Four groups of patients were evaluated in an observational retrospective analysis as follows:

A) patients affected by COVID-19, and B) patients recovered from COVID-19, in outpatient follow up; C) patients affected by flare of IMDs after COVID-19; and D) flare of IMDs or onset of IMD after vaccination anti-SARS-CoV-2.

The primary objective of this single-center retrospective study, conducted during the COVID-19 pandemic, is to assess the onset of clinical and laboratory immune manifestations related to SARS-CoV-2 infection or vaccination.

The primary endpoint of the study was the occurrence or flare of IMDs:

a) in patients affected by acute COVID-19;

b) in patients recovered and in outpatient follow up after COVID-19;

c) in patients affected by anamnestic IMD after infection by SARS-Cov2;

d) in close temporal correlation with anti-SARS-Cov2 vaccination in patients affected by an anamnestic IMD or healthy subjects.

Secondary endpoints of the study were:

▪ clinical characterization,

▪ laboratory findings, and

▪ clinical outcomes after the therapy,

of the IMDs occurring in the groups listed above.

A schematic representation of the study and the patients’ clinical characteristics are shown in Figure 1 and Table 2 , respectively.

In the cohort A we enrolled 584 patients with moderate to severe COVID-19 disease, hospitalized in the “COV-4” ward of the hospital “Ospedali Riuniti di Ancona” (AOU), Ancona, Italy, from March 2020 to June 2020 and from November 2020 to May 2021 ( Table 1 ).

Inclusion criteria were the following: a) age>18 years (yrs); b) acute infection by SARS-Cov2 detected with at least two nasopharyngeal swabs and by real-time reverse transcription polymerase chain reaction method.

Exclusion criteria were the following: absence of the inclusion criteria; trauma; neoplastic diseases; non-organ specific connective tissue diseases.

The clinical spectrum of the patients hospitalized for COVID-19 was achieved following the National Institute of Health of the United States of America (NIH) COVID-19 treatment guidelines as follows: a) Moderate Illness: Individuals who show evidence of lower respiratory disease during clinical assessment or imaging and who have an oxygen saturation (SpO2) ≥94% on room air at sea level; b) Severe Illness: Individuals who have SpO2 <94% on room air at sea level, a ratio of arterial partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) <300 mm Hg, a respiratory rate >30 breaths/min, or lung infiltrates >50%; c) Critical Illness: Individuals who have respiratory failure, septic shock, and/or multiple organ dysfunction (14).

All the patients were assessed with clinical examinations and laboratory test daily and with High-resolution computed tomography (HRCT) in the emergency department and during the hospitalization, depending on the clinical evolution.

In the cohort B we evaluated 135 outpatients from cohort A discharged out of the hospital and evaluated within 6 months of the recovery by COVID-19.

Inclusion criteria in the cohort B were the following: a) age>18 yrs; b) absence of SARS-Cov2 detected with at least two nasopharyngeal swabs and by real-time reverse transcription polymerase chain reaction method; c) absence of any respiratory symptom and fever for at least one week.

The follow up examinations were scheduled at 3 and 6 months from the discharge and included clinical examinations, laboratory test and HRCT. The scheduled follow up was anticipated in case of the occurrence of any clinical symptom.

The outcome of interest in cohort A and B was the onset of: a) symptoms of rheumatic diseases (arthralgias-myalgias) in absence of fever; b) articular pain and/or joint swelling; c) chest pain and pericarditis/myocarditis confirmed by laboratory test (elevation of erythrocyte sedimentation rate, ESR; C-reactive protein, CRP; creatin-kinase, CK; high-sensitivity troponin, hs-Tpn, electrocardiographic recording, and ultrasound-doppler echocardiography; d) clinical signs of involvement of the central and/or peripheral nervous system; e) hematological alterations, not reported in the clinical history.

The cohorts C and D were constituted by two group of patients evaluated for IMDs symptoms among the whole cohort of 1710 outpatients evaluated in our rheumatologic clinic for rheumatologic symptoms from March 2020 to November 2021.

