PRRSV RNA detection in different matrices under typical storage conditions in the UK

In the UK, approximately 40 per cent of the pig breeding herds are outdoors. To monitor their porcine reproductive and respiratory syndrome virus (PRRSV) status, blood is collected commonly from piglets around weaning. Sample collection in British outdoor pigs often occurs during the early morning hours when the piglets tend to accumulate inside sheltered areas. For practical reasons, dry cotton swabs are occasionally used for blood collection and stored at room temperature until arrival in the laboratory. Detection of PRRSV RNA is a function of viral concentration, sample type and storage condition. To evaluate a possible impact of the sampling protocol on PRRSV1 detection, experimentally spiked blood samples using three dilutions of a representative PRRSV1 strain were prepared. In addition, blood samples from pigs naturally infected with PRRSV were obtained from a PRRSV-positive British herd. Spiked blood and blood from infected pigs were used to obtain sera, dry or wet (immersed in saline) polyester or cotton swabs and FTA cards. The different samples were stored for 24 hours, 48 hours or 7 days at 4°C or 20°C and tested by a real-time reverse transcriptase PRRSV PCR assay. Under the study conditions, the best matrix was serum (96.7 per cent), followed by wet swabs (78 per cent), dry swabs (61.3 per cent) and FTA cards (51 per cent). Polyester swabs (76 per cent) showed a better performance than cotton swabs (63.3 per cent). The reduction in sensitivity obtained for swabs and FTA cards was particularly high at low viral concentrations. The results indicate that wet polyester swabs should be used whenever possible

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions

International audienceAbstractThe causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10–20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10–65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations.

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities

Saliva Alternative to Upper Respiratory Swabs for SARS-CoV-2 Diagnosis

PCR of upper respiratory specimens is the diagnostic standard for severe acute respiratory syndrome coronavirus 2 infection. However, saliva sampling is an easy alternative to nasal and throat swabbing. We found similar viral loads in saliva samples and in nasal and throat swab samples from 110 patients with coronavirus disease

Evaluation of eight lateral flow tests for the detection of anti-SARS-CoV-2 antibodies in a vaccinated population

Background: Rapid determination of an individual's antibody status can be beneficial in understanding an individual's immune response to SARS-CoV-2 and for initiation of therapies that are only deemed effective in sero-negative individuals. Antibody lateral flow tests (LFTs) have potential to address this need as a rapid, point of care test. Methods: Here we present a proof-of-concept evaluation of eight LFT brands using sera from 95 vaccinated individuals to determine sensitivity for detecting vaccination generated antibodies. Samples were analysed on eight different brands of antibody LFT and an automated chemiluminescent microparticle immunoassay (CMIA) that identifies anti-spike antibodies which was used as our reference standard. Results: All 95 (100%) participants tested positive for anti-spike antibodies by the chemiluminescent microparticle immunoassay (CMIA) reference standard post-dose two of their SARS-CoV-2 vaccine: BNT162b2 (Pfizer/BioNTech, n = 60), AZD1222 (AstraZeneca, n = 31), mRNA-1273 (Moderna, n = 2) and Undeclared Vaccine Brand (n = 2). Sensitivity increased from dose one to dose two in six out of eight LFTs with three tests achieving 100% sensitivity at dose two in detecting anti-spike antibodies. Conclusions: These tests are demonstrated to be highly sensitive to detect raised antibody levels in vaccinated individuals. RDTs are low cost and rapid alternatives to ELISA based systems

Use of the rSpaA415 antigen indicates low rates of Erysipelothrix rhusiopathiae infection in farmed cattle from the United States of America and Great Britain

Background Clinical cases of Erysipelothrix rhusiopathiae, a zoonotic gram-positive bacterium, have been reported in many ruminant species, including in cattle, deer, moose and muskoxen. Fatal cases have been repeatedly reported in cattle over the years but to date there is only one Japanese study investigating the seroprevalence of this bacterium in cattle using the growth agglutination test (GAT). This technique is subjective, time-consuming, expensive and hazardous compared to modern serological tests such as enzyme-linked immunosorbent assays (ELISA) or the newly developed fluorescent microbead-based immunoassays (FMIA). Results The FMIA based on the surface protein SpaA (rSpaA415) antigen of E. rhusiopathiae developed in this study had an almost perfect agreement with the GAT (k = 0.83) and showed a sensitivity of 89.7% and a specificity of 92.9% when compared to the GAT. Overall, detection rates of E. rhusiopathiae antibody positive samples were 13.8% (51/370) in British herds and 6% (12/200) in US herds. Positive cattle were present in 34.3% (24/70) of the investigated British farms and in 34.7% (8/23) of the US farms with an on-farm prevalence of 7.1 to 100% for the British farms and 8.3–30% for the US farms. Conclusions FMIA is a fast, safe and economic alternative to the GAT for the diagnosis of E. rhusiopathiae in cattle. This work is the first seroprevalence study of E. rhusiopathiae in healthy farmed cattle in Great Britain and the US and revealed that infection occurs at a low level. Further investigations to evaluate risks of zoonotic transmission when handling cattle are needed

Lateral flow antigen tests can sensitively detect live cultured virus of the SARS-CoV-2 B1.1.7 lineage

