A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field

Background : The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. Objectives : To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. Study design : In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 minutes. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. Results : Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). Conclusion : Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

Molecular Detection of Feline Coronavirus Based on Recombinase Polymerase Amplification Assay.

Feline coronavirus (FCoV) is endemic in cat populations worldwide. Persistently, subclinically infected cats play a significant role in spreading the infection. Testing fecal samples of cats may facilitate efforts to decrease the viral burden within a population. Real-time RT-PCR is highly sensitive and specific for the detection of FCoV but must be performed in a fully equipped laboratory. A simple and accurate assay is needed to identify FCoV at the point-of-need. The aim of this study was to develop a rapid FCoV detection assay based on isothermal amplification technology, i.e., reverse transcription-recombinase polymerase amplification (RT-RPA). Primers were designed to target the highly conserved 3' untranslated region of the 7b gene. Running on a constant temperature of 42 °C, reverse transcription as well as DNA amplification and detection was achieved in a maximum of 15 min. A probit analysis revealed a detection limit of 58.5 RNA copies/reaction. For cross-detection, nucleic acids from 19 viruses were tested. Both RT-RPA and real-time RT-PCR showed cross-detection with canine coronavirus and transmissible gastroenteritis virus, but not with other pathogens. To evaluate clinical performance, RNA was extracted from 39 fecal samples from cats. All samples were tested simultaneously with real-time RT-PCR resulting in a RT-RPA sensitivity and specificity of 90.9% and 100%, respectively. RT-RPA can be considered a promising simple method for rapid detection of FCoV.

Validation of the efficacy of air purifiers using molecular techniques.

The importance of air purifiers has increased in recent years, especially with the "coronavirus disease 2019" pandemic. The efficacy of air purifiers is usually determined under laboratory conditions before widespread application. The standard procedure for testing depends on virus cultivation and titration on cell culture. This, however, requires several days to deliver results. The aim of this study was to establish a rapid molecular assay which can differentiate between intact infectious and distorted non-infectious virus particles. Feline Coronavirus was selected as model for screening. First the samples were pretreated with enzymes (universal nuclease and RNase cocktail enzyme mixture) or viability dye (propidium monoazide) to eliminate any free nucleic acids. The ribonucleic acid (RNA) from intact virus was released via magnetic beads-based extraction, then the amount of the RNA was determined using real-time reverse transcription polymerase chain reaction (RT-PCR) or reverse transcription recombinase-aided amplification (RT-RAA). All results were compared to the infectivity assay based on the calculation of the 50% tissue culture infectious dose (TCID50). The nuclease has eliminated 100% of the free Feline Coronavirus RNA, while propidium monoazide underperformed (2.3-fold decrease in free RNA). Both RT-RAA and real-time RT-PCR produced similar results to the infectivity assay on cell culture with limit of detection of 102 TCID50/mL. Two UV-C air purifiers with prosperities of 100% inactivation of the viruses were used to validate the established procedure. Both real-time RT-PCR and RT-RAA were able to differentiate between intact virus particles and free RNA. To conclude, this study revealed a promising rapid method to validate the efficacy of air purifiers by combining enzymatic pretreatment and molecular assays.

Evaluation of a simple ultrafiltration method for concentration of infective canine parvovirus and feline coronavirus from cell culture supernatants.

Enrichment of viral infectious titers following its propagation by cell culture is desirable for various experimental studies. The performance of an ultrafiltration (UF) process to concentrate infectious titers of non-enveloped Canine parvovirus 2 (CPV-2) and enveloped Feline coronavirus (FCoV) obtained from cell culture supernatants was evaluated in this study, and compared with ultracentrifugation (UC) process. A mean gain of > 1.0 log10 TCID50/mL was obtained for CPV-2 with UF, which was comparable with the gain obtained by UC. On the other hand, the gain was lower (0.7-1.0 log10 TCID50/mL) for FCoV with UF in contrast to UC (> 2.0 log10 TCID50/mL). However, the lower retentate volume following UC (∼120 fold) compared to that following UF (∼10 fold) for either of the viruses suggests a trend of increased infectious titer retention in UF concentrates relative to UC concentrates. The simplistic UF process evaluated here thus has the potential for use in applications requiring increased infectious titers of CPV-2 and FCoV.

