A Method for Variant Agnostic Detection of SARS-CoV-2, Rapid Monitoring of Circulating Variants, and Early Detection of Emergent Variants Such as Omicron.

The rapid emergence of SARS-CoV-2 variants raised public health questions concerning the capability of diagnostic tests to detect new strains, the efficacy of vaccines, and how to map the geographical distribution of variants to understand transmission patterns and loads on healthcare resources. Next-generation sequencing (NGS) is the primary method for detecting and tracing new variants, but it is expensive, and it can take weeks before sequence data are available in public repositories. This article describes a customizable reverse transcription PCR (RT-PCR)-based genotyping approach which is significantly less expensive, accelerates reporting, and can be implemented in any lab that performs RT-PCR. Specific single-nucleotide polymorphisms (SNPs) and indels were identified which had high positive-percent agreement (PPA) and negative-percent agreement (NPA) compared to NGS for the major genotypes that circulated through September 11, 2021. Using a 48-marker panel, testing on 1,031 retrospective SARS-CoV-2 positive samples yielded a PPA and NPA ranging from 96.3 to 100% and 99.2 to 100%, respectively, for the top 10 most prevalent World Health Organization (WHO) lineages during that time. The effect of reducing the quantity of panel markers was explored, and a 16-marker panel was determined to be nearly as effective as the 48-marker panel at lineage assignment. Responding to the emergence of Omicron, a genotyping panel was developed which distinguishes Delta and Omicron using four highly specific SNPs. The results demonstrate the utility of the condensed panel to rapidly track the growing prevalence of Omicron across the US in December 2021 and January 2022.

THADA fusion is a mechanism of IGF2BP3 activation and IGF1R signaling in thyroid cancer.

Thyroid cancer development is driven by known point mutations or gene fusions found in ∼90% of cases, whereas driver mutations in the remaining tumors are unknown. The insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) plays an important role in cancer, yet the mechanisms of its activation in cancer cells remain poorly understood. Using whole-transcriptome and whole-genome analyses, we identified a recurrent fusion between the thyroid adenoma-associated (THADA) gene on chromosome 2 and the LOC389473 gene on chromosome 7 located 12 kb upstream of the IGF2BP3 gene. We show that THADA fusion to LOC389473 and other regions in the vicinity does not result in the formation of a chimeric protein but instead leads to strong overexpression of the full-length IGF2BP3 mRNA and protein, increased IGF2 translation and IGF1 receptor (IGF1R) signaling via PI3K and MAPK cascades, and promotion of cell proliferation, invasion, and transformation. THADA fusions and IGF2BP3 overexpression are found in ∼5% of thyroid cancers that lack any other driver mutations. We also find that strong IGF2BP3 overexpression via gene fusion, amplification, or other mechanisms occurs in 5 to 15% of several other cancer types. Finally, we provide in vitro and in vivo evidence that growth of IGF2BP3-driven cells and tumors may be blocked by IGF1R inhibition, raising the possibility that IGF2BP3 overexpression in cancer cells may predict an anti-IGF1R benefit.

Is calorie intake the fundamental driver of noncommunicable diseases in India - A systematic review.

BACKGROUND: Nutrition epidemiology initially focused on few nutrients thought to be responsible for noncommunicable diseases (NCDs). The database of Indian Nutrition Survey is based majorly on calorie intake. OBJECTIVE: The objective was to compare the change in the average calorie intake from 1990 to 2012 with the emerging epidemic of diabetes and hypertension (HTN) in India since 1990. METHODS: A comprehensive search was made in National Library of Medicine's PubMed database and Google Scholar from March to August 2018, on the above-mentioned subjects. Reports of national surveys (National Sample Survey Office and National Nutrition Monitoring Bureau) were included for average calorie intake among different states from year 1990 onward. Region-wise search depicted by national nutrition surveys resulted in 277 and 587 abstracts on the prevalence of HTN and diabetes mellitus, respectively. There were 51 full-text articles and abstracts on the prevalence of HTN and diabetes from the above regions. RESULTS: The average calorie intake per capita per day in the four zones of country in rural areas decreased from 1990 to 2012. An increasing trend in the prevalence of diabetes from rural areas was observed from 1994 to 2012. The per capita average calorie intake per day in urban areas from 1999 through 2011 in all zones except the eastern part of country was on rise. There was no consistent trend in the prevalence of HTN in any of the zones. CONCLUSION: It is not just an increase in calories, but a trade-off between the demand for calories and the demand for healthy lifestyles determines the prevalence of NCDs.

