DOI: 10.5061/dryad.c866t1g67
title: Investigation of Long COVID Prevalence and Its Relationship to Epstein-Barr Virus Reactivation

description: Coronavirus disease 2019 (COVID-19) patients sometimes experience
 long-term symptoms following resolution of acute disease, including
 fatigue, brain fog, and rashes. Collectively these have become known as
 long COVID. Our aim was to first determine long COVID prevalence in 185
 randomly surveyed COVID-19 patients and, subsequently, to determine if
 there was an association between occurrence of long COVID symptoms and
 reactivation of Epstein–Barr virus (EBV) in 68 COVID-19 patients recruited
 from those surveyed. We found the prevalence of long COVID symptoms to be
 30.3% (56/185), which included 4 initially asymptomatic COVID-19 patients
 who later developed long COVID symptoms. Next, we found that 66.7% (20/30)
 of long COVID subjects versus 10% (2/20) of control subjects in our
 primary study group were positive for EBV reactivation based on positive
 titers for EBV early antigen-diffuse (EA-D) IgG or EBV viral capsid
 antigen (VCA) IgM. The difference was significant (p &lt; 0.001,
 Fisher’s exact test). A similar ratio was observed in a secondary group of
 18 subjects 21–90 days after testing positive for COVID-19, indicating
 reactivation may occur soon after or concurrently with COVID-19 infection.
 These findings suggest that many long COVID symptoms may not be a direct
 result of the SARS-CoV-2 virus but may be the result of COVID-19
 inflammation-induced EBV reactivation.
description: <strong>Study Design.</strong> 357 Applicants were
 screened using a Health Insurance Portability and Accountability Act
 (HIPAA)-compliant online form under which informed consent was obtained.
 The HIPAA-compliant form, screening, and study protocol were approved by
 the ethical review committee of our Institutional Review Board prior to
 initiation (Integrity IRB Protocol identifier [ID]: 40005). As an
 assurance of confidentiality, only one investigator performed the
 recruitment, data collection and validation, kept the patient data in a
 secure database that was used only in coded form (to remove all patient
 identifiers) before the analysis of the data by the research team. Thus,
 all patient records, test data and the identity of the subjects submitting
 photos of skin manifestations has been kept by a single source.
 <strong>Patient Recruitment. </strong>Subjects were
 chosen from applicants who responded to online advertisements that we ran
 seeking recovered COVID-19 patients for this study. Each applicant was
 required to upload documentation of their COVID-19 medical history. These
 included copies of COVID-19 test results and hospitalization records, as
 well as completing an in-depth online survey in which they provided
 details related to their COVID-19 symptoms and outcomes on the
 HIPAA-compliant form. Follow-up was done by one investigator to verify
 that each subject met the study criteria, and to allow subjects
 demonstrating skin manifestations to provide images of such for the
 record, and to be kept in blinded files for evaluation by the remaining
 researchers. Subjects in the study were selected randomly from all who
 applied and were a match to the criteria for any of the study groups.
 Applicants were excluded if they were under 21 years of age, over 74 years
 of age, or if they had any of these health conditions: pregnant, had been
 given a COVID-19 vaccine, or had long-COVID-like symptoms prior to testing
 positive for COVID-19. The selection process continued until 68 qualified
 subjects had been selected. These 68 subjects provided serological samples
 to be tested for the relevant EBV parameters using a commercial
 laboratory. The selection of applicants and serological testing was
 conducted between 11 December 2020 through 11 February 2021. A small
 stipend to help defray costs associated with providing records and blood
 samples was available to subjects.
