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A new rapid and inexpensive PCR method to detect SARS-CoV-2 Omicron variant

Jacob Scott

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With the emergence of the Omicron variant of concern (VOC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent that causes the coronavirus disease 2019 (COVID-19), new and accurate diagnostic tests are needed to distinguish infection with this strain from that caused by other variants. A new study published on the preprint server medRxiv* reports a method to do this using four polymerase chain reaction (PCR) tests.

Study: Specific Detection Of SARS-CoV-2 B.1.1.529 (Omicron) Variant By Four RT-qPCR Differential Assays. Image Credit: anyaivanova / Shutterstock.com

Background

The SARS-CoV-2 Omicron variant was first described in South Africa and Botswana in November 2021. Soon after, the Omicron variant was detected in many other countries including Hong Kong and Israel.

This variant has 30 mutations in its spike protein, making it the most highly mutated variant of SARS-CoV-2 to date. Of these mutations, 15 affect the receptor-binding domain (RBD), which is a component of the spike protein that makes contact with the angiotensin-converting enzyme 2 (ACE2) receptor on host cells.

Most diagnostic tests today focus on the use of reverse transcription-polymerase chain reaction (RT-PCR) to amplify minute amounts of viral nucleic acid in the sample and thus make it possible to identify its presence and type. Using four different assays of this type the researchers in this study were able to carry out a quick identification of the new variant B.1.1.529.

The assays do this by amplifying and then binding to characteristic mutations in this variant, namely, in the non-structural protein nsp6, the viral spike, and the nucleocapsid genes. Not only are these assays simple to perform, but they are also suitable for multiplexing with a little adjustment and can be used to detect the presence of the Omicron VOC in routine test samples. This preliminary report will need to be followed up and the findings validated.

The need to find a way to quickly identify the Omicron variant spurred the development of the four assays described here by the Israel Ministry of Health Central Virology Laboratory (CVL) and the Israel Institute for Biological Research (IIBR). Based partly on existing protocols, this allows mutations already firmly linked to this variant to be detected using sequences uploaded to the Global Initiative for Sharing Avian Influenza Data (GISAID) database.

The assays were tested against the earliest Omicron samples in Israel and compared against the Delta variant which was, at that time, the only strain of SARS-CoV-2 in circulation in Israel. A few Alpha- and wild-type strains were also used to test for specificity of assay performance.

The test was modified to allow multiplexed testing based on established reactions already in use by the United States Centers for Disease Control and Prevention (CDC).

The assays can be implemented in any molecular diagnostic laboratory and do not require the use of specific brand instruments, or unique reagents.”

The mutations which these assays are designed to detect include deletion of spike gene positions 22194-22196 and nucleocapsid gene deletion 28,362-28,371 since these are found only in the Omicron variant. These were found in all Omicron sequences uploaded so far.

Negative Ndel reaction

The assay picks up N deletions 28362 using the N1 R1 reverse primer to ensure selectivity. When this deletion is present, the primer annealing is strongly inhibited 104-fold, thereby leading to the absence of any signal or a 12-15 difference in Cq values for the E-sarbeco reaction and the Ndel reaction. The former includes the deleted version and the latter does not.

By making use of the readily available primer and probe developed by the CDC, the researchers ensured the test can be easily performed in all laboratories. The assay targets the deletion at the position of reference sequence NC_045512.

Global phylogenetic analysis shows the uniqueness of the spike 22194-22196 deletion (A) and the nucleocapsid (N) 28362-28371 deletion (B). the analysis was performed using Nextstrain (https://nextstrain.org/ncov/gisaid/global), based on sequences from the GISAID database (www.epicov.org/epi3/frontend#5c1e82). The lineage in which these mutations occur is circulated with a highlighted circle.

Positive Ndel reaction

The positive Ndel reaction is caused by the annealing of the probe to the N deletion at 28362-28371 in the reference sequence. Therefore, the signal will only arise if the deletion is present.

