COVID-19 – Lablogatory

Omicron: Variant of High Significance?

Omicron is now the dominant variant in the United States and gained that title faster than any variant before it. I have been tracking variants in the North Texas region since February of this year and detected the first Alpha variant (B.1.1.7). During this time, there were multiple substrains circulating. Some like Epsilon (origin California) rose in prominence then declined to extinction. Rise in Alpha (origin U.K.) and Delta variants (B.1.617.2, origin India) were tracked over the course of weeks, but Omicron has been tracked on a daily basis, since it is rising so quickly.

Many places are using S-Gene Target Failure (SGTF) as a surrogate for Omicron variant (Yale, University of Washington below).

Photo credit @NathanGrubaugh (Yale, Left) and @pavitrarc (UW virology, right)

SGTF occurs when the TaqPath COVID-19 multiplex test has 2/3 targets successfully amplify when the S-gene target does not or “drops out.” This phenomenon was first observed in the Alpha variant, because the probe for this target overlapped a characteristic mutation: S:Del69_70 (deletion of the 69^th and 70^th amino acids in the spike protein from a 6 base pair deletion). This mutation is absent in Delta, but present in Omicron, so has been used as an early tracker of Omicron prevalence.

Most of this discussion is speculative and we won’t ever really know, but given the rate of transmission of this variant, it seems unlikely that it would have acquired so many mutations and not been detected before now. The most recent common ancestor is from over a year ago suggesting it was incubating for a long time.

We’ve seen a case of a person severely immunocompromised with no antibody response to vaccination + booster who still has an unmutated wild type strain in their system. With no immune pressure, the virus has not evolved.

However, in HIV+ patients with variable/ low immunity, there could be enough pressure to drive the immune evasion properties seen in Omicron. Southern Africa has over 30% of their HIV+ patients not on therapy who would be likely candidates for this type of host.

Did we see this coming?

Yes. Other immune evasive variants have arisen in areas with high prevalence of previous infection (Brazil/ S. Africa). Organisms evolve just enough to overcome the challenges in their environment. Thus the level of immunity provided by various immune exposures are approximately:

Previous infection < 2x Vaccine < 2x Vaccine+ previous infection ~ x3 Vaccine

Scientists theorized that either Delta would evolve more immune evasive mutations or a totally new variant would arise. However, I didn’t think it would spread this quickly.

What is the impact?

Therapies. Most antibody therapies are directed as the business end of the spike protein—the receptor binding domain (RBD). The rest of the protein is covered in glycosylation modifications that block much recognition. Thus with many mutations in Omicron compared to the wild type strain (white), most therapeutic antibodies no longer bind/ inactivate viral replication.

Source: https://biorxiv.org/content/10.1101/2021.12.12.472269v1.full.pdf

Only one monoclonal antibody—Sotrovimab from GSK—is effective, because it binds a pan-coronovirus epitope outside of the RBD. However, this antibody is in short supply.

Thus, knowing which variant someone has can direct therapy. Several hospitals in our area are out of Sotrovimab, and only people with the Delta variant can access other options. Thus, knowing the variant in a short time frame has clinical implications.
Whole genome sequencing takes too long, so the FDA has agreed to review PCR genotyping approaches for clinical use. I have described some previous approaches, but many of these methods are useful as a screening method and would not have sufficient specificity to determine whether an omicron variant is present. Next time, I will discuss variant genotyping, why it is important, how it can be done, and what clinical actions can be taken with the knowledge.

Severity. There are signs that it is less severe. Is this due to increase in immune tolerance? We now have been prepared by either previous infection or vaccination to be protected from hospitalization or severe disease.

@Jburnmurdoch https://twitter.com/jburnmurdoch/status/1478339769646166019/photo/1

Or is the decline in severity due to lower pathogenicity? A recent non-peer reviewed study indicates the virus replicates x70 faster than Delta in the upper airways (left), but infiltrates cells 10% as well as the original strain.

From: https://www.med.hku.hk/en/news/press/20211215-omicron-sars-cov-2-infection?utm_medium=social&utm_source=twitter&utm_campaign=press_release

We all hope this will continue to be better news about the severity of Omicron, but from the lab side, I’ve heard of positivity rates >50% at some places. So this can still have a broad impact.

-Jeff SoRelle, MD is Assistant Professor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His research interests include the genetics of allergy, COVID-19 variant sequencing, and lab medicine of transgender healthcare. Follow him on Twitter @Jeff_SoRelle.

A COVID Reflection

Usually, I talk about some of the more administrative happenings in the laboratory world (accreditation, competency, etc.). Today, however, as there is seemingly a glimmer of light at the end of the nation’s pandemic tunnel, I thought I would reflect on what we have collectively experienced.