 <strong>Assessments. </strong>All study participants
 volunteered to provide blood samples through a clinical reference
 laboratory (Quest Diagnostics). The samples were tested for EBV VCA
 antibody (IgM), EBV VCA antibody (IgG), EBNA antibody (IgG), and EBV EA-D
 antibody (IgG). Subjects describing long COVID symptoms who did not test
 positive for EBV VCA antibody (IgM) or for EBV EA-D antibody (IgG) were
 also tested for EBV DNA with a quantitative, real-time PCR test with a
 linear range of 200-2,000,000 copies/mL. EBV antibodies
 were measured using a Liaison analyzer system to measure chemiluminescence
 from a commercially available immunoassay (CLIA) for the qualitative
 determination of IgG and IgM antibodies in human serum specimens. The
 method for qualitative determination of specific IgG and IgM antibodies to
 EBV was a competitive (indirect) CLIA. The principal components of the EBV
 VCA (IgG) and EBV VCA (IgM) tests were magnetic particles coated with VCA
 p18 synthetic peptide, BSA, phosphate buffer containing &lt; 0.1%
 sodium azide. The principal components of the EBNA (IgG) tests were
 magnetic particles coated with EBNA-1 synthetic peptide, BSA, phosphate
 buffer containing &lt; 0.1% sodium azide. The principal components of
 the EBV EA-D (IgG) tests were magnetic particles coated with EA-D
 polypeptide (obtained in <em>E. coli</em> by recombinant DNA
 technology), BSA, phosphate buffer containing &lt; 0.1% sodium azide.
 The EBV DNA, Quantitative, Real-Time PCR test was developed in house, and
 its analytical performance validated by Quest Diagnostics.
 When EBV VCA antibody (IgM) is detectable but EBNA antibody (IgG)
 is not, this generally indicates primary EBV infection or EBV
 reactivation. When EBV VCA antibody (IgM) and EBNA antibody (IgG) are both
 detectable, this generally indicates EBV reactivation. EBV EA-D antibody
 (IgG) is generally only detectable in patients with primary infection or
 EBV reactivation. Thus, testing for the presence of EBV VCA antibody (IgM)
 or EBV EA-D antibody (IgG) has been commonly used to detect EBV
 reactivation. Some reactivation cases missed by the other tests can be
 detected through serum testing for the presence of EBV DNA circulating
 following viral release during recrudescence, by quantitative real-time
 polymerase chain reaction (PCR) of EBV. In our study, a subject was
 classified as having EBV reactivation if they exceeded any of these
 threshold values: EBV VCA antibody (IgM) &gt; 39.99 U/mL, EBV EA-D
 antibody (IgG) &gt; 9.99 U/mL, or EBV DNA, quantitative, real-time PCR
 &gt; 2.29 Log copies/mL.
description: <strong>Group:</strong><br> LongTermControl =
 30 subjects making up the long-term control group<br> LongTermPACS =
 20 subjects making up the long-term long COVID group<br>
 RecentControl = 9 subjects making up the short-term control
 group<br> RecentPACS = 9 subjects making up the short-term long
 COVID group<br> SurveyOnly = 117 additional COVID-19 positive
 applicants added to above for 185 subject survey
 <strong>ID: </strong>Unique Patient ID
 Number <strong>Age:</strong>
 18-80
 <strong>Underlying_Conditions:</strong><br>
 Total number of underlying conditions from this list (Moderate to Severe
 Asthma, Cancer, Cerebrovascular Disease, Chronic Kidney Disease, COPD
