Direct Detection of Feline Coronavirus by Three Rapid Antigen Immunochromatographic Tests and by Real-Time PCR in Cat Shelters

Billy Bennett

The aim of this study was the direct detection of feline coronavirus by real-time PCR and by three different rapid immunochromatographic (RIM) tests detecting antigens in faecal samples of shelter cats. Based on sensitivity and specificity calculated for each of the RIM tests, the utility of RIM tests was compared. Seventy faecal samples originating from shelter cats housed in quarantine were examined. Out of 70 samples analyzed by real-time PCR, 44 (62.9%) were positive. Significantly more cats (p < 0.05) tested positive than negative. Neither age nor sex of the cats played a significant role (p > 0.05) in the shedding status of the virus.

The sensitivity of the RIM tests was found to be at low (<35%; RIM tests A and C) to satisfactory level (>50%, RIM test B). The number of virus particles determined by real-time RT-PCR analysis did not significantly correlate with the results detected by any of the RIM tests (p > 0.05). The results of this study indicate that the use of rapid antigen RIM tests in routine screening of FCoV shedding status in shelter cats is limited due to the occurrence of a high number of false-negative results.

Antigen Test Positivity After COVID-19 Isolation – Yukon-Kuskokwim Delta Region, Alaska

Isolation is recommended during acute infection with SARS-CoV-2, the virus that causes COVID-19, but the duration of infectiousness varies among individual persons. Rapid antigen test results have been correlated with detection of viable virus (1-3) and might inform isolation guidance, but data are limited for the recently emerged SARS-CoV-2 B.1.1.529 (Omicron) variant. On January 5, 2022, the Yukon-Kuskokwim Health Corporation (YKHC) recommended that persons with SARS-CoV-2 infection isolate for 10 days after symptom onset (or, for asymptomatic persons, 10 days after a positive nucleic acid amplification or antigen test result).

However, isolation could end after 5-9 days if symptoms were resolving or absent, fever was absent for ≥24 hours without fever-reducing medications, and an Abbott BinaxNOW COVID-19 Ag (BinaxNOW) rapid antigen test result was negative. Antigen test results and associated individual characteristics were analyzed among 3,502 infections reported to YKHC during January 1-February 9, 2022. After 5-9 days, 396 of 729 persons evaluated (54.3%) had a positive antigen test result, with a declining percentage positive over time.

In a multivariable model, a positive antigen test result was more likely after 5 days compared with 9 days (adjusted odds ratio [aOR] = 6.39) or after symptomatic infection (aOR = 9.63), and less likely after previous infection (aOR = 0.30), receipt of a primary COVID-19 vaccination series (aOR = 0.60), or after both previous infection and receipt of a primary COVID-19 vaccination series (aOR = 0.17). Antigen tests might be a useful tool to guide recommendations for isolation after SARS-CoV-2 infection. During the 10 days after infection, persons might be infectious to others and are recommended to wear a well-fitting mask when around others, even if ending isolation after 5 days.

Impaired detection of omicron by SARS-CoV-2 rapid antigen tests

Since autumn 2020, rapid antigen tests (RATs) have been implemented in several countries as an important pillar of the national testing strategy to rapidly screen for infections on site during the SARS-CoV-2 pandemic. The current surge in infection rates around the globe is driven by the variant of concern (VoC) omicron (B.1.1.529). Here, we evaluated the performance of nine SARS-CoV-2 RATs in a single-centre laboratory study. We examined a total of 115 SARS-CoV-2 PCR-negative and 166 SARS-CoV-2 PCR-positive respiratory swab samples (101 omicron, 65 delta (B.1.617.2)) collected from October 2021 until January 2022 as well as cell culture-expanded clinical isolates of both VoCs.

In an assessment of the analytical sensitivity in clinical specimen, the 50% limit of detection (LoD50) ranged from 1.77 × 10⁶ to 7.03 × 10⁷ RNA copies subjected to the RAT for omicron compared to 1.32 × 10⁵ to 2.05 × 10⁶ for delta. To score positive in these point-of-care tests, up to 10-fold (LoD50) or 101-fold (LoD95) higher virus loads were required for omicron- compared to delta-containing samples. The rates of true positive test results for omicron samples in the highest virus load category (Ct values < 25) ranged between 31.4 and 77.8%, while they dropped to 0-8.3% for samples with intermediate Ct values (25-30). Of note, testing of expanded virus stocks suggested a comparable RAT sensitivity of both VoCs, questioning the predictive value of this type of in vitro-studies for clinical performance. Given their importance for national test strategies in the current omicron wave, awareness must be increased for the reduced detection rate of omicron infections by RATs and a short list of suitable RATs that fulfill the minimal requirements of performance should be rapidly disclosed.

