Testing for SARS-CoV-2: lessons learned and current use cases.

SUMMARYThe emergence and worldwide dissemination of SARS-CoV-2 required both urgent development of new diagnostic tests and expansion of diagnostic testing capacity on an unprecedented scale. The rapid evolution of technologies that allowed testing to move out of traditional laboratories and into point-of-care testing centers and the home transformed the diagnostic landscape. Four years later, with the end of the formal public health emergency but continued global circulation of the virus, it is important to take a fresh look at available SARS-CoV-2 testing technologies and consider how they should be used going forward. This review considers current use case scenarios for SARS-CoV-2 antigen, nucleic acid amplification, and immunologic tests, incorporating the latest evidence for analytical/clinical performance characteristics and advantages/limitations for each test type to inform current debates about how tests should or should not be used.

Comparative Diagnostic Utility of SARS-CoV-2 Rapid Antigen and Molecular Testing in a Community Setting.

BACKGROUND: SARS-CoV-2 antigen-detection rapid diagnostic tests (Ag-RDTs) have become widely utilized but longitudinal characterization of their community-based performance remains incompletely understood. METHODS: This prospective longitudinal study at a large public university in Seattle, WA utilized remote enrollment, online surveys, and self-collected nasal swab specimens to evaluate Ag-RDT performance against real-time reverse transcription polymerase chain reaction (rRT-PCR) in the context of SARS-CoV-2 Omicron. Ag-RDT sensitivity and specificity within 1 day of rRT-PCR were evaluated by symptom status throughout the illness episode and Orf1b cycle threshold (Ct). RESULTS: From February to December 2022, 5757 participants reported 17 572 Ag-RDT results and completed 12 674 rRT-PCR tests, of which 995 (7.9%) were rRT-PCR positive. Overall sensitivity and specificity were 53.0% (95% confidence interval [CI], 49.6%-56.4%) and 98.8% (95% CI, 98.5%-99.0%), respectively. Sensitivity was comparatively higher for Ag-RDTs used 1 day after rRT-PCR (69.0%), 4-7 days after symptom onset (70.1%), and Orf1b Ct ≤20 (82.7%). Serial Ag-RDT sensitivity increased with repeat testing ≥2 (68.5%) and ≥4 (75.8%) days after an initial Ag-RDT-negative result. CONCLUSIONS: Ag-RDT performance varied by clinical characteristics and temporal testing patterns. Our findings support recommendations for serial testing following an initial Ag-RDT-negative result, especially among recently symptomatic persons or those at high risk for SARS-CoV-2 infection.

Sensitive Dual-Signal ELISA Based on Specific Phage-Displayed Double Peptide Probes with Internal Filtering Effect to Assay Monkeypox Virus Antigen.

The global spread of monkeypox has become a worldwide public healthcare issue. Therefore, there is an urgent need for accurate and sensitive detection methods to effectively control its spreading. Herein, we screened by phage display two peptides M4 (sequence: DPCGERICSIAL) and M6 (sequence: SCSSFLCSLKVG) with good affinity and specificity to monkeypox virus (MPXV) B21R protein. To simulate the state of the peptide in the phage and to avoid spatial obstacles of the peptide, GGGSK was added at the C terminus of M4 and named as M4a. Molecular docking shows that peptide M4a and peptide M6 are bound to different epitopes of B21R by hydrogen bonds and salt-bridge interactions, respectively. Then, peptide M4a was selected as the capture probe, phage M6 as the detection probe, and carbonized polymer dots (CPDs) as the fluorescent probe, and a colorimetric and fluorescent double-signal capture peptide/antigen/signal peptide-displayed phage sandwich ELISA triggered by horseradish peroxidase (HRP) through a simple internal filtration effect (IFE) was constructed. HRP catalyzes H2O2 to oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB, which can further quench the fluorescence of CPDs through IFE, enabling to detect MPXV B21R in colorimetric and fluorescent modes. The proposed simple immunoassay platform shows good sensitivity and reliability in MPXV B21R detection. The limit of detection for colorimetric and fluorescent modes was 27.8 and 9.14 pg/mL MPXV B21R, respectively. Thus, the established double-peptide sandwich-based dual-signal immunoassay provides guidance for the development of reliable and sensitive antigen detection capable of mutual confirmation, which also has great potential for exploring various analytical strategies for other respiratory virus surveillance.

