Tre‐Hardy 2021 [A].

Study characteristics
Patient Sampling	Purpose: Diagnosis of current convalescent‐phase infection Design: Retrospective two‐group analysis to estimate sensitivity and specificity (n = 125) [1] Covid patients (n = 44) [2] Non‐Covid pre‐pandemic patients (n = 81) [2a] Cross‐reactivity panel (n = 75) [2b] Healthy subjects (n = 6) Recruitment: [1][2] All sera originated from blood samples taken during previous clinical requests for diagnostic purposes. [1] Blood samples positive for COVID‐19 were collected from patients with mild, severe or critical infection. Prospective or retrospective: Retrospective Sample size: 125 (44) Further detail: [1] Blood samples positive for COVID‐19 were collected from patients with mild, severe or critical infection. Patients were considered positive according to the results of the RT‐qPCR. [2a] Patients with other viral, bacterial, parasitic or auto‐immune pathologies that could be considered as confounding factors or to another strain of coronavirus, collected in 2019 [2b] No history of known auto‐immune pathologies and without any acute infection of viral or bacterial origin, collected in 2019
Patient characteristics and setting	Setting: Hospital inpatients Location: Iris Sud Hospitals (laboratory serum biobank), Brussels, Belgium Country: Belgium Dates: April 16 to 20, 2020 Symptoms and severity: Mild, severe or critical infection based on the extent of anomalies observed on CT scans: moderate (10%–25%), extensive (25%–50%), severe (> 50%) or critical > 75% and on clinical symptoms (headache, fever, fatigue, cough and sore throat, myalgia, shortness of breath or digestive signs) Demographics: Not stated Exposure history: Not stated Non‐Covid group 1: [2] Non‐Covid patients Source: 2019 prior to the pandemic Characteristics: [2a] Sera positive for the following viral, bacterial and infection from parasite origin were included to assess the possible cross‐reactivity: HBsAg (n = 7), HAV IgM (n = 3), adenovirus (n = 1), HSV IgM and CMV IgM (n = 1), IgM CMV (n = 8), IgM parvovirus B19 (n = 5), HIV (n = 1), ASLO (antistreptolysin O) (n = 4), anti‐treponema pallidum antibody (n = 1), IgG borrelia (n = 1), IgM mycoplasma pneumoniae (n = 10), toxoplasma gondii IgM (n = 16) The cross‐reactivity of the following auto‐immune pathologies was also assessed: rheumatoid factor (n = 1), anti‐TPO antibody (n = 7), irregular antibodies (n = 4), direct coombs (n = 1). Two sera from COVID‐19‐negative patients but positive to another strain of coronavirus Finally, one serum with a high level of total IgM (9.01 g/L) (normal range: 0.40–2.30 g/L), one serum with high total IgA (4.47 g/L) (normal range: 0.70–4.00 g/L) [2b] six sera from COVID‐19‐negative healthy subjects with no history of known auto‐immune pathologies and without any acute infection of viral or bacterial origin
Index tests	Test name: [A] LIAISON SARS‐CoV‐2 IgG [B] anti‐SARS‐CoV‐2 ELISA IgG Manufacturer: [A] Diasorin, Saluggia, Italy [B] Euroimmun, Medizinische Labordiagnostika, Lubeck, Germany Antibody: [A] IgG [B] IgG Antigen target: [A] S1 and S2 subunits [B] S1 subunit Evaluation setting: [A] and [B] Laboratory Test method: [A] CLIA [B] ELISA Timing of samples: >= 14 days post‐PCR + Samples used: Serum stored in the laboratory serum biobank at ≤− 20 °C Test operator: Clinical laboratory staff Definition of test positivity: Manufacturer’s cut‐off: [A] >= 15.0 AU/mL is positive, < 12.0 AU/mL is negative, in between is doubtful. [B] Ratio >= 1.1 is positive, < 0.8 is negative, in between is doubtful. ROC curve analyses cut‐off: [A] > 6.1 AU/mL [B] > 0.708 Blinding reported: Not stated Threshold predefined: Yes, using the cut‐off provided by the manufacturer
Target condition and reference standard(s)	Reference standard: RT‐qPCR, threshold not stated. Samples used: Respiratory samples. Timing of reference standard: Delay between first symptom onset and RT‐qPCR test was estimated at 4 days (± 1 days). Blinded to index test: Yes, prior Incorporated index test: No Definition of non‐COVID cases: Pre‐pandemic Samples used: NA as pre‐pandemic Timing of reference standard: NA as pre‐pandemic. Blinded to index test: Yes Incorporated index test: No
Flow and timing	Time interval between index and reference tests: >= 14 days All patients received same reference standard: No [1] PCR [2] Pre‐pandemic Missing data: not stated Uninterpretable results: not stated Indeterminate results: Thresholds for 'doubtful' results but no results recorded in this category [A] For the doubtful sample with the LIAISON®SARS‐CoV‐2 IgG kit, the sample must be retested in duplicate. If at least two of three results were doubtful, the sample was considered positive. If two of the results/three are < 12.0 AU/mL, the sample was negative. Unit of analysis: Patients
Comparative
Notes	Funding: None declared Publication status: Published paper Source: De Gruyter Clinical Chemistry & Laboratory Medicine Author COI: Authors stated no conflict of interest.
Methodological quality
Item	Authors' judgement	Risk of bias	Applicability concerns
DOMAIN 1: Patient Selection
Was a consecutive or random sample of patients enrolled?	Unclear
Was a case‐control design avoided?	No
Did the study avoid inappropriate exclusions?	Unclear
Did the study avoid inappropriate inclusions?	No
Could the selection of patients have introduced bias?		High risk
Are there concerns that the included patients and setting do not match the review question?			High
DOMAIN 2: Index Test (All tests)
DOMAIN 2: Index Test (Antibody tests)
Were the index test results interpreted without knowledge of the results of the reference standard?	Unclear
If a threshold was used, was it pre‐specified?	Yes
Could the conduct or interpretation of the index test have introduced bias?		Unclear risk
Are there concerns that the index test, its conduct, or interpretation differ from the review question?			Low concern
DOMAIN 3: Reference Standard
Is the reference standards likely to correctly classify the target condition?	Yes
Were the reference standard results interpreted without knowledge of the results of the index tests?	Yes
The reference standard does not incorporate the index test	Yes
Could the reference standard, its conduct, or its interpretation have introduced bias?		Low risk
Are there concerns that the target condition as defined by the reference standard does not match the question?			High
DOMAIN 4: Flow and Timing
Was there an appropriate interval between index test and reference standard?	Unclear
Did all patients receive the same reference standard?	No
Were all patients included in the analysis?	Yes
Did all participants receive a reference standard?	Unclear
Were results presented per patient?	Yes
Could the patient flow have introduced bias?		High risk