 (Chronic Obstructive Pulmonary Disease), Cystic Fibrosis, Dementia,
 Diabetes (Type 1), Diabetes (Type 2), Down Syndrome, Heart Condition,
 Hypertension (High Blood Pressure), Immunocompromised (from Organ
 Transplant), Immunocompromised (Other Causes), Liver Disease, Overweight
 (Moderate), Overweight (Obese), Overweight (Severely Obese), Pregnancy,
 Pulmonary Fibrosis, Sickle Cell Disease, Smoking, Thalassemia)
 <strong>Reactivated:</strong><br> 0 =
 No<br> 1 = Yes, positive for Epstein-Barr Reactivation
 <strong>Long:</strong><br> 0 = No long COVID
 symptoms<br> 1 = 1 or more long COVID symptoms
 <strong>Long_Symptoms:</strong> Total number of long
 COVID symptoms (0 to 10)
 <strong>Long_DoctorVisits:</strong> Total visits to
 doctor/clinic for long COVID symptoms (0 to 4), value of 4 signifies 4 or
 more <strong>Acute_Severity:</strong>
 (Severity of initial COVID-19 case)<br> 1 = Asymptomatic<br> 2
 = Mild<br> 3 = Moderate<br> 4 = Severe
 <strong>Acute_Symptoms:</strong> (Symptoms score of
 initial COVID-19 case computed with below formula)<br>
 1+Cough+Sore_Throat+Slight_Breathing+(Severe_Breathing*2)+(Chest_Pain*2)+Headache+Confusion+Muscle_Aches+(Smell_Taste*2)+Fever+Nausea+Diarrhea+(Hospitalized*5)+(Required_Oxygen*5)+(Intubated*5) <strong>CovAntibodyStatus:</strong><br> 0 = Subject was negative for SARS-CoV-2 antibodies at time of EBV serological testing<br> 1 = Subject was positive for SARS-CoV-2 antibodies at time of EBV serological testing <strong>EBVEAIgG: </strong>Value of EBV EA-D (IgG); value of 150 signifies &gt;150 <strong>EBVIgM:</strong> Value of EBV VCA (IgM) <strong>EBVIgG: </strong>Value of EBV VCA (IgG); value of 750 signifies &gt;750 <strong>EBVNIgG:</strong> Value of EBV Nuclear Antigen (IgG); value of 600 signifies &gt;600 <strong>EBVDNA: </strong>Value of EBV DNA, copies/mL <strong>EBVDNAL:</strong> Value of EBV DNA, Log copies/mL <em>COVID-19 Acute Disease Symptoms Reported</em> <strong>Cough: </strong>0=no, 1=yes<br> <strong>Sore_Throat:</strong> 0=no, 1=yes<br> <strong>Slight_Breathing: </strong>0=no, 1=yes<br> <strong>Severe_Breathing:</strong> 0=no, 1=yes<br> <strong>Chest_Pain:</strong> 0=no, 1=yes<br> <strong>Headache:</strong> 0=no, 1=yes<br> <strong>Confusion:</strong> 0=no, 1=yes<br> <strong>Muscle_Aches:</strong> 0=no, 1=yes<br> <strong>Smell_Taste: </strong>0=no, 1=yes<br> <strong>Fever:</strong> 0=no, 1=yes<br> <strong>Nausea: </strong>0=no, 1=yes<br> <strong>Diarrhea: </strong>0=no, 1=yes<br> <strong>Hospitalized</strong>: 0=no, 1=yes<br> <strong>Required_Oxygen:</strong> 0=no, 1=yes<br> <strong>Intubated:</strong> 0=no, 1=yes <em>Long COVID Symptoms Reported</em> <strong>LSkinRash:</strong> 0=no, 1=yes<br> <strong>LFatigue:</strong> 0=no, 1=yes<br> <strong>LWeakness: </strong>0=no, 1=yes<br> <strong>LConfusion:</strong> 0=no, 1=yes<br> <strong>LInsomnia:</strong> 0=no, 1=yes<br> <strong>LSoreThroat:</strong> 0=no, 1=yes<br> <strong>LHeadache:</strong> 0=no, 1=yes<br> <strong>LAbdominalPain:</strong> 0=no, 1=yes<br> <strong>LRingingEars:</strong> 0=no, 1=yes<br> <strong>LMuscleAches: </strong>0=no, 1=yes<br> <strong>LHearingLoss:</strong> 0=no, 1=yes<br> <strong>LFeverOver101: </strong>0=no, 1=yes<br> <strong>LSwollenLymphNodes: </strong>0=no, 1=yes