Molecular epidemiology of the SARS-CoV-2 variant Omicron BA.2 sub-lineage in Denmark, 29 November 2021 to 2 January 2022.

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Point of care testing for infectious disease: ownership and quality.

Traditionally, diagnosis of acute infections has been organism-growth based, which makes timely and actionable infection diagnosis a major challenge. In addition, traditional microbial detection methods, including direct microscopy, are not suited for outsourcing to clinical, non-laboratory-educated personnel. Optimal management of patients with known or suspected clinical infections, such as targeted (or no) antimicrobial treatment and correct use of single room contact isolation facilities, requires rapid identification of the causative infectious microorganism. We are now facing a new disruptive paradigm shift in diagnostic microbiology. The availability of small-footprint robust instruments with easy-to-use assay kits allows non-laboratory-trained nurses and physicians to perform high-quality molecular diagnostics in a near-patient setting with results available in <30 minutes. This technology is currently breaking the centralized laboratory monopoly on the delivery of gold-standard clinical microbiology diagnostics. There is clear potential for huge positive impacts on clinical patient management and antibiotic stewardship, especially in settings where access to timely laboratory test results is not possible, but there are also potentially huge risks. Moving diagnostic testing away from the controlled diagnostic laboratory environment will lead to risks such as increased risk of inappropriate use of the diagnostic tests, insufficient training of staff performing the tests, incorrect interpretation of the test results, lack of quality control procedures, failure to capture test results in electronic patient records and compromised local as well as national surveillance. To reap the upside and avoid the downside of point-of-care infectious disease testing, the diagnostic laboratory needs to maintain oversight, and each institution must have a clear strategy for implementation and execution. If we fail, the risks could outweigh the benefits.

Spotlight influenza: Influenza surveillance before and after the introduction of point-of-care testing in Denmark, season 2014/15 to 2018/19.

BackgroundIn Denmark, influenza surveillance is ensured by data capturing from existing population-based registers. Since 2017, point-of-care (POC) testing has been implemented outside the regional clinical microbiology departments (CMD).AimWe aimed to assess influenza laboratory results in view of the introduction of POC testing.MethodsWe retrospectively observed routine surveillance data on national influenza tests before and after the introduction of POC testing as available in the Danish Microbiological Database. Also, we conducted a questionnaire study among Danish CMD about influenza diagnostics.ResultsBetween the seasons 2014/15 and 2018/19, 199,744 influenza tests were performed in Denmark of which 44,161 were positive (22%). After the introduction of POC testing, the overall percentage of positive influenza tests per season did not decrease. The seasonal influenza test incidence was higher in all observed age groups. The number of operating testing platforms placed outside a CMD and with an instrument analytical time ≤ 3 h increased after 2017. Regionally, the number of tests registered as POC in the Danish Microbiological Database and the number of tests performed with an instrument analytical time ≤ 3 h or outside a CMD partially differed. Where comparable (71% of tests), the relative proportion of POC tests out of all tests increased from season 2017/18 to 2018/19. In both seasons, the percentage of positive POC tests resulted slightly lower than for non-POC tests.ConclusionPOC testing integrated seamlessly into national influenza surveillance. We propose the use of POC results in the routine surveillance of seasonal influenza.

Multicenter Evaluation of the QIAstat-Dx Respiratory Panel for Detection of Viruses and Bacteria in Nasopharyngeal Swab Specimens.

The QIAstat-Dx Respiratory Panel (QIAstat-Dx RP) is a multiplex in vitro diagnostic test for the qualitative detection of 20 pathogens directly from nasopharyngeal swab (NPS) specimens. The assay is performed using a simple sample-to-answer platform with results available in approximately 69 min. The pathogens identified are adenovirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, human metapneumovirus A and B, influenza A, influenza A H1, influenza A H3, influenza A H1N1/2009, influenza B, parainfluenza virus 1, parainfluenza virus 2, parainfluenza virus 3, parainfluenza virus 4, rhinovirus/enterovirus, respiratory syncytial virus A and B, Bordetella pertussis, Chlamydophila pneumoniae, and Mycoplasma pneumoniae This multicenter evaluation provides data obtained from 1,994 prospectively collected and 310 retrospectively collected (archived) NPS specimens with performance compared to that of the BioFire FilmArray Respiratory Panel, version 1.7. The overall percent agreement between QIAstat-Dx RP and the comparator testing was 99.5%. In the prospective cohort, the QIAstat-Dx RP demonstrated a positive percent agreement of 94.0% or greater for the detection of all but four analytes: coronaviruses 229E, NL63, and OC43 and rhinovirus/enterovirus. The test also demonstrated a negative percent agreement of ≥97.9% for all analytes. The QIAstat-Dx RP is a robust and accurate assay for rapid, comprehensive testing for respiratory pathogens.

