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The virus discovered in 2019 in the city of Wuhan, China, which was later identified as SARS-CoV-2 and which spread to the level of a pandemic, put diagnostic methods to the test. Early in the pandemic, we developed a nested PCR assay for the detection of SARS-CoV-2, which we validated and applied to detect the virus in feline samples. The present study describes the application of the nested PCR test in parallel with LAMP for the detection of the virus in 427 nasopharyngeal and oropharyngeal human samples taken between October 2020 and January 2022. Of the swabs tested, there were 43 positives, accounting for 10.1% of all samples tested, with the negatives numbering 382, i.e., 89.5%, and there were 2 (0.4%) invalid ones. The nPCR results confirmed those obtained by using LAMP, with results concordant in both methods. Nasal swabs tested using nPCR confirmed the results of oropharyngeal and nasopharyngeal swab samples tested using LAMP and nPCR. The focus of the discussion is on the two techniques: the actual practical application of the laboratory-developed assays and the diagnostic value of nasal samples. The nPCR used is a reliable and sensitive technique for the detection of SARS-CoV-2 in nasopharyngeal, oropharyngeal, and nasal swab samples. However, it has some disadvantages related to the duration of the entire process, as well as a risk of contamination. Experiments were performed to demonstrate the infectivity of the virus from the positive isolates in vitro. A discrepancy was reported between direct and indirect methods of testing the virus and accounting for its ability to cause infection in vitro. [ABSTRACT FROM AUTHOR]

Little information is presently available regarding the frequency of the silent shedders of respiratory viruses in healthy sport horses and their impact on environmental contamination. Therefore, the aim of this study was to investigate the detection frequency of selected respiratory pathogens in nasal secretions and environmental stall samples of sport horses attending a multi-week equestrian event during the summer months. Six out of fifteen tents were randomly selected for the study with approximately 20 horse/stall pairs being sampled on a weekly basis. Following weekly collection for a total of 11 weeks, all samples were tested for the presence of common respiratory pathogens (EIV, EHV-1, EHV-4, ERAV, ERBV, and Streptococcus equi ss equi (S. equi)) using qPCR. A total of 19/682 nasal swabs (2.8%) and 28/1288 environmental stall sponges (2.2%) tested qPCR-positive for common respiratory pathogens. ERBV was the most common respiratory virus (17 nasal swabs, 28 stall sponges) detected, followed by EHV-4 (1 nasal swab) and S. equi (1 nasal swab). EIV, EHV-1, EHV-4 and ERAV were not detected in any of the study horses or stalls. Only one horse and one stall tested qPCR-positive for ERBV on two consecutive weeks. All the other qPCR-positive sample results were related to individual time points. Furthermore, only one horse/stall pair tested qPCR-positive for ERBV at a single time point. The study results showed that in a selected population of sport horses attending a multi-week equestrian event in the summer, the frequency of the shedding of respiratory viruses was low and primarily restricted to ERBV with little evidence of active transmission and environmental contamination. [ABSTRACT FROM AUTHOR]

Background: In resource-poor countries, antigen-based rapid tests (Ag-RDTs) performed at primary healthcare and community settings improved access to SARS-CoV-2 diagnostics. However, the technical skills and biosafety requirements inherent to nasopharyngeal and oropharyngeal (OP) specimens limit the scale-up of SARS-CoV-2 testing. The collection of nasal-swabs is programmatically viable, but its performance has not been evaluated in resource-poor settings. Methods: We first evaluated the performance of SteriPack self-collected nasal swabs for the detection of SARS-CoV-2 by real-time PCR in 1498 consecutively enrolled patients with suspected infection. Next, we evaluated the clinical performance of three nasal swab-based Ag-RDTs against real-time PCR on OP specimens. Results: The sensitivity of nasal swabs was 80.6% [95% CI: 75.3–85.2%] compared to OP specimens. There was a good correlation (r = 0.58; p < 0.0001) between Ct values of 213 positive cases obtained using nasal and OP swabs. Our findings show sensitivities of 79.7% [95% CI: 73.3–85.1%] for Panbio COVID-19 Ag-RDT, 59.6% [95% CI: 55.2–63.8%] for COVIOS Ag-RDT, and 78.0% [95% CI: 73.5–82.0%] for the LumiraDx SARS-CoV-2 Ag-RDT. Conclusions: In our setting, the COVIOS Ag-RDT did not meet WHO requirements. Nasal swab-based Ag-RDTs for SARS-CoV-2 detection constitute a viable and accurate diagnostic option in resource-poor settings. [ABSTRACT FROM AUTHOR]

