Validation of Microchip Based RT-PCR ABC Test (InfA/B & COVID-19) in Clinical SamplesGabriel Martinez - 2022
AbstractTo contain the rapid and global spread of SARS-CoV-2, it is essential to develop an accurate and sensitive test system to address pandemic bottlenecks, simplified sample collection, and no sample prep. While meeting the demand of testing large populations, the miniaturized volume of assay reagents and offering rapid results is the need in such scenarios. Moreover, in view of the reports of co-infections and overlapping symptoms of influenza caused by Influenza A or Influenza B, and COVID-19 caused by SARS-CoV-2, a test system with three targets can be supportive for accurate clinical diagnosis. In this presentation, we evaluated the performance of a test comprising Microchip RT-PCR Influenza and COVID-19 Detection System for identifying these three viral pathogens in nasal swabs and saliva specimens. A rapid and simplified total nucleic acid extraction method was developed and validated for the reliable, high-throughput simultaneous detection of respiratory viruses causing Influenza (type A and type B viruses) and COVID-19 (SARS-CoV-2 virus) using the microchip-based AriaDNATM platform deriving the name ABC Test. The test system was evaluated using 81 nasal swab samples, 77 clinical saliva samples, 5 blind CAP reference samples, and RNA standards. The limit of detection (LoD) was assessed using SARS-CoV-2, Influenza A, and Influenza B RNA standards. The multiplex ABC Test microchip displayed LoD of 14 copies/μL for SARS-CoV-2 and approximately 26 copies/μL for influenza A, and 140 copies/μL for influenza B, respectively. The ABC Test offers rapid multiplex one-step RT-PCR in 32 minutes for 45 cycles as the miniaturized reaction of 1.2 μL offering a highly sensitive, robust, and accurate assay for the detection of influenza A/B, and SARS-CoV-2.
Novel Roles of the Nipah Virus Attachment Glycoprotein and Its Mobility in Early and Late Membrane Fusion StepsVictoria Ortega - 2022
AbstractThe Paramyxoviridae family comprises important pathogens that include measles (MeV), mumps, parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). Paramyxoviral entry into cells requires viral-cell membrane fusion, and formation of paramyxoviral pathognomonic syncytia requires cell-cell membrane fusion. Both events are coordinated by intricate interactions between the tetrameric attachment (G/H/HN) and trimeric fusion (F) glycoproteins. We report that receptor binding induces conformational changes in NiV G that expose its stalk
domain, which triggers F through a cascade from prefusion to prehairpin intermediate (PHI) to postfusion conformations, executing membrane fusion. To decipher how the NiV G stalk may trigger F, we introduced cysteines along the G stalk to increase tetrameric strength and restrict stalk mobility. While most point mutants displayed near-wild-type levels of cell surface expression and receptor binding, most yielded increased NiV G oligomeric strength, and showed remarkably strong defects in syncytium formation. Furthermore, most of these mutants displayed stronger F/G interactions and significant defects in their ability to trigger F, indicating that NiV G stalk mobility is key to proper F triggering via moderate G/F interactions. Also remarkably, a mutant capable of triggering F and of fusion pore formation yielded little syncytium formation, implicating G or G/F interactions in a late step occurring post fusion pore formation, such as the extensive fusion pore expansion required for syncytium formation. This study uncovers novel mechanisms by which the G stalk and its oligomerization/mobility affect G/F interactions, the triggering of F, and a late fusion pore expansion step—exciting novel findings for paramyxoviral attachment glycoproteins.
IMPORTANCE The important Paramyxoviridae family includes measles, mumps, human parainfluenza, and the emerging deadly zoonotic Nipah virus (NiV) and Hendra virus (HeV). The deadly emerging NiV can cause neurologic and respiratory symptoms in humans with a .60% mortality rate. NiV has two surface proteins, the receptor binding protein (G) and fusion (F) glycoproteins. They mediate the required membrane fusion during viral entry into host cells and during syncytium formation, a hallmark of paramyxoviral and NiV infections. We previously discovered that the G stalk domain is important for triggering F (via largely unknown mechanisms) to induce membrane fusion. Here, we uncovered new roles and mechanisms by which the G stalk and its mobility modulate the triggering of F and also unexpectedly affect a very late step in membrane fusion, namely fusion pore expansion. Importantly, these novel findings may extend to other paramyxoviruses, offering new potential targets for therapeutic interventions.
Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva SamplesFrancesco Saverio Tarantini - 2022
AbstractSince mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.
Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva SamplesFrancesco Saverio Tarantini - 2022
AbstractSince mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal ‘testing fatigue’ and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.
Molecular SARS-CoV-2 surveillance in Bavaria shows no Omicron transmission before the end of November 2021Jennifer Flechsler - 2022
AbstractBackground
Five SARS-CoV-2 variants are currently considered as variants of concern (VOC). Omicron was declared a VOC at the end of November 2021. Based on different diagnostic methods, the occurrence of Omicron was reported by 52 countries worldwide on December 7 2021. First notified by South Africa with alarming reports on increasing infection rates, this new variant was soon suspected to replace the currently pre-dominating Delta variant leading to further infection waves worldwide.
Methods
Using VOC PCR screening and Next Generation Sequencing (NGS) analysis of selected samples, we investigated the circulation of Omicron in the German federal state Bavaria. For this, we analyzed SARS-CoV-2 surveillance data from our laboratory generated from calendar week (CW) 01 to 49/2021.
Results
So far, we have detected 69 Omicron cases in our laboratory from CW 47–49/2021 using RT-qPCR followed by melting curve analysis. The first 16 cases were analyzed by NGS and all were confirmed as Omicron.
Conclusion
Our data strongly support no circulation of the new Omicron variant before CW 47/2021.
Extractionless Nucleic Acid Detection: A High Capacity Solution to COVID-19 TestingShairaz Baksh - 2021
AbstractWe describe an extractionless real-time reverse transcriptase-PCR (rRT-PCR) protocol for SARS‐CoV‐2 nucleic acid detection using heat as an accurate cost-effective high-capacity solution to COVID‐19 testing. We present the effect of temperature, transport media, rRT-PCR mastermixes and gene assays on SARS‐CoV‐2 gene amplification and limits of detection. Utilizing our heated methodology, our limits of detection were 12.5 and 1 genome copy/reaction for singleplex E‐ and N1‐gene assays, respectively, and 1 genome copy/reaction by utilizing an E/N1 or Orf1ab/N1 multiplex assay combination. Using this approach, we detected up to 98% of COVID‐19 positive patient samples analyzed in our various cohorts including a significant percentage of weak positives. Importantly, this extractionless approach will allow for > 2‐fold increase in testing capacity with existing instruments, circumvent the additional need for expensive extraction devices, provide the sensitivity needed for COVID‐19 detection and significantly reduce the turn‐around time of reporting COVID‐19 test results.
Comparative effects of viral-transport- medium heat inactivation upon downstream SARS-CoV- 2 detection in patient samplesJamie L. Thompson - 2021
AbstractIntroduction. The COVID-19 pandemic, which began in 2020 is testing economic resilience and surge capacity of healthcare providers worldwide. At the time of writing, positive detection of the SARS-CoV-2 virus remains the only method for diagnosing COVID-19 infection. Rapid upscaling of national SARS-CoV-2 genome testing presented challenges: (1) Unpredictable supply chains of reagents and kits for virus inactivation, RNA extraction and PCR-detection of viral genomes. (2) Rapid time to result of <24 h is required in order to facilitate timely infection control measures.Hypothesis. Extraction-free sample processing would impact commercially available SARS-CoV-2 genome detection methods.Aim. We evaluated whether alternative commercially available kits provided sensitivity and accuracy of SARS-CoV-2 genome detection comparable to those used by regional National Healthcare Services (NHS).Methodology. We tested several detection methods and tested whether detection was altered by heat inactivation, an approach for rapid one-step viral inactivation and RNA extraction without chemicals or kits.Results. Using purified RNA, we found the CerTest VIASURE kit to be comparable to the Altona RealStar system currently in use, and further showed that both diagnostic kits performed similarly in the BioRad CFX96 and Roche LightCycler 480 II machines. Additionally, both kits were comparable to a third alternative using a combination of Quantabio qScript one-step Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) mix and Centre for Disease Control and Prevention (CDC)-accredited N1 and N2 primer/probes when looking specifically at borderline samples. Importantly, when using the kits in an extraction-free protocol, following heat inactivation, we saw differing results, with the combined Quantabio-CDC assay showing superior accuracy and sensitivity. In particular, detection using the CDC N2 probe following the extraction-free protocol was highly correlated to results generated with the same probe following RNA extraction and reported clinically (n=127; R2=0.9259).Conclusion. Our results demonstrate that sample treatment can greatly affect the downstream performance of SARS-CoV-2 diagnostic kits, with varying impact depending on the kit. We also showed that one-step heat-inactivation methods could reduce time from swab receipt to outcome of test result. Combined, these findings present alternatives to the protocols in use and can serve to alleviate any arising supply-chain issues at different points in the workflow, whilst accelerating testing, and reducing cost and environmental impact.
Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroidsMarleen Botermans - 2020
AbstractPotato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the Pospisense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.
The effect of increased inoculum on oral rotavirus vaccine take amonginfants in Dhaka, Bangladesh: A double-blind, parallel group,randomized, controlled trialBenjamin Lee - 2020
AbstractBackground:Oral, live-attenuated rotavirus vaccines suffer from impaired immunogenicity and efficacyin low-income countries. Increasing the inoculum of vaccine might improve vaccine response, but thisapproach has been inadequately explored in low-income countries.Methods:We performed a double-blind, parallel group, randomized controlled trial from June 2017through June 2018 in the urban Mirpur slum of Dhaka, Bangladesh to compare vaccine take (primary out-come) among healthy infants randomized to receive either the standard dose or double the standard doseof oral Rotarix (GlaxoSmithKline) vaccine at 6 and 10 weeks of life. Infants with congenital malforma-tions, birth or enrollment weight <2000 gm, known immunocompromising condition, enrollment inanother vaccine trial, or other household member enrolled in the study were excluded. Infants were ran-domized using random permuted blocks. Vaccine take was defined as detection of post-vaccination fecalvaccine shedding by real-time reverse transcription polymerase chain reaction with sequence confirma-tion or plasma rotavirus-specific immunoglobulin A (RV-IgA) seroconversion 4 weeks following the sec-ond dose.Results:220 infants were enrolled and randomized (110 per group). 97 standard-dose and 92 high-doseinfants completed the study per-protocol. For the primary outcome, no significant difference wasobserved between groups: vaccine take occurred in 62 (67%) high-dose infants versus 69 (71%)standard-dose infants (RR 0.92, 95% CI 0.67–1.24). However, in post-hoc analysis, children with con-firmed vaccine replication had significantly increased RV-IgA responses, independent of the intervention.No significant adverse events related to study participation were detected.Conclusions:Administration of double the standard dose of an oral, live-attenuated rotavirus vaccine(Rotarix) did not improve vaccine take among infants in urban Dhaka, Bangladesh. However, improvedimmunogenicity in children with vaccine replication irrespective of initial inoculum provides further evi-dence for the need to promote in-host replication and improved gut health to improve oral vaccineresponse in low-income settings.
The effect of increased inoculum on oral rotavirus vaccine take among infants in Dhaka, Bangladesh: A double-blind, parallel group, randomized, controlled trialBenjamin Lee - 2020
AbstractBackground
Oral, live-attenuated rotavirus vaccines suffer from impaired immunogenicity and efficacy in low-income countries. Increasing the inoculum of vaccine might improve vaccine response, but this approach has been inadequately explored in low-income countries.
Methods
We performed a double-blind, parallel group, randomized controlled trial from June 2017 through June 2018 in the urban Mirpur slum of Dhaka, Bangladesh to compare vaccine take (primary outcome) among healthy infants randomized to receive either the standard dose or double the standard dose of oral Rotarix (GlaxoSmithKline) vaccine at 6 and 10 weeks of life. Infants with congenital malformations, birth or enrollment weight <2000 gm, known immunocompromising condition, enrollment in another vaccine trial, or other household member enrolled in the study were excluded. Infants were randomized using random permuted blocks. Vaccine take was defined as detection of post-vaccination fecal vaccine shedding by real-time reverse transcription polymerase chain reaction with sequence confirmation or plasma rotavirus-specific immunoglobulin A (RV-IgA) seroconversion 4 weeks following the second dose.
Results
220 infants were enrolled and randomized (110 per group). 97 standard-dose and 92 high-dose infants completed the study per-protocol. For the primary outcome, no significant difference was observed between groups: vaccine take occurred in 62 (67%) high-dose infants versus 69 (71%) standard-dose infants (RR 0.92, 95% CI 0.67–1.24). However, in post-hoc analysis, children with confirmed vaccine replication had significantly increased RV-IgA responses, independent of the intervention. No significant adverse events related to study participation were detected.
Conclusions
Administration of double the standard dose of an oral, live-attenuated rotavirus vaccine (Rotarix) did not improve vaccine take among infants in urban Dhaka, Bangladesh. However, improved immunogenicity in children with vaccine replication irrespective of initial inoculum provides further evidence for the need to promote in-host replication and improved gut health to improve oral vaccine response in low-income settings. ClinicalTrials.gov: NCT02992197.