Vaccination of Immunocompromised Cats.

Immunocompromise is a common condition in cats, especially due to widespread infections with immunosuppressive viruses, such as feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), but also due to chronic non-infectious diseases, such as tumours, diabetes mellitus, and chronic kidney disease, as well as treatment with immunosuppressive drugs, such as glucocorticoids, cyclosporins, or tumour chemotherapy. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from eleven European countries, discusses the current knowledge and rationale for vaccination of immunocompromised cats. So far, there are few data available on vaccination of immunocompromised cats, and sometimes studies produce controversial results. Thus, this guideline summarizes the available scientific studies and fills in the gaps with expert opinion, where scientific studies are missing. Ultimately, this review aims to help veterinarians with their decision-making in how best to vaccinate immunocompromised cats.

The role of toothbrush in the transmission of corona- and influenza viruses - results of an in vitro study.

OBJECTIVES: The aim of this in vitro study was to investigate viruses' stabilities on manual toothbrushes using feline coronavirus (FeCoV) as representative of coronaviruses and an Avian influenza A virus H1N1 for influenza viruses. MATERIAL AND METHODS: Two viruses, FeCoV (Strain Munich; titer 107.5 TCID50/ml) and H1N1 (RE 230/90; titer 106.5 TCID50/ml), were used in this study. Manual toothbrushes were disassembled into bristles, bristle fixation, and back of the toothbrush head, contaminated with the viruses and air-dried for 24 h. In a second experiment, whole toothbrush heads were contaminated, rinsed with water (5 ml for 15 s) and then air-dried. RESULTS: For FeCoV, immediately after contamination, the following average titers were recovered: fixation: 106.41, back of head: 106.81 and bristles: 106.63 TCID50/ml. Following air-drying of 12 (fixation) and 24 h, titers of ≤ 102.5, 103.75, and 102.72 TCID50/ml were found in the respective groups, with a detection limit of 102.5 TCID50/ml. For H1N1, immediately after contamination, the following average titers could be recovered: fixation: 105.53, back of head: 105.97 and bristles: 105.75 TCID50/ml. Following air-drying of 8 (fixation) and 24 h, titers were ≤ 102.5, 103.63, and 103.53 TCID50/ml in the respective group, again with 102.5 TCID50/ml being the detection limit. In case of water rinse, no infectious virus could be recovered after 12 h. CONCLUSION: Viral load of both viruses is reduced by air-drying, especially following water rinsing. Clinical relevance The toothbrush itself plays an insignificant role in the self-transmission of coronavirus and influenza virus.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

Anthropogenic Infection of Cats during the 2020 COVID-19 Pandemic.

COVID-19 is a severe acute respiratory syndrome (SARS) caused by a new coronavirus (CoV), SARS-CoV-2, which is closely related to SARS-CoV that jumped the animal-human species barrier and caused a disease outbreak in 2003. SARS-CoV-2 is a betacoronavirus that was first described in 2019, unrelated to the commonly occurring feline coronavirus (FCoV) that is an alphacoronavirus associated with feline infectious peritonitis (FIP). SARS-CoV-2 is highly contagious and has spread globally within a few months, resulting in the current pandemic. Felids have been shown to be susceptible to SARS-CoV-2 infection. Particularly in the Western world, many people live in very close contact with their pet cats, and natural infections of cats in COVID-19-positive households have been described in several countries. In this review, the European Advisory Board on Cat Diseases (ABCD), a scientifically independent board of experts in feline medicine from 11 European Countries, discusses the current status of SARS-CoV infections in cats. The review examines the host range of SARS-CoV-2 and human-to-animal transmissions, including infections in domestic and non-domestic felids, as well as mink-to-human/-cat transmission. It summarises current data on SARS-CoV-2 prevalence in domestic cats and the results of experimental infections of cats and provides expert opinions on the clinical relevance and prevention of SARS-CoV-2 infection in cats.