Like much of the nation, it has been a difficult journey for laboratorians. It has been particularly trying for those who were asked, who were required, to rise up and meet the unprecedented challenges of the times while suffering from the same burdens of fear, uncertainty, and physical ailments as those they were serving.

Dying Alone

One year ago, my uncle died from COVID-19. He died alone and afraid in the nursing home where he never wanted to be. We visited him after being given special permission from the president of the company operating the nursing home. After being told about how unusual it was to be allowed to see him, we dressed in full PPEs and went into his room. We found him curled in a fetal position, dead and cold to the touch. It was so unfair.

I think about all of the laboratorians who had to endure similar or worst experiences: those who lost close family members and even those who themselves suffered through the disease.

Unseen Warriors

Laboratorians have always been the silent warriors in the life-long battle to defeat pain and disease. More often, nurses and doctors received public gifts of admiration and praise for their service to patients. With quiet satisfaction, laboratory technologists, technicians, and support personnel are dedicated 24 hours a day, seven days a week, to providing the information on which 70% of medical decisions are based. Information that no other group of professionals can provide.

I think about all of the effort and skill required in the mad rush to set up tents and collection sites needed across the nation. And then, too, there were the laboratories needing to scale up testing or create entirely new testing areas with new instruments and new tests kits. The chaos was magnified by constantly changing guidelines, reagent shortages, and a lack of trained personnel.

Amid all the confusion, misinformation, and anger, laboratorians were themselves experiencing disease, death, and social isolation. Yet still, they delivered the results the nation needed to understand the pandemic’s depth and breadth.

Needless Death

Now the Delta variant has taken hold just when the nation thought the disease, if not bested, had at least been brought under some semblance of control. Unfortunately, the refusal of many to get vaccinated contributes to the virus’s persistence. More will suffer, and more will die.

How many needless deaths will the nation have to experience? Will there ever be a point when everyone who can be vaccinated will be? Or, two years later, will we be mourning preventable COVID-19 deaths. Will we still have to watch our loved ones perish with a tube down their throat, or worst, alone in a room far away surrounded by cold walls and quiet indifference?

Sigh.

Regardless of where this pandemic leads or how the nation reacts, laboratorians will continue to remain steadfast in their dedication to their profession and their patients. We have often considered ourselves the stepchild of the healthcare industry because, despite the criticality of what we do, we go unnoticed and unremarked on as long as we deliver the results our patients need. We are okay with that.

We are also tired and worn.

Conclusion

Thanks, fellow laboratorian, for reading this minor soliloquy of frustration and sadness. I will probably be back next quarter discussing inspections, competency, or some other administrative aspect of laboratory operations. I hope, also, to discuss how the nation has reached or is close to reaching the theoretical goal of herd immunity because of high vaccination levels. However, if I were honest, I know the likelihood of this happening is disappointingly low.

If you can get vaccinated, please do.

-Darryl Elzie, PsyD, MHA, MT(ASCP), CQA(ASQ), has been an ASCP Medical Technologist for over 30 years and has been performing CAP inspections for 15+ years. Dr. Elzie provides laboratory quality oversight for four hospitals, one ambulatory care center, and supports laboratory quality initiatives throughout the Sentara Healthcare system.

Pitfalls of Artificial Intelligence for COVID-19 Variant Classification

While you have surely heard about all of the SARS-CoV-2 variants and how concerning they are, I would bet that you may not know how they are classified. Sure, from my last post, the technical aspects of whole genome sequencing and targeted approaches have been described, but bioinformatic (big data) analyses are essential to assign lineages. Furthermore, the advances of machine learning have been integrated into this system for SARS-CoV-2 lineage assignment.

How VOC lineages are given

First, phylogenetic trees (circular example below) are formed to demonstrate relatedness of strains based on how many mutations they share. The more similar they are, the closer they are together. These trees are not new nor do they rely on artificial intelligence, but they can give visual clues as to whether a lineage is new. For instance, when the first variant of concern B.1.1.7 (now called Alpha) was discovered, it branched away from other limbs of the phylogenetic tree.

Within these new viral variants, there are a set of mutations that are present in most of the viral variants. For instance, there are 17 protein coding changes in Alpha variant. However, these exact 17 mutations may not be in every Alpha variant. Individually, mutations may be present in 98% of isolates or lower; the spike gene deletion of amino acids 242-244 of the Beta variant (B.1.351, South Africa origin) is only present in 88% of specimens sequenced. This could be due to issues in sequencing, data processing, or just the prevalance/biology of the virus.

As there are many mutations that fit into certain variants, it would be difficult for a human to process all of this information in a probabilistic manner to assign lineages. Thus, machine learning tools (most common SARS-CoV-2 program is Pangolin) have been added onto the end of bioinformatic analyses to assign the lineage to a sample.