Point-of-care tests for influenza A and B viruses and RSV in emergency departments - indications, impact on patient management and possible gains by syndromic respiratory testing, Capital Region, Denmark, 2018.

BackgroundPoint-of-care tests (POCT) for influenza A and B viruses and respiratory syncytial virus (RSV) were implemented in emergency departments of all hospitals in the Capital Region of Denmark in 2018.AimTo establish whether POC testing for influenza viruses or RSV is based on a valid respiratory symptom indication, whether changes in patient management based on a positive result are safe and whether syndromic POC testing may benefit patients with influenza or RSV.MethodsSamples from 180 children (< 18 years) and 375 adults tested using POCT between February and July 2018 were retested for 26 respiratory pathogens. Diagnosis, indication for POC testing, hospitalisation time, antimicrobial therapy and readmission or death within one month of testing were obtained from patient records.ResultsA valid indication for POC testing was established in 168 (93.3%) of children and 334 (89.1%) of adults. A positive POCT result significantly reduced antibiotic prescription and median hospitalisation time by 44.3 hours for adults and 14.2 hours for children, and significantly increased antiviral treatment in adults. Risk of readmission or death was not significantly altered by a positive result. Testing for 26 respiratory pathogens established that risk of coinfection is lower with increasing age and that POCT for adults should be restricted to the influenza and RSV season.ConclusionPositive POCT resulted in changed patient management for both children and adults, and was deemed safe. POCT for additional pathogens may be beneficial in children below 5 years of age and outside the influenza and RSV season.

Multicenter evaluation of the new QIAstat Gastrointestinal Panel for the rapid syndromic testing of acute gastroenteritis.

In acute gastroenteritis (AGE), identification of the infectious agent is important for patient management. Since symptoms do not reliably identify the agent, microbiological diagnostics are important. Conventional methods lack sensitivity and often take days. Multiplex PCR panels offer fast and sensitive alternatives. Our aim was to assess the performance of the new QIAstat Gastrointestinal Panel (GIP) detecting 24 different gastroenteric pathogens from stool in Cary-Blair transport medium (Adenovirus F 40/41, Astrovirus, Norovirus GI/GII, Rotavirus A, Sapovirus, Campylobacter spp., Clostridium difficile, Plesiomonas shigelloides, Salmonella spp., Vibrio cholera, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica, enteroaggregative Escherichia coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica and Giardia lamblia). We tested both prospective (n = 163) and retrospective (n = 222) stool samples sent for routine diagnostics by the QIAstat GIP comparing it to the FDA-approved BioFire FilmArray GIP. Seegene Allplex GIP was used for discrepancy testing. After discrepancy testing, QIAstat GIP detected 447 of 455 pathogens (98.2%, 95% confidence interval (CI) 96.6-99.1%). There were eight false positive detections. Multiple pathogens were detected in 32.5% of positive samples. The QIAstat GIP detected a large range of AGE pathogens with a high sensitivity. It offers an easy-to-use system for GI pathogen detection in stool within 70 min. An advantage of the QIAstat is the availability of cycle threshold (CT) values to aid in interpretation of results.

Ceftriaxone-resistant Salmonella enterica serotype Typhi in a pregnant traveller returning from Karachi, Pakistan to Denmark, 2019.

