Evaluation of four laboratory-based high-throughput SARS-CoV-2 automated antigen tests compared to RT-PCR on nasal and oropharyngeal samples.

BACKGROUND: The demand for RT-PCR testing has been unprecedented during the SARS-CoV-2 pandemic. Fully automated antigen tests (AAT) are less cumbersome than RT-PCR, but data on performance compared to RT-PCR are scarce. METHODS: The study consists of two parts. A retrospective analytical part, comparing the performance of four different AAT on 100 negative and 204 RT-PCR positive deep oropharyngeal samples divided into four groups based on RT-PCR cycle of quantification levels. In the prospective clinical part, 206 individuals positive for and 199 individuals negative for SARS-CoV-2 were sampled from either the anterior nasal cavity (mid-turbinate) or by deep oropharyngeal swabs or both. The performance of AATs was compared to RT-PCR. RESULTS: The overall analytical sensitivity of the AATs differed significantly from 42% (95% CI 35-49) to 60% (95% CI 53-67) with 100% analytical specificity. Clinical sensitivity of the AATs differed significantly from 26% (95% CI 20-32) to 88% (95% CI 84-93) with significant higher sensitivity for mid-turbinate nasal swabs compared to deep oropharyngeal swabs. Clinical specificity varied from 97% to 100%. CONCLUSION: All AATs were highly specific for detection of SARS-CoV-2. Three of the four AATs were significantly more sensitive than the fourth AAT both in terms of analytical and clinical sensitivity. Anatomical test location significantly influenced the clinical sensitivity of AATs.

Emergence of circulating influenza A H3N2 viruses with genetic drift in the matrix gene: be alert of false-negative test results.

In March 2022, we observed samples with a negative fluorescent signal (60.5%, n = 43) for the influenza A matrix gene and a stronger positive signal for subtype A(H3N2). Forty-three samples were positive in InfA (H3N2) (mean Cq 30.9, range 23.9-35.1), and 26 of the 43 samples were negative in InfA matrix (mean Cq 28.0, range 23.2-30.6). Our multiplex test is a laboratory-developed four-target, four-color influenza A reverse-transcription PCR assay targeting the matrix gene, subtypes A(H3N2) and A(H1N1)pdm09. Several samples were negative when retested on commercial influenza Point-of-Care assays. As the matrix gene is a stand-alone target in most commercial diagnostic assays, we caution against false-negative subtype A test results.

A nationwide analytical and clinical evaluation of 44 rapid antigen tests for SARS-CoV-2 compared to RT-qPCR.

BACKGROUND: The SARS-CoV-2 pandemic has resulted in massive testing by Rapid Antigen Tests (RAT) without solid independent data regarding clinical performance being available. Thus, decision on purchase of a specific RAT may rely on manufacturer-provided data and limited peer-reviewed data. METHODS: This study consists of two parts. In the retrospective analytical part, 33 RAT from 25 manufacturers were compared to RT-PCR on 100 negative and 204 positive deep oropharyngeal cavity samples divided into four groups based on RT-PCR Cq levels. In the prospective clinical part, nearly 200 individuals positive for SARS-CoV-2 and nearly 200 individuals negative for SARS-CoV-2 by routine RT-PCR testing were retested within 72 h for each of 44 included RAT from 26 manufacturers applying RT-PCR as the reference method. RESULTS: The overall analytical sensitivity differed significantly between the 33 included RAT; from 2.5% (95% CI 0.5-4.8) to 42% (95% CI 35-49). All RAT presented analytical specificities of 100%. Likewise, the overall clinical sensitivity varied significantly between the 44 included RAT; from 2.5% (95% CI 0.5-4.8) to 94% (95% CI 91-97). All RAT presented clinical specificities between 98 and 100%. CONCLUSION: The study presents analytical as well as clinical performance data for 44 commercially available RAT compared to the same RT-PCR test. The study enables identification of individual RAT that has significantly higher sensitivity than other included RAT and may aid decision makers in selecting between the included RAT. FUNDING: The study was funded by a participant fee for each test and the Danish Regions.

SARS-CoV-2 detection using reverse transcription strand invasion based amplification and a portable compact size instrument.

Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.

An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens.

BACKGROUND: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. OBJECTIVES: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. STUDY DESIGN: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. RESULTS: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08-6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. CONCLUSIONS: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.

Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype.

OBJECTIVES: To establish the prevalence of the AmpC beta-lactamase phenotype in clinical isolates of Escherichia coli and characterize the genetic resistance mechanisms causing the observed phenotype. METHODS: Clinical E. coli (n = 74) with reduced susceptibility to third-generation cephalosporins and resistance to cefoxitin were collected from the Department of Clinical Microbiology at Hvidovre Hospital, Denmark, in 2006. The AmpC disc test was used to confirm expression of AmpC, and test-positive strains were selected for further antimicrobial susceptibility testing and molecular characterization. Hyperproduction of AmpC beta-lactamase was confirmed by isoelectric focusing (IEF). The presence of a plasmid-mediated ampC gene (pAmpC) was detected by multiplex PCR. The promoter and the entire reading frame of the chromosomal ampC gene were sequenced to identify promoter mutations associated with hyperproduction and gene mutations associated with extended-spectrum AmpC (ESAC) beta-lactamase activity. RESULTS: Twenty-four isolates exhibited a positive AmpC disc test. IEF confirmed AmpC expression in all isolates except one. Four isolates contained a bla(CMY-2) gene. These were not clonally related by multilocus sequence typing (MLST). The remaining isolates all had mutations or insertions in the promoter region, which could explain increased expression of the chromosomal AmpC enzyme. Mutations in the ampC gene associated with extended activity were rare and did not cause resistance to cefepime. Sequencing of ampC showed that most isolates were not clonally related. CONCLUSIONS: E. coli expressing an AmpC phenotype occur sporadically and cause significant resistance to cephalosporins. The majority of these are hyperproducing chromosomal ampC although some isolates have acquired pAmpC.