How machine learning works

The subject of machine learning has been discussed in a previous post about Protein folding prediction. Briefly, it is helpful to remember that machine learning is a process to create algorithms that give an outcome based on training data. The more diverse, large, and well curated the data, the better the accuracy of the program. One pitfall is they are based on previous data, which works well for many situations: using AI to find a lung cancer on chest x-ray would work well, because lung cancers have consistent characteristics.

However, with COVID-19, new variants keep arising and current variants are evolving (think Delta and Delta “plus”). Furthermore, if the classifier Pangolin is trained on high quality data, then trying to interpret lower quality data (missing genome regions, few sequencing reads) may confuse Pangolin and lead to inaccurate results. What follows is an example of how this occurred at our institution.

Case study

We have been sequencing COVID-19 positive specimens at UT Southwestern for the last several months. Many of the cases have been the Alpha variant (B.1.1.7, origin U.K.). However, it was around this time that Delta (B.1.617.2, origin India) cases started to arise. In one week, we found two specimens that were classified as B.1.95. This was an unusual variant I had not heard of before. There are several “wild type” strains that are B.1.1/ B.1.2 and other derivations, but I had not seen anything like this before.

Clinical history

Two specimens sequenced belonging to Hispanic, adolescent brothers whose mother had recently been hospitalized with COVID-19. There was information on mother’s travel history.

Therefore, I performed manual review of the specific variants. Many of the diverse mutations occur in the spike protein, so this was analyzed first. Immediately, I noticed two classic mutations of the Delta variant: a 2 amino acid deletion in the spike gene (S:Del157_158) and a receptor binding site mutation (S:L452R) also seen in the variant from California (B.1.429). Other mutations could be evaluated, but the combination of these two mutations is unique to Delta variant.

One suspected cause was that the Pangolin lineage classifier had an issue. Specifically, it had not been updated since February 2021- when Delta did not exist. Thus, there was no data for the program to classify the variant properly. Upgrading to the latest version of Pangolin provided the correct lineage classification.

A Few weeks later…

Once again, I was checking the lineages reported by the classifier and there were several B.1.617.2 and B.1.617.1. Both of these are variants from India (before the helpful WHO Delta designation), but they are distinct sub-variants. It was odd to see B.1.617.1, because this was found to be less infectious compared to the dominant B.1.617.2 variant (later named Delta) and B.1.617.1 was not spreading across the globe.

Intervention:

Therefore, I once again went to the sequence data for the spike protein to compare some mutations. Although these are sub-variants from the same original variant, they have several mutually exclusive mutations in the spike protein. The figure below compares the prevalence of specific mutations in the spike protein of B.1.617.1 and B.1.617.2 (dark purple = common in a variant, white = rare).

Upon manual review, all of the spike gene mutations were specific to B.1.617.2. So why was there an issue in classification? Again, there were few sequences for either of these sub-variants at that time, so the classifier wasn’t as well trained. Updating the Pangolin version brought the benefit of new data and more accurate classifications.

Take away messages

Updating Lineage classification software (Pangolin) on a regular basis is needed for accurate results.
Manual review is essential for any abnormal findings- a typical process for pathologists, but also plays an important role in COVID-19 variant monitoring.
Know what you’re looking for and know which mutations differentiate the variants.
Delta is now the dominant strain in the U.S. (graphic below).

References

Outbreak.info
https://pangolin.cog-uk.io/

–Jeff SoRelle, MD is Assistant Instructor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His clinical research interests include understanding how lab medicine impacts transgender healthcare and improving genetic variant interpretation. Follow him on Twitter @Jeff_SoRelle.

How to Detect COVID-19 Variants of Concern

It’s a little deja-vu writing this title one year after a similar blog post on how to validate a COVID-19 assay at the start of the pandemic. In many ways, the challenges are similar: limited reagents/control material, and rising case counts. At least now, there is increasing support in the way of funding from the federal government that could help with monitoring and surveillance. I’m going to summarize the current methods available for detecting the Variants of Concern and emerging variants.

Whole Genome Sequencing

The principle method used by many is whole genome sequencing. It has the advantage of being able to comprehensively examine every letter (nucleotide) of the SARS-CoV-2 genome (30 kilobases long). At our institution, I’ve been working on the effort to sequence all of our positive specimens. While it is achievable, it is not simple nor feasible at most locations. Limitations include:

Financial: must already own expensive sequencers
Expertise: advanced molecular diagnostics personnel needed who perform NGS testing
Data Analytics: bioinformatics personnel needed to create pipelines, analyze data and report it in a digestible format.
Timing: the process usually takes a week at best and several weeks if there is a backlog or not enough samples for a sequencing run to be financially viable.
Sensitivity: the limit of detection for NGS is 30 CT cycles, which for us includes only about 1/2- 1/3 of all positive COVID19 specimens.

Bottom line: WGS is the best at detecting new/ emerging strains or mutations when cost/ time is not a concern.