Differential Effects of Anesthetics and Opioid Receptor Activation on Cardioprotection Elicited by Reactive Oxygen Species-Mediated Postconditioning in Sprague-Dawley Rat Hearts.

BACKGROUND: Despite an array of cardioprotective interventions identified in preclinical models of ischemia-reperfusion (IR) injury, successful clinical translation has not been achieved. This study investigated whether drugs routinely used in clinical anesthesia influence cardioprotective effectiveness by reducing effects of reactive oxygen species (ROS), upstream triggers of cardioprotective signaling. Effects of propofol, sevoflurane, or remifentanil were compared on postischemic functional recovery induced by ROS-mediated postconditioning with Intralipid. METHODS: Recovery of left ventricular (LV) work, an index of IR injury, was measured in isolated Sprague-Dawley rat hearts subjected to global ischemia (20 minutes) and reperfusion (30 minutes). Hearts were either untreated or were treated with postconditioning with Intralipid (1%, throughout reperfusion). Propofol (10 µM), sevoflurane (2 vol%), remifentanil (3 nM), or combinations thereof were administered peri-ischemically (before and during IR). The effects of anesthetics on ROS production were measured in LV cardiac fibers by Amplex Red assay under phosphorylating and nonphosphorylating conditions. RESULTS: Recovery of LV work (expressed as percentage of the preischemic value ± standard deviation) in untreated hearts was poor (20% ± 7%) and was improved by Intralipid postconditioning (58% ± 8%, P = .001). In the absence of Intralipid postconditioning, recovery of LV work was enhanced by propofol (28% ± 9%, P = .049), sevoflurane (49% ± 5%, P < .001), and remifentanil (51% ± 6%, P < .001). The benefit of Intralipid postconditioning was abolished by propofol (33% ± 10%, P < .001), but enhanced by sevoflurane (80% ± 7%, P < .001) or remifentanil (80% ± 9%, P < .001). ROS signaling in LV fibers was abolished by propofol, but unaffected by sevoflurane or remifentanil. We conclude that propofol abolishes ROS-mediated Intralipid postconditioning by acting as a ROS scavenger. Sevoflurane and remifentanil are protective per se and provide additive cardioprotection to ROS-mediated cardioprotection. CONCLUSIONS: These divergent effects of routinely used drugs in clinical anesthesia may influence the translatability of cardioprotective therapies such as Intralipid postconditioning.

Hearts lacking plasma membrane KATP channels display changes in basal aerobic metabolic substrate preference and AMPK activity.

Cardiac ATP-sensitive K+ (KATP) channels couple changes in cellular metabolism to membrane excitability and are activated during metabolic stress, although under basal aerobic conditions, KATP channels are thought to be predominately closed. Despite intense research into the roles of KATP channels during metabolic stress, their contribution to aerobic basal cardiac metabolism has not been previously investigated. Hearts from Kir6.2+/+ and Kir6.2-/- mice were perfused in working mode, and rates of glycolysis, fatty acid oxidation, and glucose oxidation were measured. Changes in activation/expression of proteins regulating metabolism were probed by Western blot analysis. Despite cardiac mechanical function and metabolic efficiency being similar in both groups, hearts from Kir6.2-/- mice displayed an approximately twofold increase in fatty acid oxidation and a 0.45-fold reduction in glycolytic rates but similar glucose oxidation rates compared with hearts from Kir6.2+/+ mice. Kir6.2-/- hearts also possessed elevated levels of activated AMP-activated protein kinase (AMPK), higher glycogen content, and reduced mitochondrial density. Moreover, activation of AMPK by isoproterenol or diazoxide was significantly blunted in Kir6.2-/- hearts. These data indicate that KATP channel ablation alters aerobic basal cardiac metabolism. The observed increase in fatty acid oxidation and decreased glycolysis before any metabolic insult may contribute to the poor recovery observed in Kir6.2-/- hearts in response to exercise or ischemia-reperfusion injury. Therefore, KATP channels may play an important role in the regulation of cardiac metabolism through AMPK signaling.NEW & NOTEWORTHY In this study, we show that genetic ablation of plasma membrane ATP-sensitive K+ channels results in pronounced changes in cardiac metabolic substrate preference and AMP-activated protein kinase activity. These results suggest that ATP-sensitive K+ channels may play a novel role in regulating metabolism in addition to their well-documented effects on ionic homeostasis during periods of stress.

