Montesinos 2020 [A].

Study characteristics
Patient Sampling	Purpose: Diagnosis of acute and convalescent‐phase infection using five tests for detection of SARS‐CoV‐2 IgG, IgM and IgA antibodies Design: Three‐group study to estimate sensitivity and specificity: [1] COVID‐19 patients confirmed by RT‐qPCR and CT‐ scans (n = 128) [B2 Negative controls. Stored sera from Jan 2018 to Aug 2019 (n = 62) included samples with a potential cross‐reaction to the SARS‐CoV‐2 immunoassays, namely, EBV infection (n = 5), CMV infection (n = 11), M. pneumoniae infection (n = 8), Parvovirus infection (n = 1), HBV infection (n = 1), Bartonella henselae infection (n = 1), Brucella spp infection (n = 1), auto‐immune pathologies (Anti‐DNA, n = 1; Anti‐PL12, n = 1; Anti Scl‐70, n = 1) and, [3] Sera from healthy volunteers (n = 10) obtained during the epidemic period (April 2020) Recruitment: Unclear. Prospective or retrospective: Retrospective Sample size: 200 (128) Further detail: No more details available
Patient characteristics and setting	Setting: Unclear Location: Laboratoire Hospitalier Universitaire de Bruxelles ‐ Universitair Laboratorium Brussel (LHUB‐ULB) and the Microbiology Department of Cliniques Universitaires Saint Luc‐ UCLouvain (CUSL) in Brussels, Belgium. Country: Belgium Dates: Not stated Symptoms and severity: No information Demographics: No information Exposure history: No information Non‐Covid group 1: [2] Pre‐pandemic controls Source: Stored sera from Jan 2018 to Aug 2019 (n = 62) . Laboratoire Hospitalier Universitaire de Bruxelles ‐ Universitair Laboratorium Brussel (LHUB‐ULB) and the Microbiology Department of Cliniques Universitaires Saint Luc‐ UCLouvain (CUSL) in Brussels, Belgium Characteristics: No information Non‐Covid group 2: [3] Contemporaneous healthy Source: Sera from healthy volunteers (n = 10) obtained during the epidemic period (April 2020) Characteristics: No information
Index tests	Test name: [A] 2019‐nCov Antibody IgG/IgM [B] anti‐SARS‐COV‐2 IgA [C] anti‐SARS‐COV‐2 IgG [D] anti‐SARS‐COV‐2 IgA or IgG [E] rapid test cassette [F] MAGLUMI 2019‐nCoV IgG/IgM [G] QuickZen COVID‐19 IgM/IgG Manufacturer: [A] Avioq Bio‐Tech [B] EUROIMMUN [C] EUROIMMUN [D] EUROIMMUN [E] LaboOn Time [F] Snibe Diagnostic [G] ZenTech Antibody: [A] IgG, IgM, IgG or IgM [B] to [D] IgG, IgA, IgG or IgA Antigen target: [A] magnetic microbeads coated with SARS‐CoV‐2 recombinant antigen labelled with ABEI [B to D] recombinant S1 structural protein [C] to [G] SARS‐CoV‐2 antigen Evaluation setting: All laboratory‐evaluations Test method: [A, E, G] Lateral flow immunoassays; [B to D] Enzyme‐Linked Immunosorbent Assay (ELISA), [F] chemiluminescent immunoassay (CLIA) Timing of samples: Day 0 to > 15; no further details Samples used: All evaluated using serum; 10 μL serum used for LFAs Test operator: Not stated Definition of test positivity: [B to D] Ratio of the extinction of samples over the extinction of the calibrator calculated. The ratio interpretation was as follows: < 0.8 = negative, ≥ 0.8 to < 1.1 = borderline, ≥ 1.1 = positive. [F] The thresholds of positivity for these automated immunoassays were 1.0 AU/mL for IgM and IgG [A, E, G] Visible line ‐ read and interpreted 10 min after the test Blinding reported: Not stated Threshold predefined: as per manufacturer
Target condition and reference standard(s)	Reference standard: RT‐PCR and CT scan Two RT‐qPCR kits: RealStar® SARS‐CoV‐2 RT‐PCR kit 1.0 (Altona Diagnostics, Hambourg, Germany) at LHUB‐ULB; Genesig® Real‐Time PCR Coronavirus (COVID‐19) (Primerdesign Ltd, Chandlers Ford, United Kingdom) at CUSL No further detail regarding how CT contributed to diagnosis Samples used: Not stated Timing of reference standard: Not stated Blinded to index test: Yes, since it preceded it Incorporated index test: No Definition of non‐COVID cases: [2] Pre‐pandemic stored samples with known (non‐COVID) diagnoses [3] contemporaneous healthy; no reference standard reported to confirm absence of disease Samples used: Serum Timing of reference standard: NA Blinded to index test: Yes, since it preceded it Incorporated index test: No
Flow and timing	Time interval between index and reference tests: Not stated All patients received same reference standard: Unclear; not stated whether CT used in all patients or whether +ve CT scan required to define positive Missing data: No; For ELISA and lateral flow tests all samples were reported in overall result but for CLIA, 2 cases were missing in IgG and IgM analyses, and 6 in IgG/IgM ‐ no reason given Uninterpretable results: None reported Indeterminate results: None reported Unit of analysis: Patients
Comparative
Notes	Funding: No specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors. Manufacturers offered the reagents for validation Publication status: Published paper Source: Journal of Clinical Virology Author COI: The authors declared that they had 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?	Unclear
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?	Unclear
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?		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?	Unclear
Were all patients included in the analysis?	Unclear
Did all participants receive a reference standard?	Yes
Were results presented per patient?	Unclear
Could the patient flow have introduced bias?		Unclear risk