Performance evaluation of the Panbio COVID-19/Flu A&B Panel for detection of SARS-CoV-2, influenza A, and influenza B antigens using mid-turbinate nasal swabs.

The Panbio COVID-19/Flu A&B Panel (Abbott) is an in vitro diagnostic rapid test designed for the qualitative detection of nucleocapsid proteins SARS-CoV-2 and nucleoprotein influenza A and B antigens in nasal mid-turbinate (NMT) swab specimens from symptomatic individuals meeting COVID-19 and influenza clinical and/or epidemiological criteria. This study, the largest global one to date using fresh samples, aimed to assess the diagnostic sensitivity and specificity of the Panbio COVID-19/Flu A&B Panel in freshly collected NMT swab specimens from individuals suspected of respiratory viral infection consistent with COVID-19 and/or influenza within the first 5 days of symptom onset compared with results obtained with the cobas SARS-CoV-2 and influenza A/B qualitative assay (cobas 6800/8800 systems), which were tested using nasopharyngeal swab samples. A total of 512 evaluable subjects were enrolled in the COVID-19 cohort across 18 sites, and 1,148 evaluable subjects were enrolled in the influenza cohort across 22 sites in the Asia-Pacific, Europe, and the USA. The Panbio COVID-19/Flu A&B Panel demonstrated a sensitivity of 80.4% and a specificity of 99.7% for COVID-19. For influenza A, the sensitivity and specificity rates were 80.6% and 99.3%, respectively. Likewise, for influenza B, the sensitivity and specificity rates were 80.8% and 99.4%, respectively. In conclusion, the Panbio COVID-19/Flu A&B Panel emerges as a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.4% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B. IMPORTANCE: The Panbio COVID-19/Flu A&B Panel is a suitable rapid test for detecting COVID-19 and influenza in symptomatic subjects across diverse global populations, exhibiting high sensitivity. The assay achieved a sensitivity of 94.0% in samples with Ct ≤24 for COVID-19 and 92.6% in samples with Ct ≤30 for influenza A and B.

Development of a quantum dots based immunochromatographic strip for rapid and on-site detection of African swine fever virus.

African swine fever (ASF) is a lethal disease caused by ASF virus (ASFV), severely impacting the global swine industry. Though nuclear acid-based detection methods are reliable, they are laboratory-dependent. In this study, we developed a device-independent, user friendly and cost-effective quantum dots based immunochromatographic strip (QDs-ICS) with high specificity and sensitivity for the rapid and on-site detection of ASFV antigen. For the preparation of the QDs-ICS, we generated a monoclonal antibody (mAb) mAb-8G8 and polyclonal antibody (pAb) against ASFV-p72 protein. The pAb was labelled with QDs to be used as the detection probe and the mAb-8G8 was coated on the nitrocellulose membrane as the test line. Our results proved that the strip displayed no cross-reactivity with other swine viruses and detection limit of the QDs-ICS was down to 1 ng/mL for the ASFV-p72 protein with great reproducibility. The strip also exhibited high stability with a storage period up to 12 months under room temperature. Twenty blind samples and one hundred clinical samples were examined by the QDs-ICS, conventional PCR and real-time PCR method, respectively. Results showed that the agreement rate between the QDs-ICS and PCR method was 100%, and the agreement rate between the strip and real-time PCR was 94%. The novel QDs-ICS developed here would be an effective tool for on-site detection of ASFV.

Clinical accuracy of instrument-based SARS-CoV-2 antigen diagnostic tests: a systematic review and meta-analysis.

