Comparative evaluation of assay performance for SARS-CoV-2 detection in animal oral samples, lung homogenates, and phosphate-buffered saline using the TaqPath COVID-19 Combo kit.

A One Health approach has been key to monitoring the COVID-19 pandemic, as human and veterinary medical professionals jointly met the demands for an extraordinary testing effort for SARS-CoV-2. Veterinary diagnostic laboratories continue to monitor SARS-CoV-2 infection in animals, furthering the understanding of zoonotic transmission dynamics between humans and animals. A RT-PCR assay is a primary animal screening tool established within validation and verification guidelines provided by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), World Organisation for Animal Health (WOAH), and the U.S. Food and Drug Administration (FDA). However, differences in sample matrices, RNA extraction methods, instrument platforms, gene targets, and cutoff values may affect test outcomes. Therefore, targeted validation for a new sample matrix used in any PCR assay is critical. We evaluated a COVID-19 assay for the detection of SARS-CoV-2 in feline and canine lung homogenates and oral swab samples. We used the commercial Applied Biosystems MagMAX Viral/Pathogen II (MVP II) nucleic acid isolation kit and TaqPath COVID-19 Combo kit, which are validated for a variety of human samples, including nasopharyngeal and oropharyngeal swab samples. Our masked test showed a high detection rate and no false-positive or false-negative results, supporting sample extension to include feline oral swab samples. Our study is a prime example of One Health, illustrating how a COVID-19 assay designed for human testing can be adapted and used to detect SARS-CoV-2 in oral swab samples from cats and likely dogs, but not lung homogenates.

Domestic and wild animal samples and diagnostic testing for SARS-CoV-2.

From the first cases in 2019, COVID-19 infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in over 6 million human deaths in a worldwide pandemic. SARS-CoV-2 is commonly spread from human to human through close contact and is capable of infecting both humans and animals. Worldwide, there have been over 675 animal outbreaks reported that resulted in over 2000 animal infections including domestic and wild animals. As the role of animal infections in the transmission, pathogenesis, and evolution of SARS-CoV-2 is still unfolding, accurate and reliable animal diagnostic tests are critical to aid in managing both human and animal health. This review highlights key animal samples and the three main diagnostic approaches used for animal testing: PCR, serology, and Next Generation Sequencing. Diagnostic results help inform (often difficult) clinical decision-making, but also possible ways to mitigate spread among pets, food supplies, or wildlife. A One Health approach has been key to monitoring the SARS-CoV-2 pandemic, as consistent human-animal interactions can lead to novel variants. Having multiple animal diagnostic tests for SARS-CoV-2 available is critical to ensure human, animal, and environmental health.

Diagnosis of mucormycosis using a simple duplex PCR assay: Analysis of 160 clinical samples from COVID-19 patients.

Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175 bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements.

Early diagnosis of mucormycosis, a severe complication in COVID-19 patients, is critical for reducing the mortality of the infection. In this study, a sensitive and rapid PCR assay to detect all Mucorales and delineate Rhizopus oryzae was developed and assessed to improve the diagnosis of mucormycosis.

No indication for SARS-CoV-2 transmission to pet ferrets, in five cities in Poland, 2021 - antibody testing among ferrets living with owners infected with SARS-CoV-2 or free of infection.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in China by the end of 2019 and was responsible for a pandemic in the human population that resulted in millions of deaths worldwide. Since the beginning of the pandemic, the role of animals as spill-over or reservoir hosts was discussed. In addition to cats and dogs, ferrets are becoming increasingly popular as companion animals. Under experimental conditions, ferrets are susceptible to SARS-CoV-2 and it appears that they can also be infected through contact with a SARS-CoV-2 positive owner. However, there is still little information available regarding these natural infections. Here, we serologically tested samples collected from pet ferrets (n = 45) from Poland between June and September 2021. Of the ferrets that were included in the study, 29% (13/45) had contact with owners with confirmed SARS-CoV-2 infections. Nevertheless, SARS-CoV-2-specific antibodies could not be detected in any of the animals, independent of the infection status of the owner. The obtained results suggest that ferrets cannot be readily infected with SARS-CoV-2 under natural conditions, even after prolonged contact with infected humans. However, due to the rapid mutation rate of this virus, it is important to include ferrets in future monitoring studies.