We describe a ceftriaxone-resistant Salmonella Typhi bacteraemia in a pregnant woman returning from a family visit in Pakistan. Whole genome sequencing confirmed similarity to a Pakistani outbreak clone. Pregnancy and unawareness of this outbreak delayed appropriate antibiotic therapy. Concurrently, we detected faecal carriage of a carbapenemase-producing Escherichia coli. Awareness of the ongoing outbreak should affect empiric treatment of typhoid fever and hygiene precautions in travellers returning from Pakistan. Meropenem may be warranted in severe cases.

Evaluation of the analytical and clinical performance of two RT-PCR based point-of-care tests; Cepheid Xpert® Xpress CoV-2/Flu/RSV plus and SD BioSensor STANDARD™ M10 Flu/RSV/SARS-CoV-2.

BACKGROUND: Rapid and accurate detection of viral respiratory infections is important for infection control measures. This study compares the analytical and clinical performance of the Xpert® Xpress CoV-2/Flu/RSV plus test ("Xpert", Cepheid) and the STANDARD™ M10 Flu/RSV/SARS-CoV-2 test ("M10", SD Biosensor). Both tests are quadruplex RT-PCR assays for rapid diagnosis of SARS-CoV-2, influenza A/B and RSV. STUDY DESIGN: Analytical sensitivities were determined by limit of detection for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Additionally, the clinical performance of the Xpert and the M10 tests was evaluated against standard-of-care RT-PCR by testing of 492 clinical specimens. RESULTS: The analytical sensitivities for Xpert versus M10 test was 10, 50, 50 and 300 versus 300, 200, 800 and 1500 copies/mL for SARS-CoV-2, influenza A, influenza B and RSV, respectively. Clinical sensitivity for the Xpert test was superior across all four pathogens compared to the M10 test. Xpert showed clinical sensitivity of 100 % in all Ct-ranges for all four pathogens whereas M10 showed clinical sensitivity of 100 % in the 25-30 Ct-range, 84-100 % in the 30-35 Ct-range and 47-67 % in the >35 Ct-range across the four pathogens. Translating into real-life clinical sensitivity, the Xpert would detect 100 % of all four pathogens, whereas M10 would detect 92.1, 92.4, 84.8 and 94.7 % for SARS-CoV-2, influenza A, influenza B and RSV. CONCLUSION: This study demonstrates improved analytical and clinical performance of Xpert Xpress CoV-2/Flu/RSV plus compared to STANDARD M10 Flu/RSV/SARS-CoV-2, which is important for ensuring accuracy of diagnosis at all stages of a respiratory infection.

The impact of acute SARS-CoV-2 on testicular function including insulin-like factor 3 (INSL3) in men with mild COVID-19: A longitudinal study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may affect the male reproductive system as it uses angiotensin-converting enzyme (ACE)2, which is expressed in testicular tissue, as an entry point into the cell. Few studies have evaluated the long-term effects of mild coronavirus disease 2019 (COVID-19) on testicular function, and insulin-like factor 3 (INSL3) levels have not previously been assessed during acute SARS-CoV-2 infection. OBJECTIVES: The aim of the study was to assess the impact of acute SARS-CoV-2 infection on testicular function including INSL3 and the presence of SARS-CoV-2 RNA in semen in non-hospitalised men with mild COVID-19. MATERIALS AND METHODS: This longitudinal study included 36 non-hospitalised SARS-CoV-2-positive men (median age 29 years). Inclusion was within seven days following a positive SARS-CoV-2 reverse-transcription polymerase chain reaction test. Reproductive hormone levels, semen parameters, and the presence of SARS-CoV-2 RNA in oropharyngeal and semen samples were assessed during acute SARS-CoV-2 infection (baseline) and at three- and six-month follow-up. Wilcoxon matched-pair signed-rank (two samples) test was used to assess time-related alterations in reproductive hormone levels and semen parameters. RESULTS: Lower plasma testosterone (T) (total and calculated free (c-fT)) and higher luteinising hormone (LH) concentrations were observed during acute SARS-CoV-2 infection (baseline) compared to three- and six-month follow-up. Consequently, ratios of c-fT/LH were lower at baseline compared to three- and six-month follow-up (p < 0.001 and p = 0.003, respectively). Concomitantly, lower INSL3 concentrations were observed at baseline compared to three-month follow-up (p = 0.01). The total number of motile spermatozoa was also lower at baseline compared to six-month follow-up (p = 0.02). The alterations were detected irrespective of whether the men had experienced SARS-CoV-2-related fever episodes or not. No SARS-CoV-2 RNA was detected in semen at any time point. DISCUSSION AND CONCLUSION: This study showed a reduction in testicular function, which was for the first time confirmed by INSL3, in men mildly affected by SARS-CoV-2 infection. The risk of transmission of SARS-CoV-2 RNA via semen seems to be low. Febrile episodes may impact testicular function, but a direct effect of SARS-CoV-2 cannot be excluded.