The cohort C was constituted by patients affected by IMD (among the 849 IMD patients in our center currently in follow up for at least 2 years) and satisfying the following inclusion in criteria: a) age >18 yrs; b) IMD in stable remission for at least 6 months.

The cohort D was constituted by subjects reporting IMD symptoms or patients affected by flare of IMDs in temporal close correlation with vaccine administration.

The inclusion criteria in cohort D were the following: a) age>18 yrs; b) IMD in stable remission for at least 6 months OR healthy subjects without IMD symptoms before the vaccination; c) absence SARS-Cov2 detected with at least two nasopharyngeal swabs and by real-time reverse transcription polymerase chain reaction method; d) SARS-COV2 vaccination, less than 6 weeks before the onset of the following symptoms: arthralgias-myalgias; articular pain and/or joint swelling; chest pain; dermal lesions or signs of skin inflammation; clinical signs of involvement of the central and/or peripheral nervous system.

The outcome of interest in cohorts C and D was occurrence of IMD flare or the development of new signs/symptoms of IMD. Data were collected by consulting inpatient and outpatient medical records.

The diagnosis of SARS-Cov2 acute infection in all the patients included in the study was made with at least two nasopharyngeal swabs and by real-time reverse transcription polymerase chain reaction method.

The diagnosis of IMDs was carried out by an experienced team of physicians through history, physical examination, and laboratory tests (see the following section).

Immune-Mediated Diseases (IMDs) were considered all the diseases that involve an immune response that is inappropriate or excessive, and is caused, signified, or accompanied by dysregulation of the immune system, with/without the presence of disease-specific autoantibodies (15, 16).

Given that the most common post-infectious or post-vaccinal rheumatological IMD is Reactive Arthritis, and considering that, according to the American College of Rheumatology Diagnostic Criteria (1999), it must occur within 6 weeks of the infectious trigger, we considered relevant only immunological manifestations occurred within 6 weeks from infection or vaccination.

Apart from clinical manifestations, we also collected data regarding routine laboratory tests including complete blood count, alanine transferase (ALT), aspartate transferase (AST), creatinine, uric acid, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP), as available.

If available, we also recorded results of anti-nuclear antibodies (ANAs), anti-cytoplasmic ANCA (c-ANCA) and anti-perinuclear ANCA (p-ANCA) antibodies, rheumatoid factor and anti-citrullinated peptides antibodies (ACPAs), and anti-Ro, anti-La, anti-double strand DNA and anti-Sm autoantibodies.

Any other relevant laboratory or instrumental investigation that had been required for the diagnosis was also recorded.

The study was carried out in compliance with the Declaration of Helsinki on ethical principles for medical research. Formal approval by the local Ethics Committee (Comitato Etico Regionale delle Marche) was waived due to the retrospective nature of the study.

All data were transferred to an electronic database and analyzed descriptively using the common analysis methods.

Categorical data were summarized using absolute frequencies and percentages, while continuous data were summarized using mean ± SD. Comparisons between groups were assessed by means of t test for independent samples or Chi square test, as appropriate. A p < 0.05 was considered significant.

New-onset IMDs were observed in a large group of patients affected by COVID-19 and hospitalized in a COVID ward (cohort A).

Among 584 patients non previously diagnosed with IMDs, 17 (2.9%) received a diagnosis of IMD (9 females, 52.9%; 8 males, 47.1%; mean age 60.23) ( Table 2 ).

These reactions were further subclassified as n. 2 (0.3%) peripheral arthritis, n. 3 (0.5%) pericarditis, n. 1 (0.2%) myocarditis (severe), n. 11 (1.9%) thrombocytopenia and/or pancytopenia ( Tables 2 , 3 ).

Clinical manifestations were mostly acute and observed during hospitalization, with a mean delay of symptom onset of 11 +/- 4 days. Other confounding factors such as joint trauma, additional concomitant infectious triggers or neoplasms were excluded.

Twenty-eight (4.8%) COVID-19 patients of the cohort A reported non-specific musculoskeletal symptoms (myalgia and arthralgia) but did not fulfill criteria for a defined IMD.

