Rapid surveillance platforms for key SARS-CoV-2 mutations in Denmark

Multiple mutations in SARS-CoV-2 variants of concern (VOCs) may increase, transmission, disease severity, immune evasion and facilitate zoonotic or anthoprozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K and N501Y, occur in the SARS-CoV-2 spike glycoprotein in combinations that allow detection of the most important VOCs. Here we present two flexible RT-qPCR platforms for small- and large-scale screening to detect these mutations, and schemes for adapting the platforms for future mutations. The large-scale RT-qPCR platform, was validated by pair-wise matching of RT-qPCR results with WGS consensus genomes, showing high specificity and sensitivity. Detection of mutations using this platform served as an important interventive measure for the Danish public health system to delay the emergence of VOCs and to gain time for vaccine administration. Both platforms are valuable tools for WGS-lean laboratories, as well for complementing WGS to support rapid control of local transmission chains worldwide.

The Effect of Forage-to-Concentrate Ratio on Schizochytrium spp.-Supplemented Goats: Modifying Rumen Microbiota

The inclusion of feed additives and the implementation of various nutritional strategies are studied to modify the rumen microbiome and consequently its function. Nevertheless, rumen enzymatic activity and its intermediate products are not always matched with the microbiome structure. To further elucidate such differences a two-phase trial using twenty-two dairy goats was carried out. During the first phase, both groups (20HF n = 11; high forage and 20HG n = 11; high grain) were supplemented with 20 g Schizochytrium spp./goat/day. The 20HF group consumed a diet with a forage:concentrate (F:C) ratio of 60:40 and the 20HG-diet consisted of a F:C = 40:60. In the second phase, the supplementation level of Schizochytrium spp. was increased to 40 g/day/goat while the F:C ratio between the two groups were remained identical (40HF n = 11; high forage and 40HG n = 11; high grain). By utilizing a next-generation sequencing technology, we monitored that the high microalgae inclusion level and foremost in combination with a high grains diet increased the unmapped bacteria within the rumen. Bacteroidetes and Prevotella brevis were increased in the 40HG -fed goats as observed by using a qPCR platform. Additionally, methanogens and Methanomassiliicoccales were increased in high microalgae-fed goats, while Methanobrevibacter and Methanobacteriales were decreased. Fibrolytic bacteria were decreased in high microalgae-fed goats, while cellulolytic activity was increased. Ammonia was decreased in high grains-fed goats, while docosapentaenoic and docosahexaenoic acids showed a lower degradation rate in the rumen of high forage-fed goats. The alteration of the F:C ratio in goats supplemented with Schizochytrium spp. levels modified both ruminal microbiota and enzymatic activity. However, there was no significant consistency in the relations between them.

Highly sensitive and multiplexed one-step RT-qPCR for profiling genes involved in the circadian rhythm using microparticles

Given the growing interest in molecular diagnosis, highly extensive and selective detection of genetic targets from a very limited amount of samples is in high demand. We demonstrated the highly sensitive and multiplexed one-step RT-qPCR platform for RNA analysis using microparticles as individual reactors. Those particles are equipped with a controlled release system of thermo-responsive materials, and are able to capture RNA targets inside. The particle-based assay can successfully quantify multiple target RNAs from only 200 pg of total RNA. The assay can also quantify target RNAs from a single cell with the aid of a pre-concentration process. We carried out 8-plex one-step RT-qPCR using tens of microparticles, which allowed extensive mRNA profiling. The circadian cycles were shown by the multiplex one-step RT-qPCR in human cell and human hair follicles. Reliable 24-plex one-step RT-qPCR was developed using a single operation in a PCR chip without any loss of performance (i.e., selectivity and sensitivity), even from a single hair. Many other disease-related transcripts can be monitored using this versatile platform. It can also be used non–invasively for samples obtained in clinics.

Porcine Digestible Peptides (PDP) in Weanling Diets Regulates the Expression of Genes Involved in Gut Barrier Function, Immune Response and Nutrient Transport in Nursery Pigs

This study was conducted to investigate the effects of dietary supplementation of porcine digestible peptides (PDP), spray-dried plasma (SDP), or a combination of both, on growth performance and the expression of genes related to intestinal function of weaned pigs. A total of 180 piglets (trial 1) and 198 piglets (trial 2) were used to evaluate the partial substitution of soybean ingredients with 2% SDP or 2% PDP (trial 1), and with 3% SDP or the combination of 1% SDP and 2% PDP (SDP-PDP; trial 2) during the pre-starter period (0–14 days). The gene expression of 56 genes was quantified in a qPCR platform in jejunum and ileum samples obtained from piglets 14 d after weaning (trial 2). Piglets fed SDP, PDP and SDP-PDP had a higher body weight (BW), average daily gain (ADG) and feed efficiency (G:F) than the soybean control on day 14 (p < 0.05). In addition, the combination of SDP and PDP upregulated ten genes in jejunum samples (p < 0.05) related to intestinal function. More research is needed to confirm that gene expression upregulation by PDP in combination with SDP has an impact on intestinal function and to elucidate its underlying mechanisms.