FoxO1 regulates myocardial glucose oxidation rates via transcriptional control of pyruvate dehydrogenase kinase 4 expression.

Pyruvate dehydrogenase (PDH) is the rate-limiting enzyme for glucose oxidation and a critical regulator of metabolic flexibility during the fasting to feeding transition. PDH is regulated via both PDH kinases (PDHK) and PDH phosphatases, which phosphorylate/inactivate and dephosphorylate/activate PDH, respectively. Our goal was to determine whether the transcription factor forkhead box O1 (FoxO1) regulates PDH activity and glucose oxidation in the heart via increasing the expression of Pdk4, the gene encoding PDHK4. To address this question, we differentiated H9c2 myoblasts into cardiac myocytes and modulated FoxO1 activity, after which Pdk4/PDHK4 expression and PDH phosphorylation/activity were assessed. We assessed binding of FoxO1 to the Pdk4 promoter in cardiac myocytes in conjunction with measuring the role of FoxO1 on glucose oxidation in the isolated working heart. Both pharmacological (1 µM AS1842856) and genetic (siRNA mediated) inhibition of FoxO1 decreased Pdk4/PDHK4 expression and subsequent PDH phosphorylation in H9c2 cardiac myocytes, whereas 10 µM dexamethasone-induced Pdk4/PDHK4 expression was abolished via pretreatment with 1 µM AS1842856. Furthermore, transfection of H9c2 cardiac myocytes with a vector expressing FoxO1 increased luciferase activity driven by a Pdk4 promoter construct containing the FoxO1 DNA-binding element region, but not in a Pdk4 promoter construct lacking this region. Finally, AS1842856 treatment in fasted mice enhanced glucose oxidation rates during aerobic isolated working heart perfusions. Taken together, FoxO1 directly regulates Pdk4 transcription in the heart, thereby controlling PDH activity and subsequent glucose oxidation rates.NEW & NOTEWORTHY Although studies have shown an association between FoxO1 activity and pyruvate dehydrogenase kinase 4 expression, our study demonstrated that pyruvate dehydrogenase kinase 4 is a direct transcriptional target of FoxO1 (but not FoxO3/FoxO4) in the heart. Furthermore, we report here, for the first time, that FoxO1 inhibition increases glucose oxidation in the isolated working mouse heart.

Identification of the transforming STRN-ALK fusion as a potential therapeutic target in the aggressive forms of thyroid cancer.

Thyroid cancer is a common endocrine malignancy that encompasses well-differentiated as well as dedifferentiated cancer types. The latter tumors have high mortality and lack effective therapies. Using a paired-end RNA-sequencing approach, we report the discovery of rearrangements involving the anaplastic lymphoma kinase (ALK) gene in thyroid cancer. The most common of these involves a fusion between ALK and the striatin (STRN) gene, which is the result of a complex rearrangement involving the short arm of chromosome 2. STRN-ALK leads to constitutive activation of ALK kinase via dimerization mediated by the coiled-coil domain of STRN and to a kinase-dependent, thyroid-stimulating hormone-independent proliferation of thyroid cells. Moreover, expression of STRN-ALK transforms cells in vitro and induces tumor formation in nude mice. The kinase activity of STRN-ALK and the ALK-induced cell growth can be blocked by the ALK inhibitors crizotinib and TAE684. In addition to well-differentiated papillary cancer, STRN-ALK was found with a higher prevalence in poorly differentiated and anaplastic thyroid cancers, and it did not overlap with other known driver mutations in these tumors. Our data demonstrate that STRN-ALK fusion occurs in a subset of patients with highly aggressive types of thyroid cancer and provide initial evidence suggesting that it may represent a therapeutic target for these patients.

microRNA in situ hybridization for miR-211 detection as an ancillary test in melanoma diagnosis.