These newly reported IMDs were mostly mild to moderate in severity and resolved within 2-4 weeks with restitutio ab integrum after corticosteroids therapy, except for one case of severe myocarditis and one case of severe thrombocytopenia.

In all the patients, ESR and CRP were elevated but the autoantibodies that were tested resulted negative.

Cohort B was constituted by outpatient patients of the cohort A in follow-up after the discharge from the COVID ward, evaluated for suspected IMD occurring within 6 weeks from COVID-19.

Eight patients (5.93%) reported symptoms suspect for IMDs. The following diagnoses were made: n. 4 (3.0%) peripheral arthritis, n. 1 (0.7%) polyneuropathy, n. 1 (0.7%) vasculitis, n. 1 (0.7%) myocarditis, n. 1 (0.7%) myositis ( Table 4 ).

The events were mostly mild-to-moderate and were successfully treated with low-dose corticosteroids, non-steroid anti-inflammatory drugs (NSAID), methotrexate and gabapentin ( Table 4 ).

All the cases described occurred with a mean delay of days 8.9 ± 12 from the discharge from the hospital.

The flare of an anamnestic IMD or the onset of a new one was evaluated among the whole cohort of 1710 outpatients evaluated in our rheumatologic clinic (n. 849 IMDs in follow up and n. 861 new outpatients evaluated for rheumatologic symptoms).

In the cohort C we evaluated those patients with a pre-existing diagnosis of an immuno-mediated disease and reporting symptoms of the IMDs after SARS-COV-2 infection.

Among this group, 5 patients (all females, mean age 56.8), corresponding to the 0,58% out of the 849 outpatients in follow up in our rheumatologic clinic, reported flares of the IMDs already known but in stable remission of the disease before the SARS-COV2 infection ( Table 5 ). The flares presented as follows: acute arthritis of the left knee in n. 1 psoriatic arthritis (PsA); diarrhea and arthritis of the knee or of the right wrist in n. 2 spondyloarthritis associated to inflammatory bowel disease (SpA/IBD), respectively; inflammatory back pain in n. 1 ankylosing spondylitis (AS); and symmetric polyarthritis of the small joints of the hands in n. 1 rheumatoid arthritis (RA). All the patients were successfully treated adding corticosteroid to the ongoing therapy ( Table 5 ).

In the cohort D we evaluated n. 13 outpatients with a newly diagnosed or flared IMD in close temporal correlation with SARS-CoV-2 mRNA vaccine administration ( Table 6 ). Before the onset of the symptoms, all the patients had received two boost doses of the vaccines types listed in Table 6 .

There were observed n. 8 new IMDs, corresponding to the 0.93% among the cohort of 861 outpatients evaluated for new rheumatologic symptoms. These patients showed a heterogeneous clinical spectrum ( Table 6 ), including two clinical severe presentations: one patient had autoimmune encephalitis with pericarditis and another one dermatomyositis, with the involvement of the peripheral nervous system ( Figure 2 ).

In the other patients, n. 4 patients presented a RA-like disease, characterized by symmetric polyarthritis of the wrists and of the small joints of the hands ( Figure 3 ), n. 1 had a mild vasculitis of the small vessels of the hands’ fingers, and n. 1 was diagnosed with polymyalgia rheumatica ( Table 6 ).

In this cohort, serum autoantibodies were found only in the 2 patients: case n. 4 with autoimmune encephalitis (ANA 1/320) and case n. 6 with dermatomyositis (anti-SSA-Ro-antiMDA5, 1/640).

Vaccination induced a flare of an underlying IMD in 5 patients, corresponding to the 0,58% out of the 849 IMDs in follow up in our clinic, as follows: a flare of psoriasis in one patient with PsA; arthritis of the right knee or of the left wrist in two patients with PsA, respectively; enthesitis of the left Achille’s tendon in one patient with axial-PsA; and symmetric polyarthritis of the small joints of the hands in one patient with RA.

All the patients of the cohort D were successfully treated with corticosteroids, or plus colchicine or mycophenolate mofetil (case n. 4 and n. 6, respectively), or corticosteroids added to the ongoing therapy with a bDMARD in the 2 patients (cases n. 7 and 8) already affected by an IMD ( Table 6 ).