Detection of anti-feline infectious peritonitis virus activity of a Chinese herb extract using geneLEAD VIII, a fully automated nucleic acid extraction/quantitative PCR testing system.

The geneLEAD VIII is a fully-automated nucleic acid extraction/quantitative PCR equipment developed by Precision System Science Co., Ltd., (PSS). To take advantage of its capability, we developed a quantitative assay system to measure growth of animal viruses. The system was used to assay one of the Chinese herbal extracts whose anti-malarial activities were previously reported and demonstrated its dose-dependent anti-viral activity against feline infectious peritonitis virus (FIPV), a feline coronavirus causing the fatal diseases in cats, and relatively low cell toxicity. The assay developed in this study is useful to screen antiviral drugs and the anti-FIPV activity of the herbal extract identified have a potential to lead to development of new drugs against FIPV and other coronaviruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Molecular surveillance of potential SARS-CoV-2 reservoir hosts in wildlife rehabilitation centers.

BACKGROUND: The COVID-19 pandemic, caused by SARS-CoV-2 infection, has become the most devastating zoonotic event in recent times, with negative impacts on both human and animal welfare as well as on the global economy. Although SARS-CoV-2 is considered a human virus, it likely emerged from animals, and it can infect both domestic and wild animals. This constitutes a risk for human and animal health including wildlife with evidence of SARS-CoV-2 horizontal transmission back and forth between humans and wild animals. AIM: Molecular surveillance in different wildlife rehabilitation centers and wildlife associated institutions in Chile, which are critical points of animal-human interaction and wildlife conservation, especially since the aim of wildlife rehabilitation centers is to reintroduce animals to their original habitat. MATERIALS AND METHODS: The survey was conducted in six WRCs and three wildlife associated institutions. A total of 185 samples were obtained from 83 individuals belonging to 15 different species, including vulnerable and endangered species. Each specimen was sampled with two different swabs: one oropharyngeal or nasopharyngeal according to the nostril diameter, and/or a second rectal sample. RNA was extracted from the samples and two different molecular assays were performed: first, a conventional RT-PCR with pan-coronavirus primers and a second SARS-CoV-2 qPCR targeting the N and S genes. RESULTS: All 185 samples were negative for SARS-CoV-2. CLINICAL RELEVANCE: This study constitutes the first report on the surveillance of SARS-CoV-2 from wildlife treated in rehabilitation centers in Chile, and supports the biosafety procedures adopted in those centers.

Self-Efficacy and Student Satisfaction in a Clinical-Year Diagnostic Imaging Course Using an Online Instruction Format.

Accurate interpretation of radiographic images is critical to diagnosing clinical patients. Remote instruction in radiology has become more common at veterinary colleges as academic institutions struggle to fill open veterinary radiologist positions and as a result of the COVID-19 pandemic. This study sought to gather the feedback of fourth-year veterinary students via pre- and post-study surveys (n = 45) and focus groups (n = 7) about a newly implemented 2-week long radiology rotation. Ninety-eight percent of students reported having taken an online course before, and on both pre- and post-study surveys, students commonly reported feeling interested, determined, and attentive. On average, students reported that they were neither more nor less engaged than they would have been in an in-person course and that they understood the material neither better nor worse than they would have in an in-person course. Students reported that the key to their success was primarily hard work; secondarily, instructor availability and student ability were important. Students did not rate luck as having much influence on their success. Although diagnostic imaging can be a challenging subject to master, students effectively learned this subject through online instruction. They provided feedback for the course's continued improvement; their comments centered around improved interactivity, including providing automated quiz questions' answers and increased instructor availability. Data collected in this study will help to guide further development of the radiology course.

Comparison of diagnostic performances of different serological tests for SARS-CoV-2 antibody detection in cats and dogs.