Simple Summary: Due to concerns in public health and its negative impact on the pig industry the need for Influenza A virus (IAV) surveillance is rising. The gold standard procedure for detecting IAV is to sample acutely diseased pigs. Endemic infections with unspecific clinical signs and low disease prevalence need new approaches. Our study aimed to evaluate a standardized sampling procedure for the detection of IAV in epidemically and endemically infected farms. We performed a cross-sectional study in 131 farms investigating three different age groups per farm in 12 European countries. The results of our investigation indicate that 10 nasal swabs each in suckling piglets, weaners and middle of nursery is a valuable tool for influenza detection and identification of subtypes. However, for farms with a lower prevalence than 15% it is advisable to either increase the number of nasal swabs in each age group or to use group sampling methods. Interestingly, different subtypes were found in different age groups. Thus, our study underlines that sampling of different age groups is mandatory to obtain a comprehensive overview on all circulating variants on farm. In addition, our results highlight that sampling strategies should also consider piglets without obvious clinical signs for IAV infection. Swine influenza A virus (swIAV), which plays a major role in the porcine respiratory disease complex (PRDC), is eliminated from the respiratory tract within 7–9 days after infection. Therefore, diagnosis is complicated in endemically infected swine herds presenting no obvious clinical signs. This study aimed to investigate the right time point for sampling to detect swIAV. A cross-sectional study was performed in 131 farms from 12 European countries. The sampling protocol included suckling piglets, weaners, and nursery pigs. In each age group, 10 nasal swabs were collected and further examined in pools of 5 for swIAV by Matrix rRT-PCR, followed by a multiplex RT-PCR to determine the influenza subtype. SwIAV was detected in 284 (37.9%) of the samples and on 103 (78.6%) farms. Despite the highest number of animals with clinical signs being found in the nursery, the weaners were significantly more often virus-positive compared to nursery pigs (p = 0.048). Overall, the swIAV detection rate did not significantly differ between diseased or non-diseased suckling and nursery piglets, respectively; however, diseased weaners had significantly more positive pools than the non-diseased animals. Interestingly, in 9 farms, different subtypes were detected in different age groups. Our findings indicate that to detect all circulating swIAV subtypes on a farm, different age groups should be sampled. Additionally, the sampling strategy should also aim to include non-diseased animals, especially in the suckling period. [ABSTRACT FROM AUTHOR]

Equine adenovirus 1 (EAdV-1) can cause upper respiratory disease in horses and has been reported worldwide. In this study, and for the first time in Korea, the prevalence of EAdV-1 in equine nasal swabs was investigated using a PCR to identify potential risk factors and examine the genetic diversity of its DNA sequences by a comparison with foreign strains. Nasal swabs collected from 359 horses reared at Korea Racing Authority facilities were tested using an EAdV-1 hexon-specific PCR and the associations between EAdV-1 infection and sex, age, region, breed, and activity were analyzed. Five samples (1.4%, 5/359) tested positive for EAdV-1; however, no statistically significant differences were observed with respect to any variable. Among the five EAdV-1-positive horses, a co-infection with equine influenza, equine herpesvirus 1 and 4, or Streptococcus equi was not detected; however, clinical respiratory signs were observed in one. Phylogenetic analyses based on partial EAdV-1 hexon gene sequences revealed that the Korean EAdV-1 isolates shared approximately 98.8–100% similarity among each other and with foreign strains. Three Korean isolates shared high similarity with strains from Australia and India and the remaining two isolates were separate in phylogenetic analyses. These findings highlight the molecular prevalence and genetic diversity of EAdV-1 in horses in Korea. [ABSTRACT FROM AUTHOR]

The aim of this pilot study was to assess the time-related changes in viable nasal bacteria concentrations among waste-incineration plant (WIP) workers compared to a group of office building (OB) workers outside the plant. In total, 20 volunteers participated in the study, including 14 WIP and 6 OB workers. WIP workers were divided into two sub-groups: supervisory staff (SVS) and maintenance and repair workers (MRW). Nasal swabs were collected before and after the morning work shift. Airborne bacteria were sampled with a six-stage impactor to assess the bioaerosol size distribution. The analysis showed that a significant, almost three-fold increase in nasal bacterial concentration was found only among WIP workers, and this referred mainly to anaerobic species. The load of anaerobic bacteria at the beginning of work was 12,988 CFU/mL, and after work shift 36,979 CFU/mL (p < 0.01). Significant increases in microbial concentrations was found only in the MRW subgroup, among non-smoking workers only. The results showed increased bacterial concentration in WIP nasal samples for as many as 12 bacterial species, including, e.g., Streptococcus constellatus, Peptostreptococcus spp., E. coli, and P. mirabilis. These preliminary data confirmed that the nasal swab method was helpful for assessment of the workers’ real-time exposure to airborne bacteria.