SARS-CoV-2 spike mRNA vaccine sequences circulate in blood up to 28 days after COVID-19 vaccination.

In Denmark, vaccination against Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has been with the Pfizer-BioNTech (BTN162b2) or the Moderna (mRNA-1273) mRNA vaccines. Patients with chronic hepatitis C virus (HCV) infection followed in our clinic received mRNA vaccinations according to the Danish roll-out vaccination plan. To monitor HCV infection, RNA was extracted from patient plasma and RNA sequencing was performed on the Illumina platform. In 10 of 108 HCV patient samples, full-length or traces of SARS-CoV-2 spike mRNA vaccine sequences were found in blood up to 28 days after COVID-19 vaccination. Detection of mRNA vaccine sequences in blood after vaccination adds important knowledge regarding this technology and should lead to further research into the design of lipid-nanoparticles and the half-life of these and mRNA vaccines in humans.

Whole genome sequencing reveals two genetically distinct MRSA outbreaks among people who inject drugs and homeless people in Copenhagen.

Whole genome sequencing (WGS) has greatly improved the detection of methicillin-resistant Staphylococcus aureus (MRSA) transmission between people. We describe the transmission of two unique MRSA clones among homeless people in Copenhagen using WGS and core genome MLST (cgMLST). In 2014, an accumulation of MRSA bacteremia cases among homeless people admitted to our hospital was recognized, all having the rare MRSA spa t5147/ST88. The European Typology of Homelessness and Housing Exclusion (ETHOS) categories revealed that people who inject drugs (PWID) frequently visiting the milieu but living in private accommodation accounted for most cases. Hoping to terminate the transmission, 161 homeless people were MRSA screened in 2015, but no additional cases were found. From 2009 to 2018, 60 patients with genomically related t5147/ST88 isolates were found, of these 70% were confirmed to come from the homeless setting and 17% had bacteremia. From 2017 to 2020, cgMLST revealed a smaller MRSA outbreak including 13 PWID with a completely different clone, t1476/ST8, of which 15% had bacteremia. Our study confirms that WGS and cgMLST is excellent to reveal MRSA outbreaks. The ETHOS categorization can be useful to find the primary source of spread in the homeless community.

Viral epidemic preparedness: a perspective from five clinical microbiology laboratories in Europe.

BACKGROUND: Pandemic preparedness is critical to respond effectively to existing and emerging/new viral pathogens. Important lessons have been learned during the last pandemic at various levels. This revision discusses some of the major challenges and potential ways to address them in the likely event of future pandemics. OBJECTIVES: To identify critical points of readiness that may help us accelerate the response to future pandemics from a clinical microbiology laboratory perspective with a focus on viral diagnostics and genomic sequencing. The potential areas of improvement identified are discussed from the sample collection to information reporting. SOURCES: Microbiologists and researchers from five countries reflect on challenges encountered during the COVID-19 pandemic, review published literature on prior and current pandemics, and suggest potential solutions in preparation for future outbreaks. CONTENT: Major challenges identified in the pre-analytic and post-analytic phases from sample collection to result reporting are discussed. From the perspective of clinical microbiology laboratories, the preparedness for a new pandemic should focus on zoonotic viruses. Laboratory readiness for scalability is critical and should include elements related to material procurement, training personnel, specific funding programmes, and regulatory issues to rapidly implement "in-house" tests. Laboratories across various countries should establish (or re-use) operational networks to communicate to respond effectively, ensuring the presence of agile circuits with full traceability of samples. IMPLICATIONS: Laboratory preparedness is paramount to respond effectively to emerging and re-emerging viral infections and to limit the clinical and societal impact of new potential pandemics. Agile and fully traceable methods for sample collection to report are the cornerstone of a successful response. Expert group communication and early involvement of information technology personnel are critical for preparedness. A specific budget for pandemic preparedness should be ring-fenced and added to the national health budgets.

