Multiplex degenerate PCR coupled with an oligo sorbent array for human endogenous retrovirus expression profiling

Human endogenous retroviruses (HERVs) can be divided into distinct families of tens to thousands of paralogous loci. The expression of HERV elements has been detected in all tissues tested to date, particularly germ cells, embryonic tissues and neoplastic tissues. Hence, the study of HERV expression could represent added value in cancer diagnosis. We developed a quantitative assay combining a multiplex degenerate PCR (MD-PCR) amplification, based on the relative conservation of the pol genes, and a colorimetric Oligo Sorbent Array (OLISA(®)). Nine HERV families were selected and amplification primers and capture probes were designed for each family. The features required to achieve efficient amplification of most of the elements of each HERV family and balanced co-amplification of all HERV families were analyzed. We found that MD-PCR reliability, i.e. equivalence of amplification and dose-effect relationship, relied on the adjustment of three critical parameters: the primer degeneracy, the relative concentration of each primer and the total amount of primers in the amplification mixture. The analysis of tumoral versus normal tissues suggests that this assay could prove useful in tumor phenotyping

Development of an animal-component free insect medium for the Baculovirus Expression Vector System (BEVS)

Insect cells derived from Spodoptera frugiperda have been widely used with the baculovirus expression vector system (BEVS) for the production of recombinant proteins and adeno-associated viruses (AAVs) due to their ease of culture, scalability in high cell density suspension cultures, and high protein expression levels. Traditionally, insect cells are cultured in an undefined medium containing yeast hydrolysate and cod liver oil, however, there is an increasing push to use chemically defined, animal-component free medium to minimize any potential contaminants and decrease lot-to-lot variability while maintaining high cell growth and production. In this case study, an animal-component free insect medium was developed utilizing Rational Culture Media DesignTM and evaluated with Sf9 cells. Using a traditional formulation as a starting point, the final medium was developed by optimizing multiple nutrient groups in the basal medium, replacing the animal-derived components, and screening several yeast hydrolysate sources. By utilizing multifactor design of experiment software, various nutrient groups were screened including amino acids, vitamins, and metals. The metals group was identified to have the most impact on cell growth and productivity, and therefore concentrations of metal components were further optimized. In addition, the animal-derived components in the starting formulation, cod liver oil and cholesterol, were replaced with animal-component free fatty acids and synthetic cholesterol, respectively. The concentrations of these components were optimized to achieve better growth performance and production while also sustaining formulation stability and streamlining manufacturing processes. Finally, yeast hydrolysate is a well-known, undefined component that is crucial for insect cell growth and productivity. To minimize lot-to-lot variability, the yeast hydrolysate concentration was significantly lowered, and multiple yeast hydrolysate sources and lots were evaluated to determine the highest quality source. As a result, an animal-component free insect medium was developed that had improved growth performance and comparable productivity to a widely used commercially available animal-derived medium

Development and validation of a proprietary medium formulation for recombinant subunit vaccines by the Baculovirus Expression Vector System (BEVS)

Insect cells & baculovirus expression vector system (BEVS) is an efficient platform for the production of baculovirus vectors and the expression of recombinant proteins or VLPs (Virus Like Particles). This technology is increasingly used in large-scale manufacturing of human and veterinary vaccines. The production of safe and cost-effective vaccines requires the development of animal origin free and low-cost culture media supporting strong insect cell growth, high yield of baculovirus and sustained expression of recombinant proteins or VLPs. In this context, Boehringer-Ingelheim decided to develop its own animal origin free media (with similar or better performance than the commercial one) in collaboration with NRC and suppliers. The different steps, feedback and results will be presented in this poster

A point-prevalence study on community and inpatient Clostridioides difficile infections (CDI): results from Combatting Bacterial Resistance in Europe CDI (COMBACTE-CDI), July to November 2018

Background There is a paucity of data on community-based Clostridioides difficile infection (CDI) and how these compare with inpatient CDI. Aim To compare data on the populations with CDI in hospitals vs the community across 12 European countries. Methods For this point-prevalence study (July–November 2018), testing sites sent residual diagnostic material on sampling days to a coordinating laboratory for CDI testing and PCR ribotyping (n = 3,163). Information on whether CDI testing was requested at the original site was used to identify undiagnosed CDI. We used medical records to identify differences between healthcare settings in patient demographics and risk factors for detection of C. difficile with or without free toxin. Results The CDI positivity rate was 4.4% (country range: 0–16.2) in hospital samples, and 1.3% (country range: 0–2.2%) in community samples. The highest prevalence of toxinotype IIIb (027, 181 and 176) was seen in eastern European countries (56%; 43/77), the region with the lowest testing rate (58%; 164/281). Different predisposing risk factors were observed (use of broad-spectrum penicillins in the community (OR: 8.09 (1.9–35.6), p = 0.01); fluoroquinolones/cephalosporins in hospitals (OR: 2.2 (1.2–4.3), p = 0.01; OR: 2.0 (1.1–3.7), p = 0.02)). Half of community CDI cases were undetected because of absence of clinical suspicion, accounting for three times more undiagnosed adults in the community compared with hospitals (ca 111,000 vs 37,000 cases/year in Europe). Conclusion These findings support recommendations for improving diagnosis in patients presenting with diarrhoea in the community, to guide good practice to limit the spread of CDI

Quantitative determination of escherichia coli in water using CHROMagar® E. coli

A new medium, CHROMagar® E. coli (CAEC), containing a combination of X-glucuronide and methyl-glucuronide for the detection of β-glucuronidase activity of Escherichia coli has been evaluated by the membrane filtration (MF) technique n fresh water samples. The CAEC agar was compared with conventional media, mFC agar and mLSB, for the enumeration of faecal coliforms. The variance analysis showed that CAEC was as sensitive as mFC agar and mLSB. A good correlation was found between E. coli versus those from faecal coliforms in the water sampling areas tested. Of 321 presumptively positive E. coli colonies (blue) and 154 presumptively negative E. coli colonies (white), only 8 (2.5%) false positive and 19 (12.4%) false negative colonies were found. Specificity of the CEAC agar in spanish samples was temperature dependent, false negative E. coli colonies occurred less frequently at 37°C (2.3%) than at 44.5°C (18.8%). The results of this study indicate that CAEC agar is efficient for the enumeration of E. coli from a wide range of environmental freshwater samples