China’s NMPA perspective on the clinical performance of SARS-CoV-2 antigen test reagents

The COVID-19 pandemic continues to spread all over the world. In the process of emergency use authorization, the Center for Medical Device Evaluation of the China National Medical Products Administration issued ‘Key Points of Technical Review for the Registration of SARS-CoV-2 Antigen/Antibody Detection Reagents’ as the guidance of registration of antigen and antibody test reagents for the industry. In this document, clinical evaluation requirements of antigen detection reagents are elaborated. Based on the Key Points document and the authors’ review practice, this article explains the evaluation methods and requirements of clinical performance of SARS-CoV-2 antigen-detecting rapid diagnostic tests, then analyzes the application scenarios and intended use of antigen detection reagents.

Prevalence of Pfhrp2/3 gene deletions among false negative rapid antigen test results in central India

Background &objectives: The diagnosis of Plasmodium falciparum malaria is widely dependent on the P. falciparum histidine rich protein 2 (PfHRP2) antigens based rapid diagnostic tests. There are few possible factors like Pfhrp2 polymorphism, Pfhrp2 deletion and density of malaria parasite which can affect the sensitivity of the Pf-HRP2-based RDT. The primary objective of the investigation was to check whether the Pfhrp2 gene deletion is the primary cause of RDT false negative cases.

Febrile patients from three districts of Chhattisgarh, India were screened for malaria during 2016-2017 by microscopy and RDT. All microscopy P. falciparum positive samples were validated by PCR. Microscopy positive and RDT negative samples were analyzed for the presence of Exon 2, across Exon 1-2, upstream and downstream of both the Pfhrp2 and Pfhrp3 genes fragment by PCR.

Out of 203 screened samples, 85 were detected positive for P. falciparum malaria based on microscopy and PCR. Among these 85 P. falciparum positive samples, 4 samples were observed Pf-HRP2 RDT negative. Although, it signified that the RDTs used were reliable with sensitivity of 95.3% (81/85). 3/4 PfHRP2-RDT negative samples of the P. falciparum isolates exhibited complete deletion of Pfhrp2 and Pfhrp3 genes and one sample was found RDT false negative due to high parasite density.

Interpretation & conclusion: Pfhrp2 and Pfhrp3 deletions that result in false negative RDTs were uncommon in our setting. The continued monitoring of RDTS which results in false negative tests due to Pfhrp2/3 gene deletion is

the need of the hour for an effective malaria elimination strategy.

OraSure InteliSwab Rapid Antigen Test performance with the SARS-CoV-2 Variants of Concern Alpha, Beta, Gamma, Delta, and Omicron

The emergence of SARS-CoV-2 in the human population and the resulting COVID-19 pandemic has led to the development of various diagnostic tests. The OraSure InteliSwab COVID-19 Rapid Test is a recently developed and FDA emergency use authorized rapid antigen-detecting test that functions as a lateral flow device targeting the nucleocapsid protein. Due to SARS-CoV-2 evolution, there is a need to evaluate the sensitivity of rapid antigen-detecting tests for new variants, especially variants of concern like Omicron. In this study, the sensitivity of the OraSure InteliSwab Test was investigated using cultured strains of the known variants of concern (VOCs, Alpha, Beta, Gamma, Delta, and Omicron) and the ancestral lineage (lineage A).

Based on dilution series in the cell culture medium, an approximate limit of detection for each variant was determined. The OraSure InteliSwab Test showed an overall comparable performance using recombinant nucleocapsid protein and different cultured variants with recorded limits of detection ranging between 3.77 × 10 ⁵ and 9.13 × 10 ⁵ RNA copies/mL. Finally, the sensitivity was evaluated using oropharyngeal swabs from Syrian golden hamsters inoculated with the 6 VOCs. Ultimately, the OraSure InteliSwab COVID-19 Rapid Test showed no decrease in sensitivity between the ancestral SARS-CoV-2 strain and any VOCs including Omicron.