Development of S22194 indel reaction

With this reaction, the Omicron insertion at 22207-22216 and deletion at 22194-22196 are targeted using a probe that binds only to the Omicron sequence containing this pattern,and not to other strains. It was tested along with the inclusive E-sarbeco reaction, including the Delta samples as well. This reaction showed clear specificity with clear identification of Omicron samples.

NSP6del reaction

With this reaction, the primer anneals to the deletion 11283-11292, such that if the deletion is present, the results will be equivalent to those of the E-sarbeco reaction. If not, the signal is inhibited 104-fold due to the absence of primer annealing.

Though the NSPindel mutation is common to the Alpha, Beta, and Gamma VOCs, it was attributed solely to the Delta variant in Israel since that was the only circulating variant in Israel at that time. Moreover, this deletion was found at only 0.03% in of over 3,000 sequences from Israel over the last 3 months. Thus, it was considered to be specific enough for the purpose of identifying the Omicron VOC as compared to the 1% of sequences that showed the presence of the spike gene 69-70 deletion.

When a suspected Omicron sample, along with Alpha and Delta samples, were tested, the assays showed positive Ndel for both Alpha and Omicron, negative or weakly positive Ndel for Delta, and a Cq difference of 12-14 cycles between the E and nsp6del reactions.

As expected, both the Alpha and Omicron-suspected samples were positive for the presence of the NSP6 deletion. The Delta samples were either negative for the NSP6del reaction or weakly positive, with Cq difference of 12-14 cycles between the E reaction and the NSP6del reaction.

Implications

The four assays reported in this paper are ideal for rapid diagnostic workup of a suspected case to identify the presence of the Omicron variant in a simple and cost-effective manner compared to genome sequencing. They can make up part of a scalable rapid protocol with the selected mutations providing highly accurate identification of this VOC.

Since the assays were developed based on the results of testing a small number of positive samples that had been shown to be Omicron by sequencing, their publication at this early stage could be justified only by the urgent demand for a way to distinguish this variant from other circulating strains so as to trace its spread and virulence. This is important in deciding policy measures such as travel restrictions and business closures, which could have potentially devastating economic consequences.

The contribution of the GISAID database was essential in allowing these assays to be validated on a preliminary level. Further testing using other reaction kits with required temperature adjustments will be necessary; however, the researchers expect that the results should be in agreement with this study.

We plan to conduct a far more thorough and comprehensive validation for these assays and publish our results in a proceeding report. We also welcome and comments and suggestions for improvements, should any such be available, from fellow laboratories.”

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Erster. O., Beth-Din, A., Asraf, H., et al. (2021). Specific Detection Of SARS-CoV-2 B.1.1.529 (Omicron) Variant By Four RT-qPCR Differential Assays. medRxiv. doi:10.1101/2021.12.07.21267293. https://www.medrxiv.org/content/10.1101/2021.12.07.21267293v1.

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Genetic bottlenecks could impact Wyoming toads’ ability to respond to new pathogens

Jacob Scott

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A new study from North Carolina State University examines immune system diversity in the critically endangered Wyoming toad and finds that genetic bottlenecks could impact a species’ ability to respond to new pathogens. The findings could inform captive breeding strategies for endangered animal populations.

The Wyoming toad, Anaxyrus baxteri, suffered a severe population decline throughout the latter part of the 20th century due to factors including habitat destruction and fungal infection. The toad was brought into a captive breeding program in the 1990s in order to save the species. Scientists estimate a current wild population of only 400 to 1,500 animals, meaning that the toad is considered critically endangered.

Population reduction in this species created a genetic bottleneck to begin with, meaning the level of genetic diversity is already very small. This is the first study to look specifically at genetic diversity in the immune systems of these toads and how it could impact them as a population.”