Some melanocytic tumors can be histologically ambiguous causing diagnostic difficulty, which could lead to overtreatment of benign lesions with an unwarranted psychological distress or undertreatment of malignant cancers. Previously, we demonstrated that significantly decreased miR-211 expression in melanomas compared with melanocytic nevi could accurately discriminate malignant from benign tumors. Herein we show microRNA in situ hybridization for fluorescent detection of miR-211, suitable for paraffin-embedded tissues in 109 primary melanocytic tumors. miR-211 expression was significantly lower in melanomas vs nevi (P<0.0001), and receiver operating characteristic curve (area under the curve=0.862) accurately discriminated melanomas from nevi with 90% sensitivity and 86.2% specificity. Qualitatively, all dysplastic nevi expressed miR-211 at high (86%) and low (14%) levels, independent of the degree of nuclear atypia. All 35 melanocytic tumors with Spitz morphology expressed miR-211 independent of morphological classification, where clinical follow-up of these patients showed no recurrence at the site or metastasis in mean and median of 3 (ranging 2-5) years. Moreover, a decision tree learning analysis selected age and miR-211 miRNA in situ hybridization as the predictive variables for benign or malignant outcome in 88 patients correctly classified 92% (81 out of 88) of cases as proven by receiver operating characteristic curve (area under the curve=0.9029). These results support miR-211 as a leading miRNA candidate for melanoma diagnosis and miRNA in situ hybridization as a uniquely uncomplicated ancillary test.

Myocardial mechanical dysfunction following endotoxemia: role of changes in energy substrate metabolism.

Cardiovascular depression due to endotoxemia remains a major cause of mortality in intensive care patients. To determine whether drug-induced alterations in cardiac metabolism may be a viable strategy to reduce endotoxemia-mediated cardiac dysfunction, we assessed endotoxemia-induced changes in glucose and fatty acid metabolism under aerobic and post-ischemic conditions. Endotoxemia was induced in male Sprague-Dawley rats by lipopolysaccharide (Escherichia coli 0111:B4c, 4 mg/kg, i.p.) 6 h prior to heart removal for ex vivo assessment of left ventricular (LV) work and rates of glucose metabolism (glucose uptake, glycogen synthesis, glycolysis and glucose oxidation) and palmitate oxidation. Under aerobic conditions, endotoxemic hearts had impaired LV function as judged by echocardiography in vivo (% ejection fraction, 66.0 ± 3.2 vs 78.0 ± 2.1, p < 0.05) or by LV work ex vivo (2.14 ± 0.16 vs 3.28 ± 0.16, Joules min(-1) g dry wt(-1), p < 0.05). However, rates of glucose uptake, glycogen synthesis, glycolysis, and glucose oxidation were not altered. Palmitate oxidation was lower in endotoxemic hearts in proportion to the decreased workload, thus metabolic efficiency was unaffected. In hearts reperfused following global ischemia, untreated hearts had impaired recovery of LV work (52.3 ± 9.4 %) whereas endotoxemic hearts had significantly higher recovery (105.6 ± 11.3 %, p < 0.05). During reperfusion, fatty acid oxidation, acetyl CoA production and metabolic efficiency were similar in both groups. As impaired cardiac function appeared unrelated to depression of energy substrate oxidation, it is unlikely that drug-induced acceleration of fatty acid oxidation will improve mechanical function. The beneficial repartitioning of glucose metabolism in reperfused endotoxemic hearts may contribute to the cardioprotected phenotype.

Propofol (Diprivan®) and Intralipid® exacerbate insulin resistance in type-2 diabetic hearts by impairing GLUT4 trafficking.

BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mU•L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.

Homologous chromosomes make contact at the sites of double-strand breaks in genes in somatic G0/G1-phase human cells.