Serosurveillance among animals, including pets, plays an important role in the current coronavirus disease 2019 (COVID-19) pandemic, because severe acute respiratory coronavirus 2 (SARS-CoV-2) infections in animal populations could result in the establishment of new virus reservoirs. Serological assays that offer the required sensitivity and specificity are essential. In this study, we evaluated the diagnostic performance of three different commercially available immunoassays for the detection of SARS-CoV-2 antibodies in pets, namely two ELISA tests for the detection of antibodies against SARS-CoV-2 nucleocapsid [ID Screen SARS CoV-2 double antigen multispecies (Double antigen) and ID Screen® SARS-CoV-2-N IgG indirect ELISA (Indirect)] and one test for the detection of neutralizing antibodies against SARS-CoV-2 receptor-binding-domain [surrogate virus neutralization test (sVNT)]. The obtained results were compared with those of conventional virus neutralization test (VNT), which was regarded as reference method. A total of 191 serum samples were analysed. Thirteen (6.8%) samples showed VNT-positive results. The overall sensitivity was higher for sVNT (100%) compared to nucleocapsid-based ELISA assays (23% for Double antigen and 60% for Indirect). The specificity was 100% for Indirect ELISA and sVNT, when a higher cut-off (>30%) was used compared to the one previously defined by the manufacturer (>20%), whereas the other test showed lower value (99%). The sVNT test showed the highest accuracy and agreement with VNT, with a perfect agreement when the higher cut-off was applied. The agreement between each nucleocapsid-based ELISA test and VNT was 96% for Indirect and 94% for Double antigen. Our findings showed that some commercially available serological tests may lead to a high rate of false-negative results, highlighting the importance of assays validation for the detection of SARS-CoV-2 antibodies in domestic animals.

Screening of wild deer populations for exposure to SARS-CoV-2 in the United Kingdom, 2020-2021.

Following findings in Northern America of SARS-CoV-2 infections in white-tailed deer, there is concern of similar infections in European deer and their potential as reservoirs of SARS-CoV-2 including opportunities for the emergence of new variants. UK deer sera were collected in 2020-2021 from 6 species and a hybrid with 1748 tested using anti-spike and anti-nucleocapsid serology assays. No samples were positive on both assays nor by surrogate neutralization testing. There is no evidence that spill-over infections of SARS-CoV-2 occurred from the human population to UK deer or that SARS-CoV-2 has been circulating in UK deer (over the study period). Although it cannot be ruled out, study results indicate that spill-over infections followed by circulation of SARS-CoV-2 to the most common European deer species is small.

Repetitive saliva-based mass screening as a tool for controlling SARS-CoV-2 transmission in nursing homes.

Nursing home (NH) residents and staff have been severely affected by the COVID-19 pandemic. The aim of this study was to examine the use of weekly saliva RT-qPCR testing for SARS-CoV-2 detection among NH workers as a strategy to control disease transmission within NHs in Belgium. From 16 November to 27 December 2020, a voluntary and anonymous weekly screening was implemented in a cohort of 50,000 workers across 572 NHs in the Walloon region of Belgium to detect asymptomatic cases of SARS-CoV-2 via saliva RT-qPCR testing and using the Diagenode saliva sample collection device. Positive workers were isolated to avoid subsequent infections in residents and other staff. RT-qPCR testing was based on pooled saliva sampling techniques from three workers, followed by individual testing of each positive or inconclusive pool. The majority of NHs (85%) and 55% of their workers participated. Pooling did not affect sensitivity as it only induced a very decrease in sensitivity estimated as 0.33%. Significant decreases in the prevalence (34.4-13.4%) and incidence of NHs with either single (13.8-2%) or multiple positive workers (3.7-0%) were observed over time. In addition, deaths among NH residents and NH worker absences decreased significantly over time. Weekly saliva RT-qPCR testing for SARS-CoV-2 demonstrated large-scale feasibility and efficacy in disrupting the chain of transmission. Implementation of this testing strategy in NHs could also be extended to other settings with the aim to control viral transmission for maintaining essential activities.

SARS-CoV-2 natural infection in animals: a systematic review of studies and case reports and series.