Jeff Yoder, professor of comparative immunology at NC State and co-corresponding author of paper

Yoder, with co-corresponding author Alex Dornburg of the University of North Carolina at Charlotte, performed RNA sequencing on immune tissues from three healthy, retired Wyoming toad breeders. Study co-author Michael Stoskopf, who was on the Wyoming Toad Recovery Implementation Team established in 2008, obtained the samples.

“We were focused specifically on sequences encoding toll-like receptors – TLRs – and the proteins of the major histocompatibility complex, or MHC, expressed in these tissues,” says Kara Carlson, first author of the study and current Ph.D. candidate at NC State. “These sets of genes are major components of the immune system.”

TLRs are the first responders of the immune system, and are similar, or well-conserved, between species. The MHC, on the other hand, is a large and diverse group of genes that varies between species and individuals. It can determine why one group is more resistant to a particular pathogen than another.

“MHC genes are some of the most rapidly evolving sequences in the genome,” Carlson says. “So in a healthy population there’s a lot of variety that gets passed along to descendants, enabling the species at large to adapt to different pathogens. However, if disease survivors do so because of their MHC, then that group would have a similar MHC.

“The Wyoming toads that were brought into captivity to save the species were all able to resist the fungus that had decimated the population, but that could mean that their immune diversity is reduced.”

The researchers compared the TLR and MHC of the three Wyoming toads to each other, as well as to samples from a common toad and a cane toad. Both the common toad and the cane toad showed more MHC diversity than the Wyoming toad, even though the cane toad underwent a similar genetic bottleneck.

“The small sample size in this study – which was unavoidable due to the endangered status of the toad – nevertheless lays an important framework for conservation,” Carlson says.

“Amphibians in general don’t have as many genomic resources as other organisms,” Yoder says. “And captive breeding from a small population further decreases genetic diversity. But while these toads may be better protected against the fungal infection that nearly wiped them out, they may not be equipped to deal with new pathogens down the road.”

“While we weren’t necessarily surprised by the lack of immunogenic diversity in the Wyoming toad, it does spark an important question,” Dornburg says. “How equipped are other species of conservation concern for a battle with an emergent pathogen?”

“By understanding the genetic diversity of the immune system we can inform captive breeding to increase the chance of a species to resist disease in the wild,” Yoder adds. “Studies like this one are invaluable for captive breeding practices going forward.”

Source:
Journal reference:

Carlson, K.B., et al. (2022) Transcriptome annotation reveals minimal immunogenetic diversity among Wyoming toads, Anaxyrus baxteri. Conservation Genetics. doi.org/10.1007/s10592-022-01444-8.

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Video conferencing hinders creativity

Jacob Scott

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In-person teamwork has now transformed into virtual collaboration due to the COVID-19 pandemic. But does this affect innovation and creativity?

An interesting study on virtual communication led by Dr. Melanie S. Brucks from Columbia University and Prof. Jonathan Levav from Stanford University is published in the journal Nature. The study examines whether videoconferencing affects creative idea generation.

Study: Virtual communication curbs creative idea generation. Image Credit: Andrey_Popov / Shutterstock

Communication and collaboration

Collaboration is essential for several workplace tasks. It leads to innovation and idea generation. Historically, these collaborations have been in-person and required sharing of the same physical space. The use of traditional communication technologies like letters, emails, and phone calls hinder the smooth exchange of information and limits collaboration.

Now, due to advances in audio-visual technology, face-to-face interaction is possible via videoconferencing, allowing virtual collaboration. Videoconferencing has replaced in-person interactions due to pandemic-driven social distancing. Video interaction and in-person interaction allow communication of the same information.

While videoconferencing replacing in-person interaction has been beneficial in the social scenario, does it come with a cost in the workplace scenario? For example, does it affect collaborative idea generation?

Experiments

The investigators performed a Laboratory experiment and a field experiment to test the difference between in-person interaction and videoconferencing in collaborative idea generation. First, they recruited participants and divided them into pairs: half of the pairs were assigned to an in-person setting, and the rest were assigned to a virtual setting.