Double-strand DNA breaks (DSBs) are continuously induced in cells by endogenously generated free radicals and exogenous genotoxic agents such as ionizing radiation. DSBs activate the kinase activity in sensor proteins such as ATM and DNA-PK, initiating a complex DNA damage response that coordinates various DNA repair pathways to restore genomic integrity. In this study, we report the unexpected finding that homologous chromosomes contact each other at the sites of DSBs induced by either radiation or the endonuclease I-PpoI in human somatic cells. Contact involves short segments of homologous chromosomes and is centered on a DSB in active genes but does not occur at I-PpoI sites in intergenic DNA. I-PpoI-induced contact between homologous genes is abrogated by the transcriptional inhibitors actinomycin D and α-amanitin and requires the kinase activity of ATM but not DNA-PK. Our findings provide documentation of a common transcription-related and ATM kinase-dependent mechanism that induces contact between allelic regions of homologous chromosomes at sites of DSBs in human somatic cells.

Comparison of analytical performance and economic value of two biosurveillance methods for tracking SARS-COV-2 variants of concern.

The development of biosurveillance programs with strong analytical performance and economically accessible protocols is essential for monitoring viral pathogens. Throughout the COVID-19 pandemic, whole-genome sequencing (WGS) has been the prevailing technology for SARS-CoV-2 variant of concern (VOC) detection. While WGS offers benefits, it is a lengthy process, financially and technically straining for scalable viral tracking. The aim of this study was to compare the analytical performance and economic feasibility of WGS and PCR mutation panels for distinguishing six known VOCs: Alpha (B.1.1.7 and Q.4), Gamma (P.1), Delta (B.1.617.2 and AY.4.2), and Omicron. (B.1.1.529.1). In all, 78 SARS-CoV-2-positive samples were collected from April to December 2021 at Northeastern University (Cabot Testing Site, Boston, MA, USA) for genotyping PCR and WGS analysis. MagMax Viral/Pathogen II Nucleic Acid Isolation and TaqPath COVID-19 Combo Kits were used for RNA extraction and SARS-CoV-2 confirmation. VOC discrimination was assessed using two TaqMan SARS-CoV-2 single nucleotide polymorphism (SNP) assay layouts, and Ion Torrent WGS. In November 2021, the mutation panel demonstrated marked versatility by detecting the emerging Omicron variant reported by South Africa. SNP panel analysis yielded the following 78 VOC identifications: Alpha B.1.1.7 (N = 20), Alpha Q.4 (N = 3), Gamma P.1 (N = 1), Delta B.1.617.2 (N = 30), Delta AY.4.2 (N = 3), and Omicron B.1.1.529.1 (N = 20) with one undetermined (N = 1) sample. Genotyping mutation panels designated lineages in 77 of 78 samples, 46/78 were confirmed by WGS, while 32 samples failed WGS lineage assignment. RT-PCR genotyping panels offer pronounced throughput and sensitivity and provide an economically advantageous technique for SARS-CoV-2 biosurveillance.IMPORTANCEThe results presented in our manuscript demonstrate how the value of simplistic and reliable molecular assays coupled with the core scientific principle of standardization can be overlooked by the charm of more sophisticated assays and instrumentation. This effect can often be amplified during tumultuous public health events, such as the COVID-19 pandemic. By adapting standardized PCR mutation panels to detect prominently circulating SARS-CoV-2 variants, we were able to better assess the potential health impacts of rising positivity rates and transmission clusters within the Northeastern University population. While several literature publications utilizing genotyping PCR and NGS have a similar scope to ours, many investigations lack sufficiently standardized genotyping PCR and NGS bioinformatics inclusionary/exclusionary criteria for SARS-CoV-2 variant identification. Finally, the economic benefits of standardized PCR mutation panels would allow for global implementation of biosurveillance, rather than reserving biosurveillance to more economically developed nations.

Frequency of close positioning of chromosomal loci detected by FRET correlates with their participation in carcinogenic rearrangements in human cells.