Durability and breadth of neutralisation following multiple antigen exposures to SARS-CoV-2 infection and/or COVID-19 vaccination.

BACKGROUND: Given the importance of vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the prevention of severe coronavirus disease 2019 (COVID-19), detailed long-term analyses of neutralising antibody responses are required to inform immunisation strategies. METHODS: In this study, longitudinal neutralising antibody titres to an ancestral SARS-CoV-2 isolate and cross-neutralisation to delta and omicron isolates were analysed in individuals previously infected with SARS-CoV-2, vaccinated against COVID-19, or a complex mix thereof with up to two years of follow-up. FINDINGS: Both infection-induced and vaccine-induced neutralising responses against SARS-CoV-2 appeared to follow similar decay patterns. Following vaccination in previously infected individuals, neutralising antibody responses were more durable than prior to vaccination. Further, this study shows that vaccination after infection, as well as booster vaccination, increases the cross-neutralising potential to both delta and omicron SARS-CoV-2 variants. INTERPRETATION: Taken together, these results suggest that neither type of antigen exposure is superior for neutralising antibody durability. However, these results support vaccination to increase the durability and cross-neutralisation potential of neutralising responses, thereby enhancing protection against severe COVID-19. FUNDING: This work was supported by grants from The Capital Region of Denmark's Research Foundation, the Novo Nordisk Foundation, the Independent Research Fund Denmark, the Candys Foundation, and the Danish Agency for Science and Higher Education.

An inactivated SARS-CoV-2 vaccine induced cross-neutralizing persisting antibodies and protected against challenge in small animals.

Vaccines have relieved the public health burden of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and globally inactivated vaccines are most widely used. However, poor vaccination accessibility and waning immunity maintain the pandemic, driving emergence of variants. We developed an inactivated SARS-CoV-2 (I-SARS-CoV-2) vaccine based on a viral isolate with the Spike mutation D614G, produced in Vero cells in a scalable bioreactor, inactivated with ß-propiolactone, purified by membrane-based steric exclusion chromatography, and adjuvanted with MF59-like adjuvant AddaVax. I-SARS-CoV-2 and a derived split vaccine induced persisting neutralizing antibodies in mice; moreover, lyophilized antigen was immunogenic. Following homologous challenge, I-SARS-CoV-2 immunized hamsters were protected against disease and lung pathology. In contrast with reports for widely used vaccines, hamster plasma similarly neutralized the homologous and the Delta (B.1.617.2) variant viruses, whereas the Omicron (B.1.1.529) variant was neutralized less efficiently. Applied bioprocessing approaches offer advantages regarding scalability and production, potentially benefitting worldwide vaccine coverage.

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.

Performance of PCR-based syndromic testing compared to bacterial culture in patients with suspected pneumonia applying microscopy for quality assessment.

Syndromic testing for lower respiratory tract infections with BioFire® FilmArray® Pneumonia Panel Plus (BF) detects 27 pathogens with a turn-around-time of one hour. We compared the performance of BF with culture. Samples from 298 hospitalized patients with suspected pneumonia routinely sent for culture were also analyzed using BF. Retrospectively, patients were clinically categorized as having "pneumonia" or "no pneumonia." BF and culture were compared by analytical performance, which was evaluated by pathogen concordance, and by clinical performance by comparing pathogen detections in patients with and without pneumonia. The BF results for viruses and atypical bacteria were not included in the performance analysis. In 298 patient samples, BF and culture detected 285 and 142 potential pathogens, respectively. Positive percent agreement (PPA) was 88% (125/142). In patients with community-acquired pneumonia (CAP), clinical sensitivity was 70% and 51%, and specificity was 43% and 71% for BF and culture, respectively. In patients with hospital-acquired pneumonia, the corresponding numbers were 55% and 23%, and 47% and 68%. There was no significant improvement of performance, when only high-quality sputum samples were considered. Efficacy of both BF and culture was low. Both tests are best used in CAP patients for whom the diagnosis has already been clinically established. Indiscriminate use may be clinically misleading and a cause of improper use of antibiotics.