BACKGROUND: During the COVID-19 pandemic, antigen diagnostic tests were frequently used for screening, triage, and diagnosis. Novel instrument-based antigen tests (iAg tests) hold the promise of outperforming their instrument-free, visually-read counterparts. Here, we provide a systematic review and meta-analysis of the SARS-CoV-2 iAg tests' clinical accuracy. METHODS: We systematically searched MEDLINE (via PubMed), Web of Science, medRxiv, and bioRxiv for articles published before November 7th, 2022, evaluating the accuracy of iAg tests for SARS-CoV-2 detection. We performed a random effects meta-analysis to estimate sensitivity and specificity and used the QUADAS-2 tool to assess study quality and risk of bias. Sub-group analysis was conducted based on Ct value range, IFU-conformity, age, symptom presence and duration, and the variant of concern. RESULTS: We screened the titles and abstracts of 20,431 articles and included 114 publications that fulfilled the inclusion criteria. Additionally, we incorporated three articles sourced from the FIND website, totaling 117 studies encompassing 95,181 individuals, which evaluated the clinical accuracy of 24 commercial COVID-19 iAg tests. The studies varied in risk of bias but showed high applicability. Of 24 iAg tests from 99 studies assessed in the meta-analysis, the pooled sensitivity and specificity compared to molecular testing of a paired NP swab sample were 76.7% (95% CI 73.5 to 79.7) and 98.4% (95% CI 98.0 to 98.7), respectively. Higher sensitivity was noted in individuals with high viral load (99.6% [95% CI 96.8 to 100] at Ct-level ≤ 20) and within the first week of symptom onset (84.6% [95% CI 78.2 to 89.3]), but did not differ between tests conducted as per manufacturer's instructions and those conducted differently, or between point-of-care and lab-based testing. CONCLUSION: Overall, iAg tests have a high pooled specificity but a moderate pooled sensitivity, according to our analysis. The pooled sensitivity increases with lower Ct-values (a proxy for viral load), or within the first week of symptom onset, enabling reliable identification of most COVID-19 cases and highlighting the importance of context in test selection. The study underscores the need for careful evaluation considering performance variations and operational features of iAg tests.

SARS-CoV-2 Viral Shedding and Rapid Antigen Test Performance - Respiratory Virus Transmission Network, November 2022-May 2023.

As population immunity to SARS-CoV-2 evolves and new variants emerge, the role and accuracy of antigen tests remain active questions. To describe recent test performance, the detection of SARS-CoV-2 by antigen testing was compared with that by reverse transcription-polymerase chain reaction (RT-PCR) and viral culture testing during November 2022-May 2023. Participants who were enrolled in a household transmission study completed daily symptom diaries and collected two nasal swabs (tested for SARS-CoV-2 via RT-PCR, culture, and antigen tests) each day for 10 days after enrollment. Among participants with SARS-CoV-2 infection, the percentages of positive antigen, RT-PCR, and culture results were calculated each day from the onset of symptoms or, in asymptomatic persons, from the date of the first positive test result. Antigen test sensitivity was calculated using RT-PCR and viral culture as references. The peak percentage of positive antigen (59.0%) and RT-PCR (83.0%) results occurred 3 days after onset, and the peak percentage of positive culture results (52%) occurred 2 days after onset. The sensitivity of antigen tests was 47% (95% CI = 44%-50%) and 80% (95% CI = 76%-85%) using RT-PCR and culture, respectively, as references. Clinicians should be aware of the lower sensitivity of antigen testing compared with RT-PCR, which might lead to false-negative results. This finding has implications for timely initiation of SARS-CoV-2 antiviral treatment, when early diagnosis is essential; clinicians should consider RT-PCR for persons for whom antiviral treatment is recommended. Persons in the community who are at high risk for severe COVID-19 illness and eligible for antiviral treatment should seek testing from health care providers with the goal of obtaining a more sensitive diagnostic test than antigen tests (i.e., an RT-PCR test).

Evaluation of CLINITEST® Rapid Covid-19 + Influenza antigen test in a cohort of symptomatic patients in an emergency department.