It has been well established that genes participating in oncogenic rearrangements are non-randomly positioned and frequently close to each other in human cell nuclei. However, the actual distance between these fusion partners has never been determined. The phenomenon of fluorescence resonance energy transfer (FRET) is observed when a donor fluorophore is close (<10 nm) to transfer some of it energy to an acceptor fluorophore. The aim of this study was to validate the use of FRET on directly labeled DNA molecules to assess the frequency of positioning at <10 nm distances between genes known to be involved in rearrangement and to correlate it with their probability to undergo rearrangement. In the validation experiments, the frequency of FRET-sensitized emission (SE) was found to be 93-96% between probes for the immediately adjacent chromosomal regions as compared to 0.1-0.2% between probes for the random loci located on large linear separation. Further, we found that the frequency of FRET-SE between four pairs of genes that form rearrangements in thyroid cancer was 5% for RET and CCDC6, 4% for RET and NCOA4, 2% for BRAF and AKAP9, and 2% for NTRK1 and TPR. Moreover, the frequency with which FRET was observed showed strong correlation (r = 0.9871) with the prevalence of respective rearrangements in thyroid cancer. Our findings demonstrate that FRET can be used as a technique to analyze proximity between specific DNA regions and that the frequency of gene positioning at distances allowing FRET correlates with their probability to undergo chromosomal rearrangements.

Expanded PCR Panel Testing for Identification of Respiratory Pathogens and Coinfections in Influenza-like Illness.

While COVID-19 has dominated Influenza-like illness (ILI) over the past few years, there are many other pathogens responsible for ILI. It is not uncommon to have coinfections with multiple pathogens in patients with ILI. The goal of this study was to identify the different organisms in symptomatic patients presenting with ILI using two different high throughput multiplex real time PCR platforms. Specimens were collected from 381 subjects presenting with ILI symptoms. All samples (nasal and nasopharyngeal swabs) were simultaneously tested on two expanded panel PCR platforms: Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, OpenArray™ plate (OA) (32 viral and bacterial targets); and Applied Biosystems™ TrueMark™ Respiratory Panel 2.0, TaqMan™ Array card (TAC) (41 viral, fungal, and bacterial targets). Results were analyzed for concordance between the platforms and for identification of organisms responsible for the clinical presentation including possible coinfections. Very good agreement was observed between the two PCR platforms with 100% agreement for 12 viral and 3 bacterial pathogens. Of 381 specimens, approximately 58% of the samples showed the presence of at least one organism with an important incidence of co-infections (~36-40% of positive samples tested positive for two and more organisms). S. aureus was the most prevalent detected pathogen (~30%) followed by SARS-CoV-2 (~25%), Rhinovirus (~15%) and HHV6 (~10%). Co-infections between viruses and bacteria were the most common (~69%), followed by viral-viral (~23%) and bacterial-bacterial (~7%) co-infections. These results showed that coinfections are common in RTIs suggesting that syndromic panel based multiplex PCR tests could enable the identification of pathogens contributing to coinfections, help guide patient management thereby improving clinical outcomes and supporting antimicrobial stewardship.

Cross-sectional Ct distributions from qPCR tests can provide an early warning signal for the spread of COVID-19 in communities.

Background: SARS-CoV-2 PCR testing data has been widely used for COVID-19 surveillance. Existing COVID-19 forecasting models mainly rely on case counts obtained from qPCR results, even though the binary PCR results provide a limited picture of the pandemic trajectory. Most forecasting models have failed to accurately predict the COVID-19 waves before they occur. Recently a model utilizing cross-sectional population cycle threshold (Ct-the number of cycles required for the fluorescent signal to cross the background threshold) values obtained from PCR tests (Ct-based model) was developed to overcome the limitations of using only binary PCR results. In this study, we aimed to improve on COVID-19 forecasting models using features derived from the Ct-based model, to detect epidemic waves earlier than case-based trajectories. Methods: PCR data was collected weekly at Northeastern University (NU) between August 2020 and January 2022. Campus and county epidemic trajectories were generated from case counts. A novel forecasting approach was developed by enhancing a recent deep learning model with Ct-based features and applied in Suffolk County and NU campus. For this, cross-sectional Ct values from PCR data were used to generate Ct-based epidemic trajectories, including effective reproductive rate (Rt) and incidence. The improvement in forecasting performance was compared using absolute errors and residual squared errors with respect to actual observed cases at the 7-day and 14-day forecasting horizons. The model was also tested prospectively over the period January 2022 to April 2022. Results: Rt curves estimated from the Ct-based model indicated epidemic waves 12 to 14 days earlier than Rt curves from NU campus and Suffolk County cases, with a correlation of 0.57. Enhancing the forecasting models with Ct-based information significantly decreased absolute error (decrease of 49.4 and 221.5 for the 7 and 14-day forecasting horizons) and residual squared error (40.6 and 217.1 for the 7 and 14-day forecasting horizons) compared to the original model without Ct features. Conclusion: Ct-based epidemic trajectories can herald an earlier signal for impending epidemic waves in the community and forecast transmission peaks. Moreover, COVID-19 forecasting models can be enhanced using these Ct features to improve their forecasting accuracy. In this study, we make the case that public health agencies should publish Ct values along with the binary positive/negative PCR results. Early and accurate forecasting of epidemic waves can inform public health policies and countermeasures which can mitigate spread.