We read with interest recent evaluations of SARS-CoV-2 antigen rapid diagnostic tests (Ag-RDTs), showing high sensitivity for detecting cases with higher viral loads.1 The clinical sensitivity of these tests relies on their limit of detection (LOD), and there are concerns that new SARS-CoV-2 variants may affect test performance. New variants of SARS-CoV-2 have arisen worldwide, including the lineage B.1.1.7 (Variant of Concern (VOC) strain 202,012/01), which has become the dominant circulating SARS-CoV-2 strain in the U.K., causing >95% of new infections as of April 2021.2 Lineage B.1.1.7 has now been detected in over 114 countries in Europe, America, Australia, Asia and Africa. B.1.1.7 has 17 mutations compared with the original circulating virus, including 8 in the spike protein (S), and 2 in the nucleoprotein (N).3 Most Ag-RDTs target N protein with some targeting the S protein. Therefore, these mutations may affect antibody binding in the Ag-RDT, and consequently affect the assay sensitivity. The impact of these mutations on molecular diagnostics has been demonstrated by the failure of S gene detection probes in some nucleic acid tests.4 It is essential that Ag-RDTs are re-evaluated using new variants to determine if there is any change in test sensitivity due to mutations in the target antigen. The aim of this study was to determine the LOD of seventeen commercially available RDTs using the B.1.1.7 lineage and compare results obtained with the original dominant strain (B.1). A clinically isolated SARS-CoV-2 strain from the B.1.1.7 lineage (Genbank accession number: MW980115), was used for the study. Frozen aliquots of the third passage of the virus were quantified via plaque assay as previously described.5 For the determination of LODs, a fresh aliquot was serially diluted from 1.0 × 106 plaque forming units (pfu)/ml to 1.0 × 102 pfu/ml. Each dilution was tested in triplicate. Two-fold dilutions were made below the ten-fold LOD dilution to confirm the lowest LOD. Culture media was used as negative control. Viral RNA was extracted from each dilution using QIAmp Viral RNA mini kit (Qiagen, Germany) according to the manufacturer's instructions, and quantified using TaqPath COVID-19 CE-IVD RT-PCR (ThermoFisher). Genome copy number/ml (gcn/ml) were calculated as previously described.6 We evaluated 17 commercially available Ag-RDT tests (Table 1) following the instructions for use (IFU). The LOD was defined as the lowest dilution at which all three replicates were positive. Results were interpreted by two operators, each blinded to the result of the other. If a discrepant result was obtained, a third operator read any discrepant tests as a tie-breaker. Table 1Characteristics of the Ag-RDT tested

A high-resolution melt curve toolkit to identify lineage-defining SARS-CoV-2 mutations

The emergence of severe acute respiratory syndrome 2 (SARS-CoV-2) variants of concern (VOCs), with mutations linked to increased transmissibility, vaccine escape and virulence, has necessitated the widespread genomic surveillance of SARS-CoV-2. This has placed a strain on global sequencing capacity, especially in areas lacking the resources for large scale sequencing activities. Here we have developed three separate multiplex high-resolution melting assays to enable the identification of Alpha, Beta, Delta and Omicron VOCs. The assays were evaluated against whole genome sequencing on upper-respiratory swab samples collected during the Alpha, Delta and Omicron [BA.1] waves of the UK pandemic. The sensitivities of the eight individual primer sets were all 100%, and specificity ranged from 94.6 to 100%. The multiplex HRM assays have potential as a tool for high throughput surveillance of SARS-CoV-2 VOCs, particularly in areas with limited genomics facilities

Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria

Objectives: Neonatal sepsis, a major cause of death amongst infants in sub-Saharan Africa, is often gut derived. Gut colonisation by Enterobacteriaceae producing extended spectrum beta-lactamase (ESBL) or carbapenemase enzymes can lead to antimicrobial-resistant (AMR) or untreatable infections. We sought to explore the rates of colonisation by ESBL or carbapenemase producers in two neonatal units (NNUs) in West and East Africa. Methods: Stool and rectal swab samples were taken at multiple timepoints from newborns admitted to the NNUs at the University College Hospital, Ibadan, Nigeria and the Jaramogi Oginga Odinga Teaching and Referral Hospital, Kisumu, western Kenya. Samples were tested for ESBL and carbapenemase genes using a previously validated qPCR assay. Kaplan-Meier survival analysis was used to examine colonisation rates at both sites. Results: In total 119 stool and rectal swab samples were taken from 42 infants admitted to the two NNUs. Colonisation with ESBL (37 infants, 89%) was more common than with carbapenemase producers (26, 62.4%; P = 0.093). Median survival time before colonisation with ESBL organisms was 7 days and with carbapenemase producers 16 days (P = 0.035). The majority of ESBL genes detected belonged to the CTX-M-1 (36/38; 95%), and CTX-M-9 (2/36; 5%) groups, and the most prevalent carbapenemase was blaNDM (27/29, 93%). Conclusions: Gut colonisation of neonates by AMR organisms was common and occurred rapidly in NNUs in Kenya and Nigeria. Active surveillance of colonisation will improve the understanding of AMR in these settings and guide infection control and antibiotic prescribing practice to improve clinical outcomes