OBJECTIVES: Rapid management of patients with respiratory tract infections in hospital emergency departments is one of the main objectives since the concurrent circulation of respiratory viruses following the SARS-CoV-2 pandemic. The use of new combined point-of-care antigen tests for detecting influenza A/B and SARS-CoV-2 represents an advantage in response time over the molecular tests. The objective was to evaluate the suitability of the CLINITEST® Rapid Covid-19 + Influenza Antigen test (Siemens Healthineers, Germany) (RCIA test) by measuring the sensitivity, specificity, Cohen's kappa, and cut-off values. METHODS: Nasopharyngeal samples were collected from a randomised group of symptomatic patients of all ages at emergency department during January-February 2023. In parallel, these patients were screened for influenza A/B, and SARS-CoV-2 using RT-PCR. The Ct (cycle threshold) values were collected for positive [RT-PCR (+) /RCIA test (+)] and false negative [(RT-PCR (+) /RCIA test (-)] samples. A subanalysis was performed in the paediatric population (< 16 years-old). RESULTS: We included 545 patients (55.8% females) with a median age of 7 years-old (IQR: 1-66.5). The RCIA test showed a sensitivity of 59.7% [95%CI: 46.9-67.33] for influenza A, 65.6% [95%CI: 49.5-80.3] for influenza B, and 76.9% [95%CI: 45.8-84.8] for SARS-CoV-2. The specificity was between 90.7%-99.7% with a moderate/high level of agreement with RT-PCR (kappa score: 0.6-0.8) for the three respiratory viruses included in the RCIA test. CONCLUSIONS: The sensitivity of the RCIA test is insufficient for screening of patients, including patients with low Ct values (Ct > 20). Despite its good specificity and Cohen's kappa value, its use as a screening test is not comparable to RT-PCR systems in the ED environment with a high number of false negative results.

Advances in rapid point-of-care virus testing.

Outbreaks of viral diseases seriously jeopardize people's health and cause huge economic losses. At the same time, virology provides a new perspective for biology, molecular biology and cancer research, and it is important to study the discovered viruses with potential applications. Therefore, the development of immediate and rapid viral detection methods for the prevention and treatment of viral diseases as well as the study of viruses has attracted extensive attention from scientists. With the continuous progress of science and technology, especially in the field of bioanalysis, a series of new detection techniques have been applied to the on-site rapid detection of viruses, which has become a powerful approach for human beings to fight against viruses. In this paper, the latest research progress of rapid point-of-care detection of viral nucleic acids, antigens and antibodies is presented. In addition, the advantages and disadvantages of these technologies are discussed from the perspective of practical application requirements. Finally, the problems and challenges faced by rapid viral detection methods and their development prospects are discussed.

Establishment and application of a gold nanoparticle-based immunochromatographic test strip for the detection of avian leukosis virus P27 antigen in egg white samples.

Avian leukemia is an infectious tumorous disease of chickens caused by subgroup A of the avian leukemia virus (ALV-A), which mainly causes long-term viremia, slow growth, immune suppression, decreased production performance, multi-tissue tumors, and even death. The infection rate of this disease is very high in chicken herds in China, causing huge economic losses to the poultry industry every year. We successfully expressed the specific antigen protein of ALV (P27) through recombinant protein technology and screened a pair of highly sensitive monoclonal antibodies (mAbs) through mouse immunity, cell fusion, and antibody pairing. Based on this pair of antibodies, we established a dual antibody sandwich ELISA and gold nanoparticle immunochromatographic strip (AuNP-ICS) detection method. In addition, the parameters of the dual antibody sandwich ELISA and AuNP-ICS were optimized under different reaction conditions, which resulted in the minimum detection limits of 0.2 ng mL-1 and 1.53 ng ml-1, respectively. Commonly available ELISA and AuNP-ICS products on the market were compared, and we found that our established immune rapid chromatography had higher sensitivity. This established AuNP-ICS had no cross-reactivity with Influenza A (H1N1), Influenza A (H9N2), respiratory syncytial virus (RSV), varicella-zoster virus (VZV), Listeria monocytogenes listeriolysin (LLO), and Staphylococcal enterotoxin SED or SEC. Finally, the established AuNP-ICS was used to analyze 35 egg samples, and the results showed 5 positive samples and 30 negative samples. The AuNP-ICS rapid detection method established by our group had good specificity, high sensitivity, and convenience, and could be applied to the clinical sample detection of ALV-A.

Evaluation of the diagnostic sensitivity and specificity of two pen-side tests for detecting African swine fever virus in experimentally infected pigs.