Efficient SARS-CoV-2 Surveillance during the Pandemic-Endemic Transition Using PCR-Based Genotyping Assays.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC) pose an increased risk to public health due to higher transmissibility and/or immune escape. In this study, we assessed the performance of a custom TaqMan SARS-CoV-2 mutation panel consisting of 10 selected real-time PCR (RT-PCR) genotyping assays compared to whole-genome sequencing (WGS) for identification of 5 VOC circulating in The Netherlands. SARS-CoV-2 positive samples (N = 664), collected during routine PCR screening (15 ≤ CT ≤ 32) between May-July 2021 and December 2021-January 2022, were selected and analyzed using the RT-PCR genotyping assays. VOC lineage was determined based on the detected mutation profile. In parallel, all samples underwent WGS with the Ion AmpliSeq SARS-CoV-2 research panel. Among 664 SARS-CoV-2 positive samples, the RT-PCR genotyping assays classified 31.2% as Alpha (N = 207); 48.9% as Delta (N = 325); 19.4% as Omicron (N = 129), 0.3% as Beta (N = 2), and 1 sample as a non-VOC. Matching results were obtained using WGS in 100% of the samples. RT-PCR genotyping assays enable accurate detection of SARS-CoV-2 VOC. Furthermore, they are easily implementable, and the costs and turnaround time are significantly reduced compared to WGS. For this reason, a higher proportion of SARS-CoV-2 positive cases in the VOC surveillance testing can be included, while reserving valuable WGS resources for identification of new variants. Therefore, RT-PCR genotyping assays would be a powerful method to include in SARS-CoV-2 surveillance testing. IMPORTANCE The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome changes constantly. It is estimated that there are thousands of variants of SARS-CoV-2 by now. Some of those variants, variants of concern (VOC), pose an increased risk to public health due to higher transmissibility and/or immune escape. Pathogen surveillance helps researchers, epidemiologists, and public health officials to monitor the evolution of infectious diseases agents, alert on the spread of pathogens, and develop counter measures like vaccines. The technique used for the pathogen surveillance is called sequence analysis which makes it possible to examine the building blocks of SARS-CoV-2. In this study, a new PCR method based on the detection of specific changes of those building blocks is presented. This method enables a fast, accurate and cheap determination of different SARS-CoV-2 VOC. Therefore, it would be a powerful method to include in SARS-CoV-2 surveillance testing.

One assay to test them all: Multiplex assays for expansion of respiratory virus surveillance.

Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Clinical performance of XpertXpress® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1 to 2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97 and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhance diagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections.

ONE ASSAY TO TEST THEM ALL: COMPARING MULTIPLEX ASSAYS FOR EXPANSION OF RESPIRATORY VIRUS SURVEILLANCE.

