Aim, study setting and population
The aim of this extensive prospective study was to compare the efficacy of SARS-CoV-2 antibody detection between RDT and laboratory serology, trying to identify appropriate semi-quantitative cut-offs for RDT in relation with quantitative serology values and to evaluate diagnostic accuracy of RDT compared to the NAAT gold standard in an unselected adult population.
We completed this study at Udine Hospital (Italy), a 1,000-bed tertiary-care teaching hospital identified as a regional referral centre for COVID-19 patients and serving approximately 350,000 citizens. RDT and serology samples were simultaneously collected by trained nurses from a cohort of all consecutive adult in- and out-patients (≥ 18 years) attending the Infectious Disease Department with the diagnosis of confirmed COVID-19, suspicious COVID-19, and negative for COVID-19 (March-May 2020).
The concordance between serological and rapid tests measurements were analysed considering paired measures (same patient at the same time). Samples were collected at various phases of the follow-up after onset of symptoms as explained by the patient and divided as follow: early stage (< 15 days); late stage: between day 15 and day 30; between month 1 and 2, after month 2. A SARS-CoV-2 serological test and RDT follow-up test were performed for a subset of enrolled patients with a diagnosis of COVID 19 that accepted monthly serological controls (+/- 15 days), according to a previously established protocol. The details of this prospective cohort have been provided previously [2].
Acute COVID-19 and baseline definitions
Diagnosis of COVID-19 infection was established as confirmed (positive SARS-CoV-2 NAAT in nasopharyngeal swabs or bronchoalveolar lavage) or suspected (negative SARS-CoV-2 NAAT in respiratory tract samples but suggestive laboratory or imaging findings and/or positive SARS-CoV-2 serology) during the acute phase of the disease. Remaining patients were classified as negative for COVID-19 [4].
Patients were classified using the COVID-19 Disease Severity Scale and specifically, for the analysis, patients were classified into three groups: (1) asymptomatic, (2) mild, and (3) moderate to critical disease [5].
Laboratory methods
NAAT test. Respiratory samples were tested for SARS-CoV-2 using RT-PCR targeted that investigated the E gene for screening and then the RdRp and N genes of SARS-CoV- 2 for confirmation (Roche Respiratory Panel Assay). The specimens were considered positive if the cycle threshold (CT) value for at least one of the three genes was ≤ 36 [4].
Lateral flow immunoassays. Samples for LFA were obtained from one capillary blood drop (10–20 µL) obtained from a finger stick sample. The Cellex SARS-CoV-2 IgG/IgM Rapid Test is a lateral flow qualitative chromatographic immunoassay to detect IgG/IgM s against SARS-CoV-2 N and S protein with a positive percent agreement and negative percent agreement of 93.75% and 96.40% respectively [6].
Chemiluminescent immunoassay (CLIA). Samples for SARS-CoV-2 serologies were obtained from venous blood samples. iFlash-SARS-CoV-2 (Shenzhen Yhlo Biotech Co. Ltd. China, distributed in Italy by Pantec SRL), is a paramagnetic particle CLIA for detection of IgG/IgM against SARS-CoV-2 N and (non –RBD) S protein (cut-off for IgG/ IgM positivity > 10.0 kAU/L. The test performance has been documented to have a sensitivity and specificity of 86.1% and 99.2% for IgM and 93.7% and 96.3% for IgG, respectively [7].
Identification of semi-quantitative cut-offs for RDT
An internal scientific committee consisting of three investigators (two infectious disease specialists and one laboratory medicine specialist) the hospital developed a chromatographic intensity scale (Fig. 1) and participated in the interpretation of qualitative (positive or negative) and semi-quantitative LFA results. A picture of every rapid test was taken at the manufacturer’s established time of reading (15 min), a photographic archive of LFAs was made for every test and test results were independently reviewed by three of these investigators. Results were based on full consensus among the experts. According to the expert panel chromatographic scale, the IgG and IgM band was classified as either N, Negative; WP, Weak positive; or P, Positive. Figure 1 shows three RDT displaying an example of each possible result observed in our study.
Primary and secondary outcomes and data collection
Primary endpoints of the study were assessed to compare the efficacy of SARS-CoV-2 antibody detection between RDT and CLIA serology in different phases and severity setting of COVID disease trying to identify appropriate semi-quantitative cut-offs for RDT in relation with quantitative serology values. The secondary endpoint was to evaluate the diagnostic accuracy of RDT compared to the NAAT gold standard.
Statistical analysis
Descriptive statistics for categorical variables are presented as number (percent) and for continuous variables as mean ± standard deviation (SD) or median (interquartile range (IQR)). Normality was assessed using the Shapiro-Wilk test.
The relation between serological measurements and semi-quantitative interpretation of RDT was estimated using the Kruskal-Wallis test, while the relation between serological measurements and qualitative interpretation of RDT was estimated using the Mann-Whitney U test. The agreement between serological and semi-quantitative interpretation of RDT was studied with Kendall’s tau. The performance of IgG serological measurements to discriminate between the two level of qualitative interpretation of RDT was determined using De Long’s non-parametric receiver operating characteristic (ROC) analysis. Sensitivity, specificity, positive and negative predictive values and their 95% confidence intervals (CI) were calculated to assess diagnostic performance of qualitative interpretation of RDT compared to NAAT. All analyses were performed by STATA 17 statistical software, and statistical significance was set at p < 0.05.