Mutation Screening

Other institutions have begun efforts to screen for variants of concern by detecting characteristic mutations. For instance, the N501Y mutation in the spike protein is common to the major Variants of Concern (UK B.1.1.7, Brazil P.1, and S Africa B.1.351) and E484K is present in the Brazil (P.1), S Africa (B.1.351) and New York Variant (B.1.526). Thus, several institutions (listed below) took approaches to 1) screen for these mutations and then 2) perform WGS sequentially.

Institution	Method	Targets
Hackensack Meridian Health (HMH)	Molecular Beacon Probes, melting temp	N501Y, E484K molecular beacons
Rutgers, New Jersey	Molecular Beacon Probes, melting temp	N501Y molecular beacons
Vancouver	Probe + melting curve (VirSNiP SARS-CoV-2 Mutation Assays)	N501Y screen + qPCR reflex; Probe, melt curve assay
Yale	RT-qPCR probe assay	S:144del, ORF1Adel
Columbia	RT-qPCR probe-assay	N501Y, E484K

As you can see, HMH, Rutgers and Vancouver are using assays that use probes specific to characteristic alleles combined with melting temperature curves to detect a mutation induced change. Melting curve analysis is normally performed after qPCR to ensure that a single, correct PCR product is formed. This measure is calculated based on the change in fluorescence that occurs when the fluorescent marker is able to bind to its target DNA. Thus the Tm (melting temperature) is similar to the annealing temperature. In this case where a mutation is present in the probe (DNA fragment) binding site, binding is disrupted and occurs at lower a temperature as seen by the downward shift of 5 degrees Celsius in the graph below.

Figure 1. Schematic showing the melting temperature shift for the HMH designed probe binding normal and mutant (E484K variant) sequences at decreasing concentrations.

Figure 2. Similar shift downward in melting temperature for the Rutgers assay when a wild type probe encounters a mutant vs. WT sequence.

These approaches are quick, but can only perform a 2-3 reactions per well and require much of the same expenses as diagnostic RT-qPCR assays. Most of the studies describe this method as a way of screening for samples to be NGS sequenced, however they will not be as good at detecting emerging strains. For example, the N501Y mutation is not present in the New York nor California variants.

Multiplex RT-qPCR can solve some of these problems. At Columbia and Yale, multiple targets are designed to detect B.1.1.7 (N501Y only at Columbia and S144del + ORF1A del at Yale) vs. Brazil/ S. Africa variants (N501Y & E484K at Columbia and ORF1A only at Yale). As new variants have arrived, we found the New York strain carrying both ORF1A deletion and the E484K mutation. It is now clear there are some hotspot areas for mutation within the SARS-CoV-2 genome, which can complicate interpretations. Therefore, these RT-PCR assays are still useful for screening, but do not replace the need for Whole Genome Sequencing.

Genotyping

Given the overlapping spectrum of mutations, it would be helpful to test several markers all at once in a single reaction. At a certain point, this would effectively “genotype” a variant as well as WGS. The assays above have been limited to 2 targets/ reaction due to limited light detection channels. Therefore, I’ve created a multiplex assay that can be scaled up to include 30-40 targets within a single reaction without the need for expensive probes. This method is multiplex PCR fragment analysis, which is traditionally used for forensic fingerprinting or bone marrow transplant tracking. In this method, DNA of different length is amplified by PCR, then separated by capillary electrophoresis-the same instrument that performs Sanger Sequencing.

Fragment analysis can be performed to detect deletion/ insertion mutations and single nucleotide polymorphisms (SNPs) by allele-specific primers or with restriction enzymes that only cut the WT or Mutant sequence.

I designed the assay to target 3 deletion mutations in B.1.1.7: S:D69_70, S: D144, and ORF1A: D3675_3677. Each deletion has a specific length and if 3/3 mutations are present, then there is 95% specificity for the B.1.1.7 strain. Samples from December to present were tested and in the first batch, I detected the characteristic B.1.1.7 pattern (expected pattern and observed pattern below).

Theoretical picture of what the fragment analysis assay would look like for B.1.1.7. An actual patient sample results below, which showed the expected deletions exactly as predicted:

We have tested and sequenced over 500 positive specimens, and we found increasing levels of the B.1.1.7 strain prevalence up to nearly 30% by the middle of March. All screened B.1.1.7 specimens were validated by WGS. These results and the ability to detect the New York and California variants are detailed in our recent pre-print.

Weekly prevalence of isolates consistent with B.1.1.7 in North Texas.

Implications for future Variant Surveillance

As B.1.1.7 has become the dominant strain, and sequencing efforts are increasing. I would argue that assays should be used for what they are best at. For instance, it could be considered a waste of NGS time and resources to sequence all Variants when >50% are going to be B.1.1.7 if other tests can verify the strain faster for 10-20% of the cost. Instead, I think WGS should be focused on discovering emerging variants for which it is best suited. Across the US, case numbers have been decreasing and the number of specimens testable could be expanded by using a more sensitive PCR assay that could.

