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 < 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.

Study Design. 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.

Patient Recruitment. 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.

Assessments. 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 < 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 < 0.1% sodium azide. The principal components of the EBV EA-D (IgG) tests were magnetic particles coated with EA-D polypeptide (obtained in E. coli by recombinant DNA technology), BSA, phosphate buffer containing < 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) > 39.99 U/mL, EBV EA-D antibody (IgG) > 9.99 U/mL, or EBV DNA, quantitative, real-time PCR > 2.29 Log copies/mL.

Group:
LongTermControl = 30 subjects making up the long-term control group
LongTermPACS = 20 subjects making up the long-term long COVID group
RecentControl = 9 subjects making up the short-term control group
RecentPACS = 9 subjects making up the short-term long COVID group
SurveyOnly = 117 additional COVID-19 positive applicants added to above for 185 subject survey

ID: Unique Patient ID Number

Age: 18-80

Underlying_Conditions:
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)

Reactivated:
0 = No
1 = Yes, positive for Epstein-Barr Reactivation

Long:
0 = No long COVID symptoms
1 = 1 or more long COVID symptoms

Long_Symptoms: Total number of long COVID symptoms (0 to 10)

Long_DoctorVisits: Total visits to doctor/clinic for long COVID symptoms (0 to 4), value of 4 signifies 4 or more

Acute_Severity: (Severity of initial COVID-19 case)
1 = Asymptomatic
2 = Mild
3 = Moderate
4 = Severe

Acute_Symptoms: (Symptoms score of initial COVID-19 case computed with below formula)
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)

CovAntibodyStatus:
0 = Subject was negative for SARS-CoV-2 antibodies at time of EBV serological testing
1 = Subject was positive for SARS-CoV-2 antibodies at time of EBV serological testing

EBVEAIgG: Value of EBV EA-D (IgG); value of 150 signifies >150

EBVIgM: Value of EBV VCA (IgM)

EBVIgG: Value of EBV VCA (IgG); value of 750 signifies >750

EBVNIgG: Value of EBV Nuclear Antigen (IgG); value of 600 signifies >600

EBVDNA: Value of EBV DNA, copies/mL

EBVDNAL: Value of EBV DNA, Log copies/mL

COVID-19 Acute Disease Symptoms Reported

Cough: 0=no, 1=yes
Sore_Throat: 0=no, 1=yes
Slight_Breathing: 0=no, 1=yes
Severe_Breathing: 0=no, 1=yes
Chest_Pain: 0=no, 1=yes
Headache: 0=no, 1=yes
Confusion: 0=no, 1=yes
Muscle_Aches: 0=no, 1=yes
Smell_Taste: 0=no, 1=yes
Fever: 0=no, 1=yes
Nausea: 0=no, 1=yes
Diarrhea: 0=no, 1=yes
Hospitalized: 0=no, 1=yes
Required_Oxygen: 0=no, 1=yes
Intubated: 0=no, 1=yes

Long COVID Symptoms Reported

LSkinRash: 0=no, 1=yes
LFatigue: 0=no, 1=yes
LWeakness: 0=no, 1=yes
LConfusion: 0=no, 1=yes
LInsomnia: 0=no, 1=yes
LSoreThroat: 0=no, 1=yes
LHeadache: 0=no, 1=yes
LAbdominalPain: 0=no, 1=yes
LRingingEars: 0=no, 1=yes
LMuscleAches: 0=no, 1=yes
LHearingLoss: 0=no, 1=yes
LFeverOver101: 0=no, 1=yes
LSwollenLymphNodes: 0=no, 1=yes