African swine fever virus (ASFV) has spread through many countries and regions worldwide, causing significant losses. Timely detection of ASFV-infected pigs is crucial for disease control. In this study, we assessed the performance of two pen-side tests: a portable real-time PCR (qPCR) test for detecting viral genomic DNA and a lateral flow immunoassay (LFIA) for detecting viral antigens. To determine the time from infection to the earliest detection, 10 ASFV-seronegative pigs were inoculated intramuscularly with 104.0 hemadsorption dose 50 of a highly virulent ASFV strain. Whole blood and oral swab samples were alternately collected from each group of five pigs daily until all succumbed to the infection. Samples were promptly subjected to the two pen-side tests upon collection, and a subset was transported to a veterinary diagnostic laboratory for analysis using a reference qPCR assay. Viral genomic DNA was consistently detected by the reference qPCR assay in all blood samples from 2 days postinfection (dpi), preceding the onset of clinical signs, and in oral swabs from 4 dpi onwards. The portable qPCR test demonstrated comparable performance to the reference qPCR assay for both whole blood and oral swab samples. The LFIA exhibited 100% specificity when testing with whole blood samples but showed reduced sensitivity, particularly for blood samples collected early or late after infection. The antigen test did not perform well with oral swabs.

Establishment of a Raman nanosphere based immunochromatographic system for the combined detection of influenza A and B viruses' antigens on a single T-line.

A simple and rapid system based on Raman nanosphere (R-Sphere) immunochromatography was developed in this study for the simultaneous detection of Influenza A, B virus antigens on a single test line (T-line). Two types of R-Sphere with different characteristic Raman spectrum were used as the signal source, which were labeled with monoclonal antibodies against FluA, FluB (tracer antibodies), respectively. A mixture of antibodies containing anti-FluA monoclonal antibody and anti-FluB monoclonal antibody (capture antibody) was sprayed on a single T-line and goat anti-chicken IgY antibody was coated as a C-line, and the antigen solution with known concentration was detected by the strip of lateral flow immunochromatography based on surface-enhanced Raman spectroscopy (SERS). The T-line was scanned with a Raman spectrometer and SERS signals were collected. Simultaneous specific recognition and detection of FluA and FluB were achieved on a single T-line by analyzing the SERS signals. The findings indicated that the test system could identify FluA and FluB in a qualitative manner in just 15 minutes, with a minimum detection threshold of 0.25 ng ml-1, excellent consistency, and specificity. There was no interference with the other four respiratory pathogens, and it exhibited 8 times greater sensitivity compared to the colloidal gold test strip method. The assay system is rapid, sensitive, and does not require repetitive sample pretreatment steps and two viruses can be detected simultaneously on a single T-line by titrating one sample, which improves detection efficiency, and provide a reference for developing multiplexed detection techniques for other respiratory viruses.

A high throughput immuno-affinity mass spectrometry method for detection and quantitation of SARS-CoV-2 nucleoprotein in human saliva and its comparison with RT-PCR, RT-LAMP, and lateral flow rapid antigen test.

OBJECTIVES: Many reverse transcription polymerase chain reaction (RT-PCR) methods exist that can detect SARS-CoV-2 RNA in different matrices. RT-PCR is highly sensitive, although viral RNA may be detected long after active infection has taken place. SARS-CoV-2 proteins have shorter detection windows hence their detection might be more meaningful. Given salivary droplets represent a main source of transmission, we explored the detection of viral RNA and protein using four different detection platforms including SISCAPA peptide immunoaffinity liquid chromatography-mass spectrometry (SISCAPA-LC-MS) using polyclonal capture antibodies. METHODS: The SISCAPA-LC MS method was compared to RT-PCR, RT-loop-mediated isothermal amplification (RT-LAMP), and a lateral flow rapid antigen test (RAT) for the detection of virus material in the drool saliva of 102 patients hospitalised after infection with SARS-CoV-2. Cycle thresholds (Ct) of RT-PCR (E gene) were compared to RT-LAMP time-to-positive (TTP) (NE and Orf1a genes), RAT optical densitometry measurements (test line/control line ratio) and to SISCAPA-LC-MS for measurements of viral protein. RESULTS: SISCAPA-LC-MS showed low sensitivity (37.7 %) but high specificity (89.8 %). RAT showed lower sensitivity (24.5 %) and high specificity (100 %). RT-LAMP had high sensitivity (83.0 %) and specificity (100.0 %). At high initial viral RNA loads (<20 Ct), results obtained using SISCAPA-LC-MS correlated with RT-PCR (R2 0.57, p-value 0.002). CONCLUSIONS: Detection of SARS-CoV-2 nucleoprotein in saliva was less frequent than the detection of viral RNA. The SISCAPA-LC-MS method allowed processing of multiple samples in <150 min and was scalable, enabling high throughput.

