Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

Combined nasal- and oropharyngeal self-swab provides equivalent performance compared to professionally collected oropharyngeal swabs in detecting SARS-CoV-2 in a real-life setting.

PURPOSE: To investigate the performance of a combined nasal midturbinate- and oropharyngeal (NAOP) self-swab compared to a deep oropharyngeal (OP) swab by health care workers (HCW) in detecting SARS-CoV-2 in a real-life setting. METHODS: Paired swabs from 1119 participants were included. RT-PCR were used to detect SARS-CoV-2 in both swab samples. RESULTS: 330 participants tested positive. The sensitivity of the combined self-swab and OP swab was 96.9 % and 95.4 % respectively, whereas the Ct-values for self-swabs were significantly lower compared to OP swabs. CONCLUSION: The combined NAOP self-swab outperformed the OP swab and thus, the NAOP self-swab may be an alternative sampling method under the given circumstances.

SARS-CoV-2 RNA stability in dry swabs for longer storage and transport at different temperatures.

During the current COVID-19 pandemic, different methods have been used to evaluate patients with suspected SARS-CoV-2 infection. In this study, we experimentally evaluate the ability of spiked saliva-moist swabs and spiked swabs without any transport medium to retain SARS-CoV-2 for storage and transport at different environmental settings during different incubation time periods. Our results show that at ambient temperature of 20°C, SARS-CoV-2 RNA remains stable for up to 9 days allowing a long-time span for transport and storage without compromising clinical results. Additionally, this study demonstrates that saliva-moist swabs can also be stored at -20°C and +4°C for up to 26 days without affecting RT-qPCR results. Our data are relevant for low-and middle-income countries, which have limited access to rapid refrigerated transport and storage of samples representing an economical alternative. Finally, our study demonstrates the practical and economic advantage of using swabs without transport medium.

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.

Late regulation of immune genes and microRNAs in circulating leukocytes in a pig model of influenza A (H1N2) infection.

MicroRNAs (miRNAs) are a class of short regulatory RNA molecules which are implicated in modulating gene expression. Levels of circulating, cell-associated miRNAs in response to influenza A virus (IAV) infection has received limited attention so far. To further understand the temporal dynamics and biological implications of miRNA regulation in circulating leukocytes, we collected blood samples before and after (1, 3, and 14 days) IAV challenge of pigs. Differential expression of miRNAs and innate immune factor mRNA transcripts was analysed using RT-qPCR. A total of 20 miRNAs were regulated after IAV challenge, with the highest number of regulated miRNAs seen on day 14 after infection at which time the infection was cleared. Targets of the regulated miRNAs included genes involved in apoptosis and cell cycle regulation. Significant regulation of both miRNAs and mRNA transcripts at 14 days after challenge points to a protracted effect of IAV infection, potentially affecting the host's ability to respond to secondary infections. In conclusion, experimental IAV infection of pigs demonstrated the dynamic nature of miRNA and mRNA regulation in circulating leukocytes during and after infection, and revealed the need for further investigation of the potential immunosuppressing effect of miRNA and innate immune signaling after IAV infection.

Reduced response to Epstein-Barr virus antigens by T-cells in systemic lupus erythematosus patients.

OBJECTIVE: Epstein-Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. METHODS: T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. RESULTS: SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. CONCLUSIONS: These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed.

Transcriptional profiling at different sites in lungs of pigs during acute bacterial respiratory infection.