References

Clark AE et al. Multiplex Fragment Analysis Identifies SARS-CoV-2 Variants. https://www.medrxiv.org/content/10.1101/2021.04.15.21253747v1
Zhao Y et al. A Novel Diagnostic Test to Screen SARS-CoV-2 Variants Containing E484K and N501Y Mutations. A Novel Diagnostic Test to Screen SARS-CoV-2 Variants Containing E484K and N501Y Mutations | medRxiv
Banada P et al. A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants. A Simple RT-PCR Melting temperature Assay to Rapidly Screen for Widely Circulating SARS-CoV-2 Variants | medRxiv
Annavajhala MK et al. A Novel SARS-CoV-2 Variant of Concern, B.1.526, Identified in New York. A Novel SARS-CoV-2 Variant of Concern, B.1.526, Identified in New York | medRxiv
Matic N et al. Rapid detection of SARS-CoV-2 variants of concern identifying a cluster of B.1.1.28/P.1 variant in British Columbia, Canada. Rapid detection of SARS-CoV-2 variants of concern identifying a cluster of B.1.1.28/P.1 variant in British Columbia, Canada | medRxiv
Vogels CBF et al. PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern. PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern | medRxiv

Lab Inspections in a COVID World

The current pandemic has highlighted the importance of the laboratory in the delivery of healthcare. Patients and families depend on the laboratory to delivery accurate and timely results. Regulations have been written to ensure laboratories meet society’s expectations. Medical laboratories are one of the most highly regulated industries requiring biennial inspections by accrediting agencies. Despite operating under the COVID-19 testing pressures, laboratories still need to be inspection-ready.

New Inspection Process

As a result of social-distancing mandates and state-level restrictions, laboratories need to adjust to a new inspection environment. The College of American Pathologists (CAP) is temporarily allowing virtual inspections and has created information on its website about a few laboratories’ experiences with the virtual inspection process.

The CAP has also shared some expectations laboratories should be aware of when discussing inspection aspects.

Currently, in states where there are travel restrictions with quarantine requirements, a greater than 5% positivity rate, or where the institutions have travel/visitor restrictions, the laboratory medical director may choose to have a virtual inspection. However, the laboratory should be aware that they will still be required to have an in-person on-site inspection within 4-6 months if virtually inspected.

In addition, the laboratory director and the inspection team must both agree to perform a virtual inspection.

Virtual Aspects

Laboratories should take into consideration some of the aspects of a virtual inspection. In-person inspections for many small to medium laboratories often consist of inspectors being on-site for only one day. Conversely, virtual inspections can be weeks or even a month in duration depending on the laboratory’s size, the number of specialties, and the inspectors’ availability.

Virtual inspections also require a lot of document handling. Laboratories utilizing manual worksheets, quality control and troubleshooting logs will need to upload these documents for review. The CAP has created a secure website for this purpose, but it still requires personnel to scan each document individually.

Technical Priorities

There is also the risk of technical issues hampering the virtual process. Laboratories must have reliable Wi-Fi, electronic communication devices (laptops, tablets, cameras) and have personnel comfortable with the challenges inherent in managing multiple requests simultaneously. Having a dedicated IT person for an inspection is a great but difficult to get asset.

Mult-day Inspection

If there are no limiting COVID restrictions, laboratories may still opt for an in-person inspection.

Some inspection teams (in agreement with the laboratory medical director) have modified the in-person inspection process so that it is conducted over a 3-4 day time period. In this process, only a few inspectors come on each day to inspect specific disciplines. Usually, one inspector will return the next day to provide some continuity to the inspection process.

Instead of an intense one-day process, spreading an in-person inspection out to 3-4 days allows the team and facility to practice social distancing, reduces the level of stress, and gives the laboratory more time to provide evidence or have a deficiency changed to “corrected on-site.”

Competency Note

Laboratories need to be reminded that regardless of COVID, the requirements for competency still apply. New hires must still have semi-annual competencies performed at the required frequencies, and the laboratory must be able to provide competency documentation during an inspection. There are no exceptions to the competency mandate.

Conclusion

It is expected laboratory administrators and managers may have a bit of angst regarding the uncertainty that comes with a new inspection process affecting the entire laboratory. Amid the COVID crises, the laboratory has been tasked to deliver high-quality results efficiently. Laboratories across the nation have met the COVID challenge and are able to adapt to the demands inspections require. Virtual inspections are just another example of the laboratory adapting to meet its regulatory and accrediting requirements.