Comparative examination of a rapid immunocytochemical test for the detection of highly pathogenic avian influenza virus in domestic birds in field outbreaks.

The quantitative real-time reverse polymerase chain reaction (RRT-PCR) is the preferred test method for the diagnosis of avian influenza (AI), but can be performed only in specialized laboratories. Different antigen detection methods for the diagnosis of AI were previously reported to be specific and sensitive in field outbreaks. These tests can be performed in basic countryside labs. Brain smears of domestic birds (n = 105) collected during AI field outbreaks were examined with immunocytochemistry (IC). The results were statistically analysed by comparing IC to brain histology (BH), and immunohistochemistry (IHC), to gross pathological examination (GP) (n = 105), and RRT-PCR (n = 91). AI was diagnosed with RRT-PCR in 66 cases. IC and IHC were positive in 59/66 (90%) and 60/66 (91%) cases, respectively. Lesions suspicious for AI were detected with GP and HP in 66/66 (100%) and 61/66 (92%) cases, respectively. An almost perfect agreement was found between RRT-PCR, IC, IHC, and HP. Substantial agreement was found between IC and GP, between IHC and GP, between HP and GP, and between RRT-PCR and GP. The chromogen-based IC test presented in this study produces durable staining, which can be evaluated using a simple brightfield microscope. The test is rapid (can be completed in 2 h), sensitive (90%), specific (100%), and cost-effective, which makes the method suitable for routine diagnostic tests in AI epidemics.RESEARCH HIGHLIGHTSAvian influenza virus (AIV) antigen detection was examined in field outbreaks.Bird brain smears were tested using immunocytochemistry (IC).IC results strongly correlated with real-time RT-PCR results.The IC method was rapid, specific, sensitive, and cost-effective in AIV field outbreaks.

Quantitative detection of mpox antigen using time-resolved fluorescence immunochromatography.

Recently, concern has been raised about the spread of human mpox virus, and the demand for rapid detection reagents for mpox virus has increased. This study aims to establish a time-resolved fluorescence immunochromatography (TRFICO) method for qualitative/quantitative detection of mpox virus. A double-antibody sandwich TRFICO method was optimized and established using mpox recombinant fusion antigen and its paired monoclonal antibody. The test performance of the method was evaluated using mpox fusion antigen and control serum, including sensitivity, linearity range, specificity, precision, and reference interval. We successfully established a TRFICO method for qualitative/quantitative detection of mpox antigen, its linearity range 0-100 ng/mL, analytical sensitivity 0.017 ng/mL, and reference intervals greater than 0.045 ng/mL. No cross-reaction was detected with various poxvirus and clinical negative controls, with good specificity. All average recoveries of the intra- and inter-batch ranged from 81.33% to 97.83%, and all CVs were below 10%. Additionally, the TRFICO strips can be stably stored at 37°C for 7 days without significant changes in the fluorescence intensity. This TRFICO method, with high sensitivity, linearity range, specificity, precision, and stability with 16-min detection time, provides a new option for qualitative/quantitative and convenient testing of mpox virus.

Establishment and application of PDCoV antigen-specific DAS-ELISA detection method.