COVID-19 pandemic is essentially a zoonotic disease. In this context, early in 2020, transmission from humans to certain animals began reporting; the number of studies has grown since. To estimate the pooled prevalence of SARS-CoV-2 natural infection in animals and to determine differences in prevalence between countries, years, animal types and diagnostic methods (RT-PCR or serological tests). A systematic literature review with meta-analysis using eight databases. Observational studies were included but analyzed separately. We performed a random-effects model meta-analysis to calculate the pooled prevalence and 95% confidence interval (95% CI) for prevalence studies and case series. After the screening, 65 reports were selected for full-text assessment and included for qualitative and quantitative analyses. A total of 24 reports assessed SARS-CoV-2 infection by RT-PCR, combining a total of 321,785 animals, yielding a pooled prevalence of 12.3% (95% CI 11.6%-13.0%). Also, a total of 17 studies additionally assessed serological response against SARS-CoV-2, including nine by ELISA, four by PRTN, one by MIA, one by immunochromatography (rest, two studies, the method was not specified), combining a total of 5319 animals, yielding a pooled prevalence of 29.4% (95% CI 22.9%-35.9%). A considerable proportion of animals resulted infected by SARS-CoV-2, ranking minks among the highest value, followed by dogs and cats. Further studies in other animals are required to define the extent and importance of natural infection due to SARS-CoV-2. These findings have multiple implications for public human and animal health. One Health approach in this context is critical for prevention and control.

Data standardization implementation and applications within and among diagnostic laboratories: integrating and monitoring enteric coronaviruses.

Every day, thousands of samples from diverse populations of animals are submitted to veterinary diagnostic laboratories (VDLs) for testing. Each VDL has its own laboratory information management system (LIMS), with processes and procedures to capture submission information, perform laboratory tests, define the boundaries of test results (i.e., positive or negative), and report results, in addition to internal business and accounting applications. Enormous quantities of data are accumulated and stored within VDL LIMSs. There is a need for platforms that allow VDLs to exchange and share portions of laboratory data using standardized, reliable, and sustainable information technology processes. Here we report concepts and applications for standardization and aggregation of data from swine submissions to multiple VDLs to detect and monitor porcine enteric coronaviruses by RT-PCR. Oral fluids, feces, and fecal swabs were the specimens submitted most frequently for enteric coronavirus testing. Statistical algorithms were used successfully to scan and monitor the overall and state-specific percentage of positive submissions. Major findings revealed a consistently recurrent seasonal pattern, with the highest percentage of positive submissions detected during December-February for porcine epidemic diarrhea virus, porcine deltacoronavirus, and transmissible gastroenteritis virus (TGEV). After 2014, very few submissions tested positive for TGEV. Monitoring VDL data proactively has the potential to signal and alert stakeholders early of significant changes from expected detection. We demonstrate the importance of, and applications for, data organized and aggregated by using LOINC and SNOMED CTs, as well as the use of customized messaging to allow inter-VDL exchange of information.

Lower respiratory tract samples are reliable for severe acute respiratory syndrome coronavirus 2 nucleic acid diagnosis and animal model study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease 2019 (COVID-19) continue to impact countries worldwide. At present, inadequate diagnosis and unreliable evaluation systems hinder the implementation and development of effective prevention and treatment strategies. Here, we conducted a horizontal and longitudinal study comparing the detection rates of SARS-CoV-2 nucleic acid in different types of samples collected from COVID-19 patients and SARS-CoV-2-infected monkeys. We also detected anti-SARS-CoV-2 antibodies in the above clinical and animal model samples to identify a reliable approach for the accurate diagnosis of SARS-CoV-2 infection. Results showed that, regardless of clinical symptoms, the highest detection levels of viral nucleic acid were found in sputum and tracheal brush samples, resulting in a high and stable diagnosis rate. Anti-SARS-CoV-2 immunoglobulin M (IgM) and G (IgG) antibodies were not detected in 6.90% of COVID-19 patients. Furthermore, integration of nucleic acid detection results from the various sample types did not improve the diagnosis rate. Moreover, dynamic changes in SARS-CoV-2 viral load were more obvious in sputum and tracheal brushes than in nasal and throat swabs. Thus, SARS-CoV-2 nucleic acid detection in sputum and tracheal brushes was the least affected by infection route, disease progression, and individual differences. Therefore, SARS-CoV-2 nucleic acid detection using lower respiratory tract samples alone is reliable for COVID-19 diagnosis and study.