Making Lemonade from Lemons: Our Laboratory Lives after COVID-19

Laboratorians struggled through 2020 but successfully navigated a difficult situation while maintaining and improving our high-quality service to our patients. By laboratorian, I mean all of us—medical, public health, research, industry, etc.—because, across all sectors, anyone working in a laboratory (our family) was pushed to the limits to do more with less, work harder with fewer people, provide results with challenging procedure standard, and save lives while risking our own. It is quite easy to go into a clinical laboratory that is providing COVID-19 testing and find heroes that were there before, excelled during this pandemic, and will be there tomorrow. But there were heroes in every laboratory. Our public health laboratorians spent tireless hours trying to provide testing, coordinate testing, disseminate information, and relay the best current epidemiology to leadership to keep the country running. Our research laboratorians developed and delivered data, new information, novel biology, and potential interventions for the novel coronavirus. Our industry laboratorians were crucial components to vaccine development and delivery. And, unlike most of the country, our laboratorians were not able to “work from home” because, well, there are laws against having certain things in your house that might escape and kill your neighbors. It is good to be essential, but it has it pain points. Our laboratorians have felt that pain by still commuting to their benches to get the work done every day. But they did it and did it well! And what is often forgotten is that every single one of these laboratorians already had a “day job” in delivering a full catalog of laboratory-based services to which they added a successful COVID-19 response. If you see a laboratorian after you read this blog, you should want to hug them and say thank you.

Vaccination is spreading and will overtake and conquer this virus in parallel with our continued social distancing, hand washing, and mask wearing. In the background, testing will continue and will drive how our leaders make decisions more than anything else. We can see an end to this bedlam and are now facing, perhaps, one of the most difficult questions we have ever faced as a global laboratory community: “What do we do now?”

Our pathologists, long awaiting the day when digital telepathology was the norm, were thrust headfirst into that practice during the pandemic under emergency conditions. Many of them had already started (sometimes in a big way) but others were pushing glass routinely. Many of us have leapfrogged to a place from which we cannot return. We need to evaluate the virtual practice of the past year to determine the error rates and see if it is comparable (or better) than our routine glass slide practice. Is eBay or LetGo going to be overwhelmed with microscopes while high resolution monitors go into backorder? We must still contend with the requirement of “presence” and the moniker of “CLIA”, which was temporarily separated from a pathologist’s role in care during the pandemic. These new digital practices may address our long-standing workforce shortages. Working from home was not a possibility but a requirement for much of the last year. Care continued and work was done. What evidence would argue that working in an office is “better” than working from home when we consider the practice of pathologists? The financial implications of cost per square foot of overhead when taking up space in an academic medical facility is more than sufficient for a CFO to argue that pathologists working from home is great. But this is assuming that the workstation, the workflow, and the outputs were optimized. Not all pathology laboratories went fully digital and there was a great deal of slide shipping/couriering. On the other side of this pandemic, much like the 6 to 10 different platforms found in a clinical lab to perform a COVID-19 test, we will find that many practices are not sustainable, can be replaced and optimized, and will require more upheaval and pivot from our pathologists. To clarify, before COVID-19, pathologists practiced basically the same anywhere in the world; namely, review of glass slides in slide folders with a connected case file. During COVID-19, a whole new set of options emerged for how we would do that routine work that were uncontrolled and ad hoc. Now on the other side, we must separate the practices that are best for patient care from those that got the work done in a crisis to find our way forward. If the optimal model is (and I am not saying that it is) digital telepathology from anywhere, we must work hard to define “anywhere” for the sake of our patient’s care and safety. Monitor or other devices standards, which have long been the bane of the telepathology community, are still not standards. CLIA is specific about what constitutes a laboratory and its four walls. Accreditation teams do not inspect people’s home offices. On the other side of this pandemic, how do we find a common, best practice in a virtual age? We must return to a state of highest possible quality for our patients without giving up the advances we made in this crisis.

I once wrote up a laboratory revision plan for a firm that had 9 hospitals. Each had its own pathology laboratory employing 1 to 3 pathologist and similar staff for grossing, histology, and admin. Each laboratory had a volume of less than 3000 samples per year (and referred complex cases to a tertiary care center out of network). Based on our revision, in formalin concentration and recycling alone, the system would save $100,000. With a centralized laboratory (easily capable of handling 30,000 samples per year) and a digital pathology strategy, the work could be done by half the number of pathologists. Most importantly, the reagent/supply savings from having one laboratory rather than 9 was astronomical. The bottom line was an increase in revenue of nearly $1,000,000 with a cost savings of more than 75%. The key element of this plan that is important here is the digital telepathology component that reduces the number of staff needed and the office space needed which, at the time of the revision proposal, was “innovative” but thought too new to be reasonable. COVID-19 has tested that one aspect of the model and found it to be more than reasonable. More importantly, laboratory management and organizational leadership has had to take a hard look at costs, cost centers, and fixed expenses in such a way that the model above now becomes not lucrative but essential to staying in operation. We are trained in the laboratory to always be working on quality improvement, but COVID-19 has pushed us to always be working on fiscal improvement as well.

