Kowitdamrong 2020 [A].

Study characteristics
Patient Sampling	Purpose: Diagnosis of current acute‐phase infection, current convalescent‐phase infection Design: Multi‐group study to estimate sensitivity and specificity [1] Confirmed COVID cases (118 patients with 213 samples); [2] Non‐COVID controls (n = 171); [2a] COVID suspects with negative PCR (n = 49); [2b] Concurrent patients with other respiratory infections (n = 20); [2c] Healthy volunteers (pre‐pandemic) (n = 20); [2d] Pre‐pandemic healthy blood donors (n = 82). Recruitment: Not stated Prospective or retrospective: [1] Prospective [2a] and [2b] Prospective [2c] Unclear (possibly retrospective) [2d] Retrospective Sample size: 289 (118) participants with 384 (213) samples Further detail: Inclusion: [1] Confirmed COVID‐19 cases defined as those that tested positive for SARS‐CoV‐2 RNA using real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) testing of combined nasopharyngeal and throat swab (NT) samples [2a] Plasma samples collected from May 1 to May 31, 2020, from patients under investigation (PUI) for COVID‐19 with RT‐PCR results that were negative for SARS‐CoV‐2 [2b] Serum specimens collected from May 1 to May 31, 2020 from patients with other infections (dengue, HBV, HCV, HIV, mumps, measles, rubella, EBV, CMV, VZV, HSV, and treponema) [2c] Plasma samples collected from healthy volunteers in the laboratory (prior to February 2020) [2d] Plasma samples leftover from healthy blood donors prior to February 2020 No exclusion criteria reported
Patient characteristics and setting	Setting: Not stated Location: Thai Red Cross Emerging Infectious Diseases Clinical Center (TRC‐EIDCC, King Chulalongkorn Memorial Hospital) and the Faculty of Medicine at Chulalongkorn University, Bangkok, Thailand Country: Thailand Dates: March 10 to May 31, 2020. Symptoms and severity: mild (upper respiratory symptoms) 59/118, moderate (pneumonia without hypoxia) 27/118, severe (pneumonia with hypoxia) 32/118. Demographics: Adult patients; median age of 38 years (IQR: 27–48); 47 (40%) male Exposure history: Not stated Non‐Covid group 1: [2a] Covid suspects with negative‐PCR Source: [2a] collected from May 1 to May 31, 2020, from patients under investigation (PUI) for COVID‐19 with RT‐PCR results that were negative for SARS‐CoV‐2 Characteristics: Median age 47 (IQR 28–65) years; 25 (51%) male Non‐Covid group 2: [2b] Concurrent patients with other diseases [2c] Pre‐pandemic healthy controls [2d] Pre‐pandemic healthy controls Source: [2b] Serum specimens collected from May 1 to May 31, 2020 from patients with other infections [2c] healthy volunteers in the laboratory prior to February 2020 [2d] healthy blood donors prior to February 2020 Characteristics: [2b] Patients with other infections (dengue, HBV, HCV, HIV, mumps, measles, rubella, EBV, CMV, VZV, HSV, and treponema). [2c] healthy volunteers in the laboratory [2d] healthy blood donors.
Index tests	Test name: [A] anti‐SARS‐CoV‐2 ELISA IgA kit [B] anti‐SARS‐CoV‐2 ELISA IgG kit Manufacturer: [A] and [B] EUROIMMUN Antibody: [A] IgA [B] IgG Antigen target: [A] and [B] S1‐protein Evaluation setting: [A] and [B] Lab test performed in lab Test method: [A] and [B] ELISA Timing of samples: 0‐3 days pso: 37/213 4‐7 days pso: 49/213 8‐14 days pso: 45/213 15‐28 days pso: 21/213 > 28 days pso: 61/213 Samples used: Plasma and serum were aliquoted and stored at ‐20˚C prior to serological testing. Test operator: Not stated Definition of test positivity: Semi‐quantitative results were evaluated by calculating the ratio of extinction at 450 nm of each sample over the calibrator. [A] A cut‐off ratio of 1.1 was used for SARS‐CoV‐2 IgA, as suggested by the package insert. [B] The borderline cut‐off ratio of 0.8 for SARS‐CoV‐2 IgG was assigned as positive. Blinding reported: Not stated Threshold predefined: Manufacturer's threshold but unclear why they used the borderline threshold for IgG
Target condition and reference standard(s)	Reference standard: SARS‐CoV‐2 RNA using real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) testing performed in the Department of Microbiology of the Faculty of Medicine at Chulalongkorn University. SARS‐CoV‐2 RNA was detected using the cobas1 SARS‐CoV‐2 kit (Roche Diagnostics, Basel, Switzerland) on a fully automated cobas1 6800 system (Roche Diagnostics, Basel, Switzerland) according to the manufacturer’s recommendations. Nucleic acid was automatically extracted from 400 μL of the NT specimens in viral transport medium (VTM) along with added internal control RNA (RNA IC). Subsequent real‐time RT‐PCR was performed automatically by the system, targeting ORF1a/b and E genes specific to SARS‐CoV‐2 and pan‐Sarbecovirus, respectively. Samples used: combined nasopharyngeal and throat swab (NT) samples. Timing of reference standard: Not stated Blinded to index test: yes, prior to index test Incorporated index test: no Definition of non‐COVID cases: [2a] RT‐PCR results that were negative for SARS‐CoV‐2 [2b] Not stated/ None? [2c] Pre‐pandemic (prior February 2020) [2d] Pre‐pandemic (prior February 2020) Samples used: [2a] Not stated (possibly as for cases) [2b] None [2c] Pre‐pandemic [2d] Pre‐pandemic Timing of reference standard: Not stated Blinded to index test: yes, prior to index test Incorporated index test: no
Flow and timing	Time interval between index and reference tests: Not stated All patients received same reference standard: No Missing data: Not stated Uninterpretable results: Not stated Indeterminate results: Borderline results for IgG classed as positive Unit of analysis: [1] A total of 213 samples collected from 118 patients were tested for antibodies against SARS‐CoV‐2, with 36 patients having 1 sample, 69 patients having 2 samples, and 13 patients having 3 samples. [2] Patients
Comparative
Notes	Funding: This work was supported by funding to support Biobank from Ratchadapisek Sompoch Fund, Faculty of Medicine, Chulalongkorn University. Publication status: Published paper Source: PLOS One Author COI: The authors declared that no competing interests existed.
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?	Unclear
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?	No
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?		High 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?	No
Were results presented per patient?	No
Could the patient flow have introduced bias?		High risk