Laboratory experiment

A total of 602 participants were recruited for the laboratory experiment and divided into pairs. The participants were in separate rooms in the virtual setting and communicated through videoconferencing. The pairs were allotted five minutes to generate creative uses for a frisbee (150 pairs) or bubble wrap (151 pairs) and then one minute to select their most innovative idea.

The pairs were evaluated by counting the number of creative ideas and ideas they generated. The virtual pairs generated significantly fewer total and creative ideas compared to in-person pairs.

So, virtual collaboration hampered creative ideas. This could be because the virtual space narrows the visual scope, which in turn narrows the cognitive scope. To assess the visual focus, two methods were used. Firstly, the participants had to recollect the individual props in the room and point them on a worksheet. Secondly, the participants’ eye gaze was recorded during the experiment.

The virtual pairs narrowed their focus to the screen. Compared to in-person pairs, they spent significantly more time looking directly at their partner and less time looking at the surrounding room, and remembered significantly fewer props in the surrounding room.

As a consequence, the virtual medium narrowed the visual focus and inhibited the generation of ideas.

However, these results are in the context of a controlled laboratory setting.

Field experiment

The experiment was repeated in ‘the field’ under actual work conditions within a large multinational telecommunications company to see if these results could be extrapolated to the real world. The field experiment was conducted in five country sites – in Europe, the Middle East, and South Asia. In this setting, the participants knew their partners and used video conferencing regularly for work. Moreover, it included domain experts highly invested in the outcome of the collaboration.

A total of 1,490 engineers were recruited to participate in an ideation workshop and randomly divided into pairs. The pairs were allotted an hour to generate product ideas and submit one idea as future product innovation for the company.

The engineer pairs who worked on the task virtually generated fewer total ideas and creative ideas than in-person pairs at all five sites.

However, the decision quality was not affected by virtual collaboration. The in-person pairs generated a significantly higher top-scoring idea, but the selected idea did not significantly differ in quality between the virtual pairs and in-person pairs.

Other reasons for reduced creativity

The could be other reasons why virtual collaboration negatively affected idea generation. Therefore, the investigators explored the alternative explanations.

More ideas

Since the in-person collaborators generated more total and creative ideas than the virtual collaborators, they could generate additional ideas similar to each other. However, upon semantic analysis, it was observed that they generated diverse and disconnected ideas.

Feelings of connection and trust

Studies have shown that feelings of connection and trust can foster team creativity. The virtual pairs may have reduced feelings of connection and trust toward their partner.

However, when assessed for subjective feelings of closeness, verbal and non-verbal behaviors, and mimicry, the virtual pairs were similar to in-person pairs in the laboratory experiment.

Thus, virtual and in-person interactions are very similar in terms of social connection or social behavior.

Communication coordination

Usually, there is a lack of coordination in a conversation in virtual interactions due to the absence of eye contact. However, it could not wholly explain the effect of virtual interaction on idea generation.

Interpersonal processes

This study also assessed the effect of interpersonal processes on idea generation. Interpersonal processes fear of evaluation, dominance, social facilitation, social loafing, social sensitivity, perceptions of performance, and production blocking were affected in virtual collaborations and these, in turn, affected idea generation.

Implications of the study

This study supports previous research suggesting that pairs perform better than large groups, both in-person and online. Therefore, this study recommends ideation in pairs and in person. Also, this study suggests that larger videoconferencing screens would not impact idea generation.

In-person collaborations offer a cognitive advantage. Now several workplaces are moving towards a hybrid setup. This study indicates that the creative idea generation should be reserved for in-person meetings.

