « Ecole Supérieure des Techniques Biologiques et Alimentaires (ESTBA) », University of Lomé.

The biological material was serum samples collected: 1) on COVID-19 infected patients admitted at the reference center for COVID-19 care named “Centre Hospitalier Regional Lomé Commune” (CHR-LC) during COVID-19 break in Togo; and 2) on patients who visited “Institut National d’Hygiène” (INH) before year 2019, considered as “COVID-19 negative”.

Samples from male and female were randomly selected according to these criteria: age equal or more than 18 years, HIV negative, COVID-19 positive admitted at CHR-LC during COVID-19 break, and COVID-19 negative sampled before year 2019 at INH stored at -80°C. HIV positive, pregnant women and subjects aged less than 18 years were not included.

Definition

COVID-19 positive individuals were diagnosed by Real-Time Polymerase Chain Reaction (RT-PCR) in the molecular biology laboratory of INH, detecting 2 molecular markers of SARS-CoV-2 (N gene and ORF1ab) in the sera. They were subdivided in 2 groups by the medical practicians trained for COVID-19 diagnosis following WHO recommandations.

COVID-19 symptomatic: individuals diagnosed positive to SARS-CoV-2 and who had COVID-19 related symptoms confirmed by medical practicians following WHO guidelines.

COVID-19 asymptomatic: individuals for who no symptoms were declared or registered following medical consultation. It was about individuals who have been in close contact with symptomatic COVID-19 subjects or have been diagnosed during a screening test for a travel purpose.

Total Protein Assay

The total protein was measured by Biuret colorimetric method using TP2 Total Protein Gen.2 kit (Roche Diagnostics, Indianapolis, USA) on the fully automated biochemical analyzer COBAS c311 (Roche Diagnostics), according to manufacturer’s instructions. Thus, copper ions (Cu2+) react in alkaline solution with the peptide bonds of proteins, forming a characteristic purple complex (biuret reaction). The intensity of the colour is directly proportional to the concentrations of protein in the milieu and is measured by photometry at 546nm.

C-Reactive Protein Assessment

C-Reactive Protein (CRP) was assayed by immunoturbidimetry on latex particles coupled to human anti-CRP antibodies, using CRP4 Tina-quant C-Reactive Protein kit (Roche Diagnostics) and the fully automated biochemical analyzer COBAS c311.

The CRP in the serum, link specifically to the anti-CRP, forming an agglutination of latex particles, this latter agglutination increase the turbidity. The level of CRP in the sample is proportional to the quantity of light absorbed in the turbidimeter.

Determining the Level of Protein Fractions

Protein fractions assay was performed by electrophoresis on the fully automated MiniCap Flex piercing (Sebia, Lisses, France) using Minicap Protein(e) 6-Electrophoresis kit (Sebia) following the manufacturer’s instructions. In brief, proteins were separated in silica capillaries by their electrophoretic mobility and electroosmotic flow at high voltage in an alkaline buffer. Serum proteins submitted to an electric flow in an alcalin buffer are negatively charged and move from cathode (negative side) to anode (positive). Each protein fraction (albumin, a-1 and a-2 globulins, β-1 and β-2 globulins, γ-globulins) was directly detected during migration by UV absorbance which is proportional to the concentration of the protein.

Cytokines Measurement

The Level of cytokines, IL-6 and TNFa was determined in the serum samples by ELISA sandwich technique, using InvitrogenTM Human IL-6 ELISA kit and InvitrogenTM Human TNFa ELISA kit (Thermo Fisher Scientific, Bender MedSysthems GmBH, Vienna, Australia) following manufacturer’s instructions. Cytokines concentrations were measured at 450nm on a Huma Reader HS plate reader (Human Diagnostics Worldwide, Wiesbaden, France).

Statistical Analysis

Data were anlysed using SPSS software (IBM SPSS Statistics 21; Armonk, NY) and GraphPad Prism version 9 (GraphPad Software, San Diego California USA). Student's t-test and χ²/Fisher's exact tests were used, respectively, to compare means and frequencies; with a significance level if p-value<0.05. Binary logistic regression was performed by associating each of the inflammation biomarker with a positive COVID-19 on one hand, and with the severity of the COVID-19 disease on the other hand. Odds Ratio (OR) and adjusted odds ratio were considered significant at 95% confidence interval, with p-Value≤0.05 in the univariate model and multivariate model. Also when OR/aOR = 1, there was no association; when OR/aOR>1 the study variable was associated with COVID-19 or COVID-19 severity; and when OR/aOR<1, the study variable was a protective factor against COVID-19 or COVID-19 severity. GraphPad Prism version 9 was essentially used to screen the profile of biomarkers in COVID-19 positive patients on the one hand, and in symptomatic and asymptomatic patients on the other hand. The distribution of values was tested using the d’Agostino-Pearson Omnibus normality test. Since the distribution of our variables was non-parametric, the Mann-Whitney U test was used to compare the medians between 2 groups; and the Kruskal-wallis followed by Dunn’s multiple analysis to compare medians between 3 groups. Differences were considered significant when the p-value<0.05.

Ethical Considerations

This study received the approval of the ethical board (“Comité de Bioéthique pour la Recherche en Santé”, CBRS) of the Ministry of Health of Togo (N°035/2022/CBRS). All participants were adults and already provided written consent for further use of their samples for research purpose.

Results

Characteristics of the Study Population

Our study population comprised 74 individuals. Among them, 26 were symptomatic, 18 asymptomatic and 30 negative. The mean age was 46.31±15.60 years, 29.50±8.27 and 29.30±9.70 (p=0.025) respectively for symtomatics, asymptomatics and negatives. Most of COVID-19 positive were male (Table 1).