630. A 5-mRNA host response whole-blood classifier trained using patients with non-COVID-19 viral infections accurately predicts severity of COVID-19

Background While major progress has been made to establish diagnostic tools for the diagnosis of SARS-CoV-2 infection, determining the severity of COVID-19 remains an unmet medical need. With limited hospital resources, gauging severity would allow for some patients to safely recover in home quarantine while ensuring sicker patients get needed care. We discovered a 5 host mRNA-based classifier for the severity of influenza and other acute viral infections and validated the classifier in COVID-19 patients from Greece. Methods We used training data (N=705) from 21 retrospective clinical studies of influenza and other viral illnesses. Five host mRNAs from a preselected panel were applied to train a logistic regression classifier for predicting 30-day mortality in influenza and other viral illnesses. We then applied this classifier, with fixed weights, to an independent cohort of subjects with confirmed COVID-19 from Athens, Greece (N=71) using NanoString nCounter. Finally, we developed a proof-of-concept rapid, isothermal qRT-LAMP assay for the 5-mRNA host signature using the QuantStudio 6 qPCR platform. Results In 71 patients with COVID-19, the 5 mRNA classifier had an AUROC of 0.88 (95% CI 0.80-0.97) for identifying patients with severe respiratory failure and/or 30-day mortality (Figure 1). Applying a preset cutoff based on training data, the 5-mRNA classifier had 100% sensitivity and 46% specificity for identifying mortality, and 88% sensitivity and 68% specificity for identifying severe respiratory failure. Finally, our proof-of-concept qRT-LAMP assay showed high correlation with the reference NanoString 5-mRNA classifier (r=0.95). Figure 1. Validation of the 5-mRNA classifier in the COVID-19 cohort. (A) Expression of the 5 genes used in the logistic regression model in patients with (red) and without (blue) mortality. (B) The 5-mRNA classifier accurately distinguishes non-severe and severe patients with COVID-19 as well as those at risk of death. Conclusion Our 5-mRNA classifier demonstrated very high accuracy for the prediction of COVID-19 severity and could assist in the rapid, point-of-impact assessment of patients with confirmed COVID-19 to determine level of care thereby improving patient management and healthcare burden. Disclosures ljubomir Buturovic, PhD, Inflammatix Inc. (Employee, Shareholder) Purvesh Khatri, PhD, Inflammatix Inc. (Shareholder) Oliver Liesenfeld, MD, Inflammatix Inc. (Employee, Shareholder) James Wacker, n/a, Inflammatix Inc. (Employee, Shareholder) Uros Midic, PhD, Inflammatix Inc. (Employee, Shareholder) Roland Luethy, PhD, Inflammatix Inc. (Employee, Shareholder) David C. Rawling, PhD, Inflammatix Inc. (Employee, Shareholder) Timothy Sweeney, MD, Inflammatix, Inc. (Employee)

On-site eDNA detection of species using ultra-rapid mobile PCR

Molecular methods, including environmental DNA (eDNA) methods, provide essential information for biological and conservation sciences. Molecular measurements are often performed in the laboratory, which limits their scope, especially for rapid on-site analysis. eDNA methods for species detection provide essential information for the management and conservation of species and communities in various environments. We developed an innovative novel method for on-site eDNA measurements using an ultra-rapid mobile PCR platform. We tested the ability of our method to detect the distribution of silver carp, Hypophthalmichthys molitrix, an invasive fish in Japanese rivers and lakes. Our method reduced the measurement time to 30 min and provided high detectability of aquatic organisms compared to the national observation surveys using multiple fishing nets and laboratory extraction/detection using a benchtop qPCR platform. Our on-site eDNA method can be immediately applied to various taxa and environments.

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77–100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Multicenter Evaluation of Independent High-Throughput and RT-qPCR Technologies for the Development of Analytical Workflows for Circulating miRNA Analysis

Background: Among emerging circulating biomarkers, miRNA has the potential to detect lung cancer and follow the course of the disease. However, miRNA analysis deserves further standardization before implementation into clinical trials or practice. Here, we performed international ring experiments to explore (pre)-analytical factors relevant to the outcome of miRNA blood tests in the context of the EU network CANCER-ID. Methods: Cell-free (cfmiRNA) and extracellular vesicle-derived miRNA (EVmiRNA) were extracted using the miRNeasy Serum/Plasma Advanced, and the ExoRNeasy Maxi kit, respectively, in a plasma cohort of 27 NSCLC patients and 20 healthy individuals. Extracted miRNA was investigated using small RNA sequencing and hybridization platforms. Validation of the identified miRNA candidates was performed using quantitative PCR. Results: We demonstrate the highest read counts in healthy individuals and NSCLC patients using QIAseq. Moreover, QIAseq showed 15.9% and 162.9% more cfmiRNA and EVmiRNA miRNA counts, respectively, in NSCLC patients compared to healthy control samples. However, a systematic comparison of selected miRNAs revealed little agreement between high-throughput platforms, thus some miRNAs are detected with one technology, but not with the other. Adding to this, 35% (9 of 26) of selected miRNAs in the cfmiRNA and 42% (11 of 26) in the EVmiRNA fraction were differentially expressed by at least one qPCR platform; about half of the miRNAs (54%) were concordant for both platforms. Conclusions: Changing of (pre)-analytical methods of miRNA analysis has a significant impact on blood test results and is therefore a major confounding factor. In addition, to confirm miRNA biomarker candidates screening studies should be followed by targeted validation using an independent platform or technology.