Van Elslande 2020b [A].

Study characteristics
Patient Sampling	Purpose: Diagnosis of current acute and convalescent‐phase infection Design: Multi‐group study to assess sensitivity and specificity [1] Covid‐positive (n = 233 samples, 114 patients) [2] Covid‐negative, pre‐pandemic (n = 113) [2a] Pre‐pandemic respiratory infection (n = 49) [2b] Pre‐pandemic coronavirus (n = 24) [2c] Pre‐pandemic other infections (n = 40) Recruitment: [1] Patients PCR‐positive for COVID‐19 [2a] Pre‐pandemic serum samples from consecutive patients with a respiratory infection who had a PCR test for respiratory pathogens between September and November 2019 [2b] Pre‐pandemic patients with a confirmed non‐SARS‐CoV‐2 coronavirus infection collected 12‐42 days after the positive PCR, not stated [2c] Pre‐pandemic patients with antibodies against other pathogens (e.g. cytomegalovirus, Epstein Barr virus, human immunodeficiency virus) from routine serology testing Prospective or retrospective: Retrospective Sample size: 346 (233) Further detail: Inclusion [1] PCR‐confirmed SARS‐CoV‐2 infection [2a] Respiratory infection [2b] Non‐SARS‐CoV‐2 coronavirus infection [2c] Antibodies against other pathogens Exclusion: [1] Immunocompromised patients (e.g. acute leukaemia, treatment with azathioprine) excluded [2a] [2b][2c] Not stated
Patient characteristics and setting	Setting: Hospital inpatients Location: University Hospitals Leuven, Leuven Country: Belgium Dates: Not stated Symptoms and severity: All symptomatic, 36/114 patients were classified as critical (needed mechanical ventilation or fatal infection), 78 non‐critical (moderate) Demographics: 81 male, 33 female; median age 66.5 years (rage 23‐90 years) Exposure history: Not stated Non‐Covid group 1: [2a] Respiratory infections, pre‐pandemic Source: September to November 2019, University Hospitals Leuven Characteristics: Confirmed respiratory infection: HSV n = 19, CMV n = 13, entero/rhinovirus n = 8, S. pneumoniae n = 7, RSV n = 3, parainfluenza virus n = 2, HMPV n = 1, P. jirovecii n = 1, bocavirus n = 1, L. pneumophila n = 1 Non‐Covid group 2: [2b] Other human coronaviruses, pre‐pandemic [2c] Antibodies against various viruses, pre‐pandemic Source: [2b] [2c] Pre‐pandemic (before January 2020), University Hospitals Leuven Characteristics: [2b] Non‐SARS‐CoV‐2 coronavirus infection: a‐Cov HCoV‐229E n = 7, a‐Cov HCoV‐NL63 n = 6, B‐Cov HCoV‐OC43 n = 7, B‐CoV HCoV‐HKU1 n = 4 [2c] Antibodies against other pathogens: CMV n = 21, EBV n = 15, VZV IgG n = 10, HIV‐1 n = 8, HSV IgG n = 7, HAV/HBV/HCV n = 14
Index tests	Test name: [A] Roche Ig anti‐N [B] Abbott IgG anti‐N [C] Euro NCP IgG anti‐N [D] Mikrogen IgG anti‐N [E] Maglumi IgG anti‐N/S [F] Diasorin IgG anti‐S [G] Euro S1 IgG anti‐S Manufacturer: [A] Roche Diagnostics, Basel, Switzerland [B] Abbott Diagnostics, Lake Forest, Illinois [C] Euroimmun, Lubeck, Germany [D] Mikrogen, Neuried, Germany [E] Snibe, Shenzen, China [F] Diasorin, Saluggia, Italy [G] Euroimmun, Lubeck, Germany Antibody: [A] Total Ig antibodies [B]‐[G] IgG Antigen target: [A] N‐protein [B] N‐protein [C] N‐protein [D] N‐protein [E] N and S‐protein [F] S‐protein (S1 and S2) [G] S1‐protein Evaluation setting: Laboratory performed in laboratory Test method: [A] CLIA [B] CLIA [C] ELISA [D] ELISA [E] CLIA [F] CLIA [G] ELISA Timing of samples: 0‐6 days pso, n = 43 7‐13 days pso, n = 98 14‐17 days pso, n = 42 18‐21 days pso, n = 16 22‐27 days pso, n = 13 28‐37 days pso, n = 11 Samples used: Serum Test operator: Staff at University Hospitals Leuven (technical assistants) Definition of test positivity: Cut‐off [A] >= 1.0 [B] >= 1.4 [C] >= 0.8 positive, equivocal zone 0.8/1.1 [D] >= 20 positive, equivocal zone 20/24 [E] >= 1.0 [F] >= 12 positive, equivocal zone 12/15 [G] >= 0.8 positive, equivocal zone 0.8/1.1 Blinding reported: Not stated Threshold predefined: Yes, according to manufacturer
Target condition and reference standard(s)	Reference standard: RT‐PCR; described as 'in‐house method complying with the WHO guidelines', threshold not stated Samples used: Nasopharyngeal swabs (UTM, Copan, Italy) Timing of reference standard: 83.3% of patients were admitted the day of the first PCR‐positive result. Blinded to index test: Yes, prior Incorporated index test: No Definition of non‐COVID cases: Pre‐pandemic Samples used: NA pre‐pandemic Timing of reference standard: Pre‐pandemic Blinded to index test: yes, prior to index test Incorporated index test: No
Flow and timing	Time interval between index and reference tests: 83.3% of patients were admitted the day of the first PCR‐positive result. The median time between onset of symptoms and admission to the hospital was 7 days. 0‐6 days pso, n = 43 7‐13 days pso, n = 98 14‐17 days pso, n = 42 18‐21 days pso, n = 16 22‐27 days pso, n = 13 28‐37 days pso, n = 11 All patients received same reference standard: No Missing data: Not stated Uninterpretable results: Not stated Indeterminate results: Equivocal results [C][D][F][G] treated as positive Unit of analysis: Samples, only one sample included per patient per time frame
Comparative
Notes	Funding: Pieter Vermeersch reported personal fees from Roche, outside the submitted work. Katrien Lagrou reported personal fees and non‐financial support from Pfizer, personal fees and non‐financial support from MSD, personal fees from SMB Laboratoires, personal fees from Gilead, and personal fees from FUJIFILM Wako, outside the submitted work. The research did not receive any specific grant from funding agencies in the public, commercial or not‐for‐profit sectors. Publication status: Published paper Source: Clinical and Microbiology and Infection Author COI: Pieter Vermeersch reported personal fees from Roche, outside the submitted work. Katrien Lagrou reported personal fees and nonfinancial support from Pfizer, personal fees and non‐financial support from MSD, personal fees from SMB Laboratoires, personal fees from Gilead, and personal fees from FUJIFILM Wako, outside the submitted work. The other authors stated no conflicts of interests.
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?			Unclear
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?	Unclear
The reference standard does not incorporate the index test	Yes
Could the reference standard, its conduct, or its interpretation have introduced bias?		Unclear 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?	Unclear
Did all participants receive a reference standard?	Yes
Were results presented per patient?	Yes
Could the patient flow have introduced bias?		High risk