The local transcriptional response was studied in different locations of lungs from pigs experimentally infected with the respiratory pathogen Actinobacillus pleuropneumoniae serotype 5B, using porcine cDNA microarrays. This infection gives rise to well-demarcated infection loci in the lung, characterized by necrotic and haemorrhagic lesions. Lung tissue was sampled from necrotic areas, from visually unaffected areas and from areas bordering on necrotic areas. Expression pattern of these areas from infected pigs was compared to healthy lung tissue from un-infected pigs. Transcription of selected genes important in the innate defence response were further analysed by quantitative real-time reverse-transcriptase PCR. A clear correlation was observed between the number of differentially expressed genes as well as the magnitude of their induction and the sampling location in the infected lung, with the highest number of differentially expressed genes, and the most highly induced genes found in necrotic areas. Interestingly, a group of differentially regulated genes was represented in all three areas, comprising genes encoding cytokines, acute phase proteins, and factors related to regulation of apoptosis and the complement system. Interferon-γ was downregulated in both necrotic and bordering areas. Evidence of neutrophil recruitment was seen by the up-regulation of chemotactic factors for neutrophils. In conclusion, we found subsets of genes expressed at different levels in the three selected areas of the infected lung as compared to the control group. Thus it is demonstrated that an infection with clearly defined infected loci leads to a rapid disseminated intra-organ response in neighbouring seemingly unaffected tissue areas of the infected organ. Within the lung, we found a clear division of induced genes as, in unaffected areas a large part of differently expressed genes were involved in systemic reactions to infections, while differently expressed genes in necrotic areas were mainly concerned with homeostasis regulation.

Hepatic gene expression changes in pigs experimentally infected with the lung pathogen Actinobacillus pleuropneumoniae as analysed with an innate immunity focused microarray.

Knowledge on gene expression in the liver during respiratory infections is limited although it is well-established that this organ is an important site of synthesis of several systemic innate immune components as response to infections. In the present study, the early transcriptional hepatic response of genes associated with innate immune responses was studied in pigs 14-18 h after intranasal inoculation with Actinobacillus pleuropneumoniae, using innate immune focused microarrays and quantitative real-time PCR (qPCR). The microarray analysis of liver tissue established that 51 genes were differentially expressed. A large group of these genes encoded proteins involved in the acute phase response, including serum amyloid A, C-reactive protein, fibrinogen, haptoglobin and tumor necrosis factor-α the expression of which were all found to be up-regulated and glutathione S-transferase, transthyretin, transferrin and albumin which were down-regulated. Additional genes associated with innate immune responses were investigated using qPCR; genes encoding interleukin-(IL-)1, IL-6, IL-8, lipopolysaccharide binding protein, lactotransferrin, and PigMAP were up-regulated and interferon 1α, α1-acid glycoprotein, mannan-binding lectin A, surfactant protein D, and surfactant protein A1 were down-regulated in the liver of infected animals. Down-regulation of α1-acid glycoprotein during infection has not been described previously in any species. These results confirm that the liver plays an important role in initiating and orchestrating the innate immune response to A. pleuropneumoniae infection.

Rapid and widely disseminated acute phase protein response after experimental bacterial infection of pigs.

The acute phase protein response is a well-described generalized early host response to tissue injury, inflammation and infection, observed as pronounced changes in the concentrations of a number of circulating serum proteins. The biological function of this response and its interplay with other parts of innate host defence reactions remain somewhat elusive. In order to gain new insight into this early host defence response in the context of bacterial infection we studied gene expression changes in peripheral lymphoid tissues as compared to hepatic expression changes, 14-18 h after lung infection in pigs. The lung infection was established with the pig specific respiratory pathogen Actinobacillus pleuropneumoniae. Quantitative real-time PCR based expression analysis were performed on samples from liver, tracheobronchial lymph node, tonsils, spleen and on blood leukocytes, supplemented with measurements of interleukin-6 and selected acute phase proteins in serum. C-reactive protein and serum amyloid A were clearly induced 14-18 h after infection. Extrahepatic expression of acute phase proteins was found to be dramatically altered as a result of the lung infection with an extrahepatic acute phase protein response occurring concomitantly with the hepatic response. This suggests that the acute phase protein response is a more disseminated systemic response than previously thought. The current study provides to our knowledge the first example of porcine extrahepatic expression and regulation of C-reactive protein, haptoglobin, fibrinogen, pig major acute phase protein, and transferrin in peripheral lymphoid tissues.

Validation of putative reference genes for qRT-PCR normalization in tissues and blood from pigs infected with Actinobacillus pleuropneumoniae.