As we return to our “new normal” after COVID-19, the lessons we learned from this pandemic are going to translate into mergers, acquisitions, consolidations, closings, and restructurings of all types of businesses and services with the laboratory being no exception. The concept of surge capacity, for example, for testing of a new infectious agent that has emerged, has been a trial by fire, and there are many important lessons to learn from this as well. Should our approach to the next pandemic be to divert our staff from regular laboratory operations and bring into our facilities 6 to 10 new platforms for testing? Perhaps we should consider using temporary warehouse space offsite from our existing laboratory as well as backfill or relocating staffing for this crisis management to prevent complete disruption of our workflow and our policies. This is the type of solution that can exist when contingency planning is a routine part of operations. Those many facilities that were forced to bring in extra platforms are going to be facing a different crisis as test volumes crash; namely, what to do with the equipment. The firms that produce and sell that equipment have a similar challenge of expanding their platform beyond COVID-19 testing and making it relevant and competitive for the laboratories that have their extra platforms. Although I am not sure eBay or LetGo will be full of microscopes just yet, I am sure you are going to be able to pick up some nifty analyzers for an incredibly low price very soon. Will the memorial to the half-a-million we have lost in this country to COVID-19 be the useless bodies of laboratory devices that we so desperately needed in 2020? I think we owe them a lot more than that. Let us actively rethink our strategies in the laboratory and across our healthcare system so that such memorials are never needed again.

-Dan Milner, MD, MSc, spent 10 years at Harvard where he taught pathology, microbiology, and infectious disease. He began working in Africa in 1997 as a medical student and has built an international reputation as an expert in cerebral malaria. In his current role as Chief Medical officer of ASCP, he leads all PEPFAR activities as well as the Partners for Cancer Diagnosis and Treatment in Africa Initiative.

COVID Variants

Since my last post on the B.1.1.7 (UK) variant, several other variants have arisen. I wanted to describe what makes some Variants of Interest and other Variants of Concern. While a “variant” is often synonymous with a mutation in genetic terms, in the context of SARS-CoV-2, variant means an alternative strain of the virus.

To become a Variant of Interest (VOI), the World Health Organization (WHO) or Centers for Disease Control (CDC) has the following characteristics:

Evidence of variants that affect transmission, resistance to vaccines/ therapeutics, mortality, or diagnostic tests
Evidence that the variants is contributing to a rise in the proportion of cases in an area.
However, limited geographical spread.

Examples: P.2 (from Brazil) B.1.525 (New York), and B.1.526 (New York).

Variants of Concern have increased problems with the same characteristics listed above:

Evidence of reduced vaccine protection from severe disease
Evidence of substantially reduced response to neutralizing antibodies or therapeutics
Evidence of widespread spread
Increased Transmissibility or disease severity

Current VOCs: B.1.1.7 (UK), B.1.351 (South Africa), P.1 (Brazil), and B.1.427/ B.1.429 (California).

The initial VOC of B.1.1.7, B.1.351 and P.1 were identified from having increased spread and more mutations than expected, especially in the Spike gene region (Figure 1).

The N501Y mutation in the Spike protein is present in each VOC. It is located at the tip of the protein that binds the ACE2 receptor, increasing binding strength.

So far, vaccines react against the B.1.1.7 variant. However, B.1.351 pseudovirus shows decreased neutralization by both Moderna and Pfizer sera. Specifically, the E484K mutation in the Spike protein confers resistance to neutralizing antibodies. Thus, the strains B.1.351 and P.1 are more likely to be resistant as would any other strain with the E484K variant.

Lastly, the California variant arose as it was found to rise in prevalence from November to February. The key mutations include W152C and L452R, but the significance of this variant is uncertain. However, this variant has begun to spread over much of Southern California and Nevada.

References

Wu K, Werner AP, Moliva JI, Koch M, Choi A, Stewart-Jones GBE, Bennett H, Boyoglu-Barnum S, Shi W, Graham BS, Carfi A, Corbett KS, Seder RA, Edwards DK. mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv [Preprint]. 2021 Jan 25:2021.01.25.427948. doi: 10.1101/2021.01.25.427948. PMID: 33501442; PMCID: PMC7836112.
Tada T, Dcosta BM, Samanovic-Golden M, et al. Neutralization of viruses with European, South African, and United States SARS-CoV-2 variant spike proteins by convalescent sera and BNT162b2 mRNA vaccine-elicited antibodies. Preprint. bioRxiv. 2021;2021.02.05.430003. Published 2021 Feb 7. doi:10.1101/2021.02.05.430003
Gangavarapu, Karthik; Alkuzweny, Manar; Cano, Marco; Haag, Emily; Latif, Alaa Abdel; Mullen, Julia L.; Rush, Benjamin; Tsueng, Ginger; Zhou, Jerry; Andersen, Kristian G.; Wu, Chunlei; Su, Andrew I.; Hughes, Laura D. outbreak.info. Available online: https://outbreak.info/ (2020)
https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html

ASCP Releases Two Evidence-Based Recommendations for COVID-19 Testing

COVID-19 testing can be a bit confusing. Recently, ASCP released two recommendations for COVID-19 testing to help clinicians and laboratories sort through the noise and order the right test at the right time. In addition, ASCP has a plethora of COVID-19 resources, including Town Halls, podcasts, journal articles, and more.