BACKGROUND: Porcine deltacoronavirus (PDCoV) is a swine enteropathogenic coronavirus that affects young pigs, causing vomiting, acute diarrhea, dehydration, and even death. There is growing evidence that PDCoV can undergo cross-species as well as zoonotic transmissions. Due to the frequent outbreaks of this deadly virus, early detection is essential for effective prevention and control. Therefore, developing a more convenient and reliable method for PDCoV detection is the need of the hour. RESULTS: This study utilized a high-affinity monoclonal antibody as the capture antibody and a horseradish peroxidase labeled polyclonal antibody as the detection antibody to develop an enzyme-linked immunosorbent assay (DAS-ELSA) for PDCoV detection.Both antibodies target the PDCoV nucleocapsid (N) protein. The findings of this study revealed that DAS-ELISA was highly specific to PDCoV and did not cross-react with other viruses to cause swine diarrhea. The limit of detection of the virus titer using this method was 103 TCID50/mL of PDCoV particles. The results of a parallel analysis of 239 known pig samples revealed a coincidence rate of 97.07% (κ = 0.922) using DAS-ELISA and reverse transcriptase PCR (RT-PCR). The DAS-ELISA was used to measure the one-step growth curve of PDCoV in LLC-PK cells and the tissue distribution of PDCoV in infected piglets. The study found that the DAS-ELISA was comparable in accuracy to the TCID50 method while measuring the one-step growth curve. Furthermore, the tissue distribution measured by DAS-ELISA was also consistent with the qRT-PCR method. CONCLUSION: The developed DAS-ELISA method can be conveniently used for the early clinical detection of PDCoV infection in pigs, and it may also serve as an alternative method for laboratory testing of PDCoV.

Lesions and viral antigen distribution in bald eagles, red-tailed hawks, and great horned owls naturally infected with H5N1 clade 2.3.4.4b highly pathogenic avian influenza virus.

An epidemic of highly pathogenic avian influenza (HPAI) began in North America in the winter of 2021. The introduced Eurasian H5N1 clade 2.3.4.4b virus subsequently reassorted with North American avian influenza strains. This postmortem study describes the lesions and influenza A virus antigen distribution in 3 species of raptors, including bald eagles (Haliaeetus leucocephalus, n = 6), red-tailed hawks (Buteo jamaicensis, n = 9), and great horned owls (Bubo virginianus, n = 8), naturally infected with this virus strain based on positive reverse transcriptase polymerase chain reaction and sequencing results from oropharyngeal swabs. The birds presented with severe neurologic signs and either died or were euthanized because of the severity of their clinical signs and suspected influenza virus infection. Gross lesions were uncommon and included forebrain hemorrhages in 2 eagles, myocarditis in 1 hawk, and multifocal pancreatic necrosis in 3 owls. Histological lesions were common and included encephalitis, myocarditis, multifocal pancreas necrosis, multifocal adrenal necrosis, histiocytic splenitis, and anterior uveitis in decreasing frequency. Influenza A viral antigen was detected in brain, heart, pancreas, adrenal gland, kidney, spleen, liver, and eye. In conclusion, bald eagles, red-tailed hawks, and great horned owls infected with the HPAI clade 2.3.4.4b virus strain and showing neurological signs of illness may develop severe or fatal disease with histologically detectable lesions in the brain that are frequently positive for viral antigen.

A sensitive gold nanoflower-based lateral flow assay coupled with gold staining technique for the detection of SARS-CoV-2 antigen.

An enhanced lateral flow assay (LFA) is presented for rapid and highly sensitive detection of acute respiratory syndrome coronavirus-2 (SARS-CoV-2) antigens with gold nanoflowers (Au NFs) as signaling markers and gold enhancement to amplify the signal intensities. First, the effect of the morphology of gold nanomaterials on the sensitivity of LFA detection was investigated. The results showed that Au NFs prepared by the seed growth method showed a 5-fold higher detection sensitivity than gold nanoparticles (Au NPs) of the same particle size, which may benefit from the higher extinction coefficient and larger specific surface area of Au NFs. Under the optimized experimental conditions, the Au NFs-based LFA exhibited a detection limit (LOD) of 25 pg mL-1 for N protein using 135 nm Au NFs as the signaling probes. The signal was further amplified by using a gold enhancement strategy, and the LOD for the detection of N protein achieved was 5 pg mL-1. The established LFA also exhibited good repeatability and stability and showed applicability in the diagnosis of SARS-CoV-2 infection.