The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive and very efficient technique for quantification of gene expression. However, qRT-PCR relies on accurate normalization of gene expression data, as RNA recovery and cDNA synthesis efficiency might vary from sample to sample. In the present study, six putative reference genes were validated for normalization of gene expression in three different tissues and in white blood cells from pigs experimentally infected with the common respiratory pathogen Actinobacillus pleuropneumoniae. Two dedicated validation programs (geNorm and Normfinder) were used to rank the six reference genes from best to worst. qRT-PCR data for the proinflammatory cytokine IL-6 was normalized using the proposed genes from geNorm and Normfinder as well as the commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). IL-6 expression was quantified in white blood cells, liver, lymph nodes and tonsils from 10 infected pigs and 5 control pigs. After normalization using either geNorm or Normfinder IL-6 was shown to be significantly up-regulated (P<0.05) in all of the tissues from infected animals compared to non-infected control animals with a good agreement of expression differences between the two programs. On the contrary, normalization of IL-6 expression data from blood using GAPDH rendered the difference between infected and non-infected groups non-significant, and resulted in significantly different values compared to geNorm (P=0.01). Based on these results, we recommend to validate putative reference genes before normalization.

Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays.

BACKGROUND: The bacterium Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after A. pleuropneumoniae infection is still not clarified. The objective of this study was hence to characterise the transcriptional response, measured by using cDNA microarrays, in pigs 24 hours after experimental inoculation with A. pleuropneumoniae. METHODS: Microarray analyses were conducted to reveal genes being differentially expressed in inflamed versus non-inflamed lung tissue sampled from inoculated animals as well as in liver and tracheobronchial lymph node tissue sampled from three inoculated animals versus two non-inoculated animals. The lung samples were studied using a porcine cDNA microarray with 5375 unique PCR products while liver tissue and tracheobronchial lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products. RESULTS: A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue, genes encoding immune activating proteins and other pro-inflammatory mediators of the innate immune response were found to be up-regulated. Genes encoding different acute phase reactants were found to be differentially expressed in the liver. CONCLUSION: The obtained results are largely in accordance with previous studies of the mammalian immune response. Furthermore, a number of differentially expressed genes have not previously been associated with infection or are presently unidentified. Determination of their specific roles during infection may lead to a better understanding of innate immunity in pigs. Although additional work including more animals is clearly needed to elucidate host response to porcine pleuropneumonia, the results presented in this study demonstrate three subsets of genes consistently expressed at different levels depending upon infection status.

Transcriptional gene expression profiling of small cell lung cancer cells.

A global gene expression analysis using oligonucleotide microarrays was performed on many human small cell lung cancer (SCLC) cell lines in cell culture and/or as xenografts. The expression was compared with the expression profiles of 18 normal tissues. In a hierarchical cluster analysis the cell lines clustered distinctly from normal tissues and grouped into four clusters. One cluster consisted of two related cell lines and was markedly different from the other SCLC cell lines, whereas the rest of the clusters grouped together. Two subclusters contained the classical SCLC types and one subcluster the variant SCLC type, thus identifying many genes with differential expression between the two variants of SCLC. All of the xenografts clustered closest to the cell lines from which they originated and had the same expression levels as the cells grown in culture for the majority of genes. The analysis confirmed the high expression of many genes identified previously as highly expressed in SCLC cells including neuroendocrine markers, oncogenes, and genes involved in cell proliferation and division. The analysis furthermore identified a number of molecules not identified previously as expressed in SCLC. Several of these are expressed in low or undetectable amounts in the majority of normal tissues and, therefore, are potential targets for new therapeutic approaches. By including the published array profiles of six ressected SCLC tumors from Bhattacharjee et al. (A. Bhattacharjee et al., Proc. Natl. Acad. Sci. USA, 98: 13790-13795, 2001.), the analysis revealed that most of the novel potential targets expressed by SCLC cell lines and xenografts were also expressed in the tumors. This analysis demonstrates the value of using cell lines and xenografts for expression profiling, when a limited quantity of tumor material is available.