Background: Molecular multiplex assays (MPAs) for simultaneous detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza and respiratory syncytial virus (RSV) in a single RT-PCR reaction reduce time and increase efficiency to identify multiple pathogens with overlapping clinical presentation but different treatments or public health implications. Methods: Clinical performance of XpertXpress ® SARS-CoV-2/Flu/RSV (Cepheid, GX), TaqPath™ COVID-19, FluA/B, RSV Combo kit (Thermo Fisher Scientific, TP), and PowerChek™ SARS-CoV-2/Influenza A&B/RSV Multiplex RT-PCR kit II (KogeneBiotech, PC) was compared to individual Standards of Care (SoC). Thirteen isolates of SARS-CoV-2, human seasonal influenza, and avian influenza served to assess limit of detection (LoD). Then, positive and negative residual nasopharyngeal specimens, collected under public health surveillance and pandemic response served for evaluation. Subsequently, comparison of effectiveness was assessed. Results: The three MPAs confidently detect all lineages of SARS-CoV-2 and influenza viruses. MPA-LoDs vary from 1-2 Log10 differences from SoC depending on assay and strain. Clinical evaluation resulted in overall agreement between 97% and 100%, demonstrating a high accuracy to detect all targets. Existing differences in costs, testing burden and implementation constraints influence the choice in primary or community settings. Conclusion: TP, PC and GX, reliably detect SARS-CoV-2, influenza and RSV simultaneously, with reduced time-to-results and simplified workflows. MPAs have the potential to enhancediagnostics, surveillance system, and epidemic response to drive policy on prevention and control of viral respiratory infections. IMPORTANCE: Viral respiratory infections represent a major burden globally, weighed down by the COVID-19 pandemic, and threatened by spillover of novel zoonotic influenza viruses. Since respiratory infections share clinical presentations, identification of the causing agent for patient care and public health measures requires laboratory testing for several pathogens, including potential zoonotic spillovers. Simultaneous detection of SARS-CoV-2, influenza, and RSV in a single RT-PCR accelerates time from sampling to diagnosis, preserve consumables, and streamline human resources to respond to other endemic or emerging pathogens. Multiplex assays have the potential to sustain and even expand surveillance systems, can utilize capacity/capability developed during the COVID-19 pandemic worldwide, thereby strengthening epidemic/pandemic preparedness, prevention, and response.

Performance of the TaqMan COVID-19 Pooling Kit for detection of SARS-CoV-2 in asymptomatic and symptomatic populations.

Clinical evidence for asymptomatic cases of coronavirus disease (COVID-19) has reinforced the significance of effective surveillance testing programs. Quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays are considered the 'gold standard' for detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. However, the labor and resource requirements can be prohibitive with respect to large testing volumes associated with the pandemic. Pooled testing algorithms may serve to increase testing capacity with more efficient resource utilization. Due to the lack of carefully curated cohorts, there is limited evidence for the applicability of RT-PCR pooling in asymptomatic COVID-19 cases. In this study, we compared the analytical sensitivity of the TaqMan™ SARS-CoV-2 Pooling Assay to detect one positive sample in a pool of five anterior nares swabs in symptomatic and asymptomatic cohorts at an institute of higher education. Positive pools were deconvoluted and each individual sample was retested using the TaqPath™ COVID-19 Combo Kit. Both assays target the open reading frame (ORF) 1ab, nucleocapsid (N), and spike (S) gene of the strain that originated in Wuhan, Hubei, China. Qualitative results demonstrated absolute agreement between pooled and deconvoluted samples in both cohorts. Independent t-test performed on Ct shifts supported an insignificant difference between cohorts with p-values of 0.306 (Orf1ab), 0.147 (N), and 0.052 (S). All negative pools were correctly reported as negative. Pooled PCR testing up to five samples is a valid method for surveillance testing of students and staff in a university setting, especially when the prevalence is expected to be low.

SARS-CoV-2 variants of concern surveillance including Omicron using RT-PCR-based genotyping offers comparable performance to whole genome sequencing.

Known SARS-CoV-2 variants of concern (VOCs) can be detected and differentiated using an RT-PCR-based genotyping approach, which offers quicker time to result, lower cost, higher flexibility, and use of the same laboratory instrumentation for detection of SARS-CoV-2 when compared with whole genome sequencing (WGS). In the current study, we demonstrate how we applied a genotyping approach for identification of all VOCs and that such technique can offer comparable performance to WGS for identification of known SARS-CoV-2 VOCs, including more recent strains, Omicron BA.1 and BA.2.