An Agreement of Antigen Tests on Oral Pharyngeal Swabs or Less Invasive Testing With Reverse Transcription Polymerase Chain Reaction for Detecting SARS-CoV-2 in Adults: Protocol for a Prospective Nationwide Observational Study.

BACKGROUND: The SARS-CoV-2 pandemic has resulted in an unprecedented level of worldwide testing for epidemiologic and diagnostic purposes, and due to the extreme need for tests, the gold-standard Reverse Transcription Polymerase Chain Reaction (RT-PCR) testing capacity has been unable to meet the overall worldwide testing demand. Consequently, although the current literature has shown the sensitivity of rapid antigen tests (RATs) to be inferior to RT-PCR, RATs have been implemented on a large scale without solid data on performance. OBJECTIVE: This study will compare analytical and clinical sensitivities and specificities of 50 lateral flow- or laboratory-based RATs and 3 strand invasion-based amplification (SIBA)-RT-PCR tests from 30 manufacturers to RT-PCR testing of samples obtained from the deep oropharynx. In addition, the study will compare sensitivities and specificities of the included RATs as well as RT-PCR on clinical samples obtained from the deep oropharynx, the anterior nasal cavity, saliva, the deep nasopharynx, and expired air to RT-PCR on deep oropharyngeal samples. METHODS: In the prospective part of the study, 200 individuals found SARS-CoV-2 positive and 200 individuals found SARS-CoV-2 negative by routine RT-PCR testing will be retested with each RAT, applying RT-PCR as the reference method. In the retrospective part of the study, 304 deep oropharyngeal cavity swabs divided into 4 groups based on RT-PCR quantification cycle (Cq) levels will be tested with each RAT. RESULTS: The results will be reported in several papers with different aims. The first paper will report retrospective (analytical sensitivity, overall and stratified into different Cq range groups) and prospective (clinical sensitivity) data for RATs, with RT-PCR as the reference method. The second paper will report results for RAT based on anatomical sampling location. The third paper will compare different anatomical sampling locations by RT-PCR testing. The fourth paper will focus on RATs that rely on central laboratory testing. Tests from 4 different manufacturers will be compared for analytical performance data on retrospective deep oropharyngeal swab samples. The fifth paper will report the results of 4 RATs applied both as professional use and as self-tests. The last paper will report the results from 2 breath tests in the study. A comparison of sensitivity and specificity between RATs will be conducted using the McNemar test for paired samples and the chi-squared test for unpaired samples. Comparison of the positive predictive value (PPV) and negative predictive value (NPV) between RATs will be performed by the bootstrap test, and 95% CIs for sensitivity, specificity, PPV, and NPV will be calculated as bootstrap CIs. CONCLUSIONS: The study will compare the sensitivities of a large number of RATs for SARS-CoV-2 to with those of RT-PCR and will address whether lateral flow-based RATs differ significantly from laboratory-based RATs. The anatomical test locations for both RATs and RT-PCR will also be compared. TRIAL REGISTRATION: ClinicalTrials.gov NCT04913116; https://clinicaltrials.gov/ct2/show/NCT04913116. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/35706.

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.

[Presence of SARS-CoV-2 in semen and the impact of COVID-19 on the male reproductive system].

This article reviews the current studies published on the transmission of SARS-CoV-2 in semen and the potential effect of COVID-19 on male fertility. The aim was to provide insight into different possible mechanisms of involvement of the male reproductive system by SARS-CoV-2 infection and to evaluate the studies investigating the presence of virus in semen. It is concluded that the likelihood of SARS-CoV-2 transmission through semen is low and that COVID-19 may negatively affect semen parameters, but that the impairment is probably short-termed.