Diagnostic performance of rapid antigen tests (RAT) for COVID-19 and factors associated with RAT-negative results … – BMC Infectious Diseases

Study design, setting, and participants

We conducted a retrospective study among individuals who were tested for SARS-CoV-2 using RAT and RT-PCR during the same clinical encounter between 9 May 2022 and 21 November 2022. Under the Acute Respiratory Infection (ARI) surveillance programme at 11 polyclinics in Singapore which served as sentinel outpatient primary care sites, individuals presenting with acute respiratory symptoms of cough, runny nose, sore throat and/or fever were randomly selected and offered to join the surveillance programme by their clinician. Individuals who were willing to participate then underwent testing with a healthcare-administered RAT and RT-PCR. We evaluated the two RAT kits predominantly used in the sentinel clinics, Flowflex SARS-CoV-2 Antigen Rapid Test (Acon Laboratories) and STANDARD Q COVID-19 Ag Home Test (SD Biosensor).

Specimens for RT-PCR were tested using the BioFire Respiratory 2.1 (RP2.1) Panel (bioMrieux, France), a multiplex PCR which allows the simultaneous detection of multiple viral and bacterial respiratory organisms, including SARS-CoV-2 [14]. RT-PCR-positive cases were those which were SARS-CoV-2-positive on RP2.1 panel. The SARS-CoV-2-positive specimens were then tested using TaqPath COVID-19 Combo Kit (ThermoFisher, USA) and selected for whole genome sequencing (WGS) based on the cycle threshold (Ct) and S-gene target failure (SGTF) status. 40 PCR cycles were run as recommended by the manufacturer, and specimens with Ct value<30 could be selected for WGS.

WGS was conducted on specimens using the ARTIC nCoV-2019 amplicon panel (Integrated DNA Technologies) and the Nextera XT DNA Library Preparation Kit (Illumina) on the Illumina MiSeq platform in accordance with the manufacturers instructions. The viral genome sequences were assembled from raw data by in-house pipelines and the viral lineages were determined by Pangolin.

Data were collected from databases maintained by the National Public Health Laboratory and Ministry of Health, Singapore. Paired RAT and RT-PCR results for SARS-CoV-2, as well as age, sex, ethnicity, brand of RAT kit, presence of co-infection with other viral respiratory pathogen(s), clinic attended, vaccination status at time of infection, previous infection status, days between symptom onset and sample collection and SARS-CoV-2 lineage (if sequenced) were collected. Ct value was used as a proxy indicator of viral load, based on the lowest Ct value of the three targets in the TaqPath COVID-19 Combo Kit assay. Swab collection site for RT-PCR (nasopharyngeal, nasal, oropharyngeal and mid-turbinate, or throat) was recorded by healthcare staff and collected, but anatomical collection site of RAT was not known. Completion of the primary vaccination series was defined as two doses of Pfizer-BioNTech/Comirnaty or Moderna-Spikevax, three doses of Sinovac-CoronaVac or Sinopharm BBIBP-CorV, or two doses of non-mRNA vaccines approved under the World Health Organization (WHO) Emergency Use Listing besides Sinovac-CoronaVac and Sinopharm BBIBP-CorV [15]. Individuals who received additional vaccine doses after the primary vaccination series were considered boosted.

Those who had a SARS-CoV-2 infection notified to the Ministry of Health at least 90 days before the date of study inclusion were considered to have a previous documented infection [16]. In January 2022, Omicron overtook Delta as the predominant strain in Singapore and comprised over 91% of local cases which were sequenced [17]. Hence, individuals with a documented infection episode before 1 January 2022 were considered to have a previous pre-Omicron infection, while those with a previous documented infection from 1 January 2022 were assumed to have an Omicron infection. Individuals who tested RT-PCR-positive during the study period and did not have a previous documented infection were considered first infections.

RAT sensitivity, specificity, negative predictive value, and positive predictive value were calculated using BioFire RP2.1 Panel RT-PCR as the reference standard, with 95% CI calculated. Logistic regression models were used to estimate odds ratios (OR) of factors with negative RAT results among those who were SARS-CoV-2-positive by RT-PCR. Two multivariable logistic regressions were constructed. Model 1 adjusted for demographics, COVID-19 vaccination status, RAT brand, presence of co-infection with other respiratory pathogens, previous known SARS-CoV-2 infection, days between symptom onset and sample collection, SARS-CoV-2 lineage, PCR sample type and clinic visited, while Model 2 adjusted for factors in Model 1 and Ct value, to elucidate the effect of Ct values on the associations. Adjusted ORs of Models 1 and 2 were labelled aOR1 and aOR2 respectively. All data analysis was performed using Stata version 15.0(StataCorp, College Station, TX, USA).A p-value of <0.05 was considered statistically significant.

The study received ethics approval from the NHG Domain Specific Review Board (DSRB Ref: 2023/00131) with waiver of informed consent.

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Diagnostic performance of rapid antigen tests (RAT) for COVID-19 and factors associated with RAT-negative results ... - BMC Infectious Diseases