Bean Extract-Based Gargle for Efficient Diagnosis of Active COVID-19 Infection Using Rapid Antigen Tests

The antigen-based rapid diagnostic test (Ag-RDT) using saliva specimens is fast, noninvasive, and suitable for SARS-CoV-2 self-testing, unlike nasopharyngeal swab (NPS) testing. We evaluated a novel Beanguard gargle (BG)-based virus collection method that can be applied to Ag-RDT as an alternative to the current RT-PCR with an NPS for early diagnosis of COVID-19. This clinical trial comprised 102 COVID-19-positive patients hospitalized after a governmental screening process and 100 healthy individuals. Paired NPS and BG-based saliva specimens from COVID-19 patients and healthy individuals were analyzed using NPS-RT-PCR, BG-RT-PCR, and BG-Ag-RDTs, whose diagnostic performance for detecting SARS-CoV-2 was compared. BG-Ag-RDTs showed high sensitivity (97.8%) and specificity (100%) in 45 patients within 6 days of illness and detected all cases of SARS-CoV-2 Alpha and Delta variants.

NATtrol Zika Virus Stock (Qualitative) (1 mL)
TEST	Zeptometrix	1 mL	1327.97 EUR
CA199 (Cancer antigen) ELISA test
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CA153 (Cancer antigen) ELISA test
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CA125 (Cancer antigen) ELISA test
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CA50 (Cancer antigen) ELISA test
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NOVATest Antigen Rapid Test Kit (For Single Use) (NOVA Test)
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PSA (Prostate-specific antigen) ELISA test
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NOVATest Antigen Rapid Kit (NOVA Test)
nCov-500	Atlas Link Technology	20 Tests	162 EUR
HEV-Ag hepatitis E antigen ELISA test
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SARS-CoV-2 Rapid Antigen Test Nasal
9901-NCOV-03G	Roche Diagnostics	25 Tests/Kit	112.8 EUR
HBeAg hepatitis B E antigen ELISA test
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Human Streptococcus Pneumoniae (SP) Antigen Rapid Test Kit
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Avian Influenza Virus Antigen Rapid Test Kit (Colloidal gold)
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Newcastle Disease Virus Antigen Rapid Test Kit (Colloidal gold)
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Human Chlamydia Trachomatis Antigen Rapid Test Kit (Colloidal gold)
abx092049-20tests	Abbexa	20 tests	276 EUR
Coronavirus (SARS-Cov-2) Antigen Rapid Test Device (Saliva)
IOV87952	INVBIO	20T/kit	46.8 EUR

In 11 asymptomatic active COVID-19 cases, both BG-Ag-RDTs and BG-RT-PCR showed sensitivities and specificities of 100%. Sensitivities of BG-Ag-RDT and BG-RT-PCR toward salivary viral detection were highly concordant, with no discrimination between symptomatic (97.0%), asymptomatic (100%), or SARS-CoV-2 variant (100%) cases. The intermolecular interactions between SARS-CoV-2 spike proteins and truncated canavalin, an active ingredient from the bean extract (BE), were observed in terms of physicochemical properties. The detachment of the SARS-CoV-2 receptor-binding domain from hACE2 increased as the BE concentration increased, allowing the release of the virus from hACE2 for early diagnosis. Using BG-based saliva specimens remarkably enhances the Ag-RDT diagnostic performance as an alternative to NPS and enables noninvasive, rapid, and accurate COVID-19 self-testing and mass screening, supporting efficient COVID-19 management.

An Ag-RDT is less likely to be accepted as an initial test method for early diagnosis owing to its low sensitivity. However, our self-collection method, Ag-RDT using BG-based saliva specimens, showed significantly enhanced detection sensitivity and specificity toward SARS-CoV-2 including the Alpha and Delta variants in all patients tested within 6 days of illness. The method represents an attractive alternative to nasopharyngeal swabs for the early diagnosis of symptomatic and asymptomatic COVID-19 cases. The evidence suggests that the method could have a potential for mass screening and monitoring of COVID-19 cases.