Porcine respirovirus 1 (PRV1) is also known as porcine parainfluenza virus 1 (PPIV1). The prevalence and the role of PRV1 infections for pig health is largely unknown. In order to assess the PRV1 prevalence in Poland, nasal swabs and oral fluids collected from pigs from 30 farms were examined with RT real-time PCR. Additionally, IAV and PRRSV infection statuses of PRV1-positive samples were examined. The results showed that the virus is highly prevalent (76.7% farms positive) and different patterns of PRV1 circulation in herds with mild–moderate respiratory disease were observed. Co-infections with IAV and PRRSV were infrequent and detected in 8 (23.5%) and 4 (11.8%) out of 34 PRV1-positive nasal swab pools from diseased pens, respectively. In one pen PRV1, IAV, and PRRSV were detected at the same time. Interestingly, PRV1 mean Ct value in samples with co-infections was significantly lower (29.8 ± 3.1) than in samples with a single PRV1 infection (32.5 ± 3.6) (p < 0.05), which suggested higher virus replication in these populations. On the other hand, the virus detection in pig populations exhibiting respiratory clinical signs, negative for PRRSV and IAV, suggests that PRV1 should be involved in differential diagnosis of respiratory problems. [ABSTRACT FROM AUTHOR]

Madin–Darby canine kidney (MDCK) cells are commonly used for the isolation of mammalian influenza A viruses. The goal of this study was to compare the sensitivity and suitability of the original MDCK cell line in comparison with MDCK-derived cell lines, MDCK.2, MDCK SIAT-1 and MDCK-London for isolation of swine-origin influenza A viruses (IAV-S) from clinical specimens. One-hundred thirty clinical specimens collected from pigs in the form of nasal swabs, lung tissue and oral fluids that were positive by PCR for the presence of IAV-S RNA were inoculated in the cell cultures listed above. MDCK-SIAT1 cells yielded the highest proportion of positive IAV-S isolations from all specimen types. For nasal swabs, 58.62% of the specimens were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (36.21%), and conventional MDCK and MDCK.2 cells (27.5%). For lung specimens, 59.38% were IAV-S positive in MDCK-SIAT1 cells, followed by MDCK-London (40.63%), and conventional MDCK and MDCK.2 cells (18.75–31.25%). Oral fluids yielded the lowest number of positive virus isolation results, but MDCK-SIAT1 cells were still had the highest rate (35%) of IAV-S isolation, whereas the isolation rate in other cells ranged from 5–7.5%. Samples with lower IAV-S PCR cycle threshold (Ct) values were more suitable for culturing and isolation. The isolated IAV-S represented H1N1-β, H1N2-α, H1N1pdm and H3N2 cluster IV and cluster IVB viruses. The result of the current study demonstrated the importance of using the most appropriate MDCK cells when isolating IAV-S from clinical samples. [ABSTRACT FROM AUTHOR]

This paper discusses the application of two approaches (direct and inverse) to the identification of volatile substances by means of a gas sensor array in a headspace over nasal mucus swab samples taken from calves with differing degrees of respiratory damage. We propose a unique method to visualize sensor array data for quality analysis, based on the spectra of cross mass sensitivity parameters. The traditional method, which requires an initial sensor array trained on the vapors of the individual substances (database accumulation)—with their further identification in the analyzed bio-samples through the comparison of the analysis results to the database—has shown unsatisfactory performance. The proposed inverse approach is more informative for the pattern recognition of volatile substances in the headspace of mucus samples. The projection of the calculated parameters of the sensor array for individual substances in the principal component space, acquired while processing the sensor array output from nasal swab samples, has allowed us to divide animals into groups according to the clinical diagnosis of their lung condition (healthy respiratory system, bronchitis, or bronchopneumonia). The substances detected in the gas phase of the nasal swab samples (cyclohexanone, butanone-2,4-methyl-2-pentanone) were correlated with the clinical state of the animals, and were consistent with the reference data on disease markers in exhaled air established for destructive organism processes. [ABSTRACT FROM AUTHOR]

The main reservoir of Coxiella (C.) burnetii are ruminants. They shed the pathogen through birth products, vaginal mucus, faeces and milk. A direct comparison of C. burnetii excretions between naturally infected sheep and goats was performed on the same farm to investigate species-specific differences. The animals were vaccinated with an inactivated C. burnetii phase I vaccine at the beginning of the study period for public health reasons. Vaginal and rectal swabs along with milk specimens were taken monthly during the lambing period and once again at the next lambing season. To estimate the environmental contamination of the animals' housings, nasal swabs from every animal were taken simultaneously. Moreover, dust samples from the windowsills and straw beddings were collected. All samples were examined by qPCR targeting the IS1111 gene and the MLVA/VNTR typing method was performed. Whole genome sequencing was applied to determine the number of IS1111 copies followed by a calculation of C. burnetii genome equivalents of each sample. The cattle-associated genotype C7 was detected containing 29 IS1111 copies. Overall, goats seem to shed more C. burnetii through vaginal mucus and in particular shed more and for longer via the rectal route than sheep. This is supported by the larger quantities of C. burnetii DNA detected in caprine nasal swabs and environmental samples compared to the ovine ones. Transmission of C. burnetii from cattle to small ruminants must also be considered. [ABSTRACT FROM AUTHOR]