Journal reference:
Brucks, M.S., Levav, J. (2022) Virtual communication curbs creative idea generation. Nature. https://doi.org/10.1038/s41586-022-04643-y, https://www.nature.com/articles/s41586-022-04643-y

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Gum Health Day 2022 calls for prevention, early detection, and effective treatment of gum diseases

Jacob Scott

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“Treat your gums” is the slogan for Gum Health Day 2022, a worldwide awareness campaign organized by the European Federation of Periodontology (EFP). The event aims to inform the public of the detrimental effects of gum diseases – gingivitis, periodontitis, peri-implant mucositis, and peri-implantitis – on both oral and overall health. The campaign calls for the prevention, early detection, and – where necessary – effective treatment of gum diseases.

Although still poorly acknowledged by the public, gum diseases are chronic inflammatory conditions affecting a high proportion of adults worldwide, causing tooth loss and other problems in the mouth. Crucially, gum diseases are also linked to major systemic health issues including diabetes mellitus, cardiovascular diseases, chronic kidney disease, adverse pregnancy outcomes, rheumatoid arthritis, Alzheimer’s disease, erectile dysfunction, certain forms of cancer, and more severe Covid-19 outcomes. This means that gum diseases and their prevention and treatment are of major importance not only for oral health, but also for the whole body.

This year’s campaign focuses heavily on the treatment part – we know that millions of people suffer from gum diseases that can be treated effectively. ‘Treat your gums’ calls for this treatment – with all the documented positive effects for the mouth and whole body – to actually happen.”

Moritz Kebschull, coordinator of Gum Health Day 2022

That is why the hashtag for the campaign is #TreatYourGums, and why the recent EFP-produced clinical practice guidelines on the treatment of periodontitis are a major part of the Gum Health Day 2022 initiative.

“The new EFP-produced clinical practice guidelines on the treatment of all four stages of periodontitis are a crucial development, as they are the first high-quality international guidelines to outline a structured and easily implemented pathway for the efficient and effective treatment of gum disease,” Prof. Kebschull says. “In a nutshell, gum disease treatment that works!”

He adds: “It is important to underline that gum disease is one of the most widespread chronic diseases in the worldwide adult population, and that it is usually painless, so its early detection and successful treatment depends heavily on how fast the patient takes action.”

A major innovation of Gum Health Day 2022 is an EFP-designed “customized content generator”, a feature that allows the federation’s 37 affiliated national societies of periodontology, their individual members – as well as practices, hospitals, and members of the public – to customise their own Gum Health Day 2022 materials, based on a series of graphic templates and catchphrases.

In the framework of Gum Health Day 2022, the EFP encourages periodontists, dentists, researchers, and other health-related professionals to sign and disseminate the EFP Manifesto: Perio and General Health, an international call to action for the prevention, early detection, and treatment of gum disease. Individuals and organisations are invited to endorse it and join the 1,200+ professionals, dental practices, companies, and universities that have so far supported it.


The European Federation of Periodontology (EFP, ww.efp.org) is a non-profit organization dedicated to promoting awareness of periodontal science and the importance of gum health. Its guiding vision is “periodontal health for a better life.”

Founded in 1991, the EFP is a federation of 37 national periodontal societies that represents more than 16,000 periodontists, dentists, researchers, and oral-health professionals from Europe and around the world. It supports evidence-based science in periodontal and oral health, and it promotes events and campaigns aimed at both professionals and the public.

The EFP organizes EuroPerio, the world’s leading congress in periodontology and implant dentistry, as well as other important professional and expert events such as Perio Master Clinic and Perio Workshop. The annual Gum Health Day on May 12, organized by the EFP and its member societies, brings key messages on gum health to millions of people across the world.

The EFP also organizes workshops and outreach campaigns with its partners: projects to date have covered the relationship between periodontal disease and diabetes, cardiovascular disease, and caries, as well as women’s oral health during pregnancy.

The EFP’s Journal of Clinical Periodontology is the most authoritative scientific publication in this field. The federation also publishes JCP Digest, a monthly digest of research, and the Perio Insight magazine, which features experts’ views and debates.

The EFP’s work in education is also highly significant, notably its accreditation programme for postgraduate education in periodontology and implant dentistry.

The EFP has no professional or commercial agenda.

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