Assessment of DNA/RNA Defend Pro: An Inactivating Sample Collection Buffer for Enhanced Stability, Extraction-Free PCR, and Rapid Antigen Testing of Nasopharyngeal Swab Samples.

This study comprehensively evaluated the DNA/RNA Defend Pro (DRDP) sample collection buffer, designed to inactivate and stabilize patient samples. The primary objectives were to assess DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing (RAT). Ninety-five diagnostic nasopharyngeal swab samples tested for influenza virus (influenza A), respiratory syncytial virus (RSV A), and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were 10-fold diluted with DRDP and anonymized. Initial characterization and retesting of these samples using cobas Liat confirmed 88 samples as positive, validating the presence of viral targets. Results from rapid antigen testing showed lower sensitivity compared to nucleic acid amplification testing (NAAT) but maintained perfect specificity, with 40 out of 88 positive samples by cobas Liat also testing positive for RAT. Direct RT-qPCR of DRDP-diluted samples demonstrated robust compatibility, with 72 out of 88 samples positive for cobas Liat also testing positive by direct RT-qPCR. Non-concordant results could be explained by the 200-fold lower input of extraction-free NAAT. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with extraction-free NAAT. DRDP guaranteed viral RNA stability at all temperatures for influenza A, SARS-CoV-2, and RSV A, showing stability up to 7 days at 4 °C. In conclusion, DRDP is an effective stabilizing medium compatible with direct RT-qPCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.

[Evaluation of Viral Subgenomic RNAs and Antigen Presence in SARS-CoV-2 PCR Positive Cases]. / SARS-CoV-2 PCR Pozitif Olgularda Viral Subgenomik RNA'larin ve Antijen Varliginin Degerlendirilmesi.

Polymerase chain reaction (PCR) and antigen test (AgT) are frequently used in the diagnosis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Routine PCR tests that detect the virus genome cannot determine whether the virus is infectious or not. However, detection of subgenomic RNA (sgRNA) produced during the replication period may indicate active viral infection. Active virus detection can offer various health and economic benefits from isolation time to treatment. Antigen tests are also considered as indicators of infectiousness since they can detect viruses above a certain load amount. The aim of this study was to use two different subgenomic RNAs and antigen test instead of genomic RNA to examine the relationship with each other and the clinic in terms of infectiousness. Evaluating the antigen test together with subgenomic RNA as an indicator of infectiousness may show the importance of this test. SARS-CoV-2 PCR positive 109 naso/oropharyngeal swab samples stored at -80 °C were included in the study. In order to confirm the PCR positivity of these samples, E gene PCR was performed and AgT, and E and N sgRNA quantitative real-time reverse transcription-PCR (RT-qPCR) detection was performed. Of the 109 SARSCoV-2 PCR positive samples, 83 (76.14%) had antigen test positivity, 88 (80.73%) had E gene sgRNA, 96 (88.07%) had N gene sgRNA and 97 (89%) had at least one sgRNA positivity.The antigen test was found positive in 77.3% of the samples in which at least one sgRNA was detected and in 66.7% of the negative samples and this difference was not statistically significant (p= 0.475). The difference between E sgRNA and AgT positivity was significant (p= 0.023). N sgRNA was positive in 98.9% of E sgRNA positive samples and 42.9% of the negative samples and this difference was statistically significant (p= 0.0001). The AgT positivity rate was found to be 98.15% (53/54) for cycle threshold (Ct) value ≤ 25, 57.14% (12/21) for Ct 25-30, and 52.94% (18/34) for Ct ≥ 30. The difference in antigen test positivity between E gRNA Ct value ≤ 25 and > 25, ≤ 29 and > 29, < 30 and ≥ 30 was statistically significant (p= 0.0001). Antigen test positivity appears to be associated with viral load and infectivity, as expected. In our study, it has been shown that sgRNAs and AgT which are indicators of infectiousness can be detected at least 10 days after the symptom period. Using these two tests together could detect infective individuals with higher accuracy and shorten the duration of hospital stay and isolation.