Will the B.1.1.7 variant evade the Vaccine/Tests?

Will the B.1.1.7 variant evade the vaccine/tests?

This question came up recently and I wanted to share some cutting edge information the addresses this. This was in part adapted from Akiko Iwasaki’s (Yale HHMI immunologist) Twitter discussion of this subject.¹

Will B.1.1.7 evade our tests?

The UK variant commonly called lineage B.1.1.7 (officially Variant of Concern 202012/01) has 23 genetic variants that result in 17 protein coding changes.² Most tests including the ones at our institution (Abbott) are not currently affected (see below). Only the ThermoFisher assay has declared a target that covers the 69-70del variant in the S gene (in green). This conversely makes the TaqPath^® assay one way to detect a potential B.1.1.7 variant.

Figure 1. A picture of the SARS-CoV-2 genome with red lines indicating mutation sites and different assays and relative location of their qPCR targets.

Will the vaccine protect against the B.1.1.7 variant?

The Pfizer and Moderna RNA vaccines create an immune response against the spike protein. We don’t know the exact sequences or reactivity of the vaccines’ spike protein. However, a recent study looked at the antibody reactivity to linear epitopes of COVID-19 in 579 patients who were naturally infected with COVID-19. For the antibodies against the spike, the major reactive linear epitopes are indicated in Red at the bottom. None of the B.1.1.7 mutations (Orange) overlap with these major reactive epitopes.³

For a closer look, see below.

A limitation of these analyses is the use of only linear epitopes. Mutations might impact a 3D epitope affecting Ab binding. However, people make multiple antibodies to the spike protein.⁴ So, broad coverage should arise after exposure to the either the vaccine or natural infection with COVID-19.

The vaccine should induce a polyclonal antibody response that recognizes multiple parts of the spike protein, making it effective, even against novel variants. Also, there should be few to no False Negative COVID-19 tests due to the new variant, but we will continue to monitor and test this experimentally.

References

Prof. Akiko Iwasaki @VirusesImmunity
Chand, Meera et al. Investigation of novel SARS-COV-2 variant: Variant of Concern 202012/01 Public Health England.
Haynes WA et al. High-resolution mapping and characterization of epitopes in COVID-19 patients. MedRxiv. https://www.medrxiv.org/content/10.1101/2020.11.23.20235002v1#p-5
Shrock E et al. Viral epitope profiling of COVID-19 patients reveals cross-reactivity and correlates of severity. Science 2020 370(6520). https://science.sciencemag.org/content/370/6520/eabd4250

Critical Values: The Burden, Promise and Realization of Virtual Interviews for Pathology Residency During a Pandemic

The SARS-CoV-2 virus continues to cause increased infections and deaths around the world with considerable impact on clinical and laboratory medicine communities. Meanwhile, medical students and the medical community are also undertaking the yearly tribulation of residency interview season. Following the May announcement by the Coalition for Physician Accountability’s Work Group on Medical Students,¹ the 2020 interview season will be entirely conducted utilizing virtual interviews. In pointed response to this change in format, residency programs rapidly scrambled to bolster websites, increase their social media presence, add virtual tours and prepare for the virtual interview format prior to the start of interview season. Now, at the midpoint of interview season, it is evident that some burdens of traditional on-site interviews are indeed being alleviated. Whether or not online resident socials and virtual tours can sufficiently substitute for all aspects of on-site visits and if the promise of increased spread of geographic and cultural diversity can be realized remains to be accurately assessed. The survival of the virtual format may even depend on this assessment.

The average cost of traditional on-site pathology interviews has continued to increase for medical students from a per person average of $3400 in 2015 to $4000 in 2020.² Much of this expense comes from travel/transportation while some pathology programs provided accommodations. Additionally, interview season required about 20 total days away from medical school. To cover these expenses, about half (49%) of medical students borrow money for interviews . Not surprisingly, the majority of them agree that travel (79%) and lodging (65%) are overly burdensome components of interview season.² Beyond accounting, the salient impact of these time and financial investments is that they were influencing the majority (58%) of interview decisions.

While the rising time and financial burdens of traditional on-site residency interviews were well-known and there was and continues to be a myriad of ideas³ on how to best address these concerns and the match overall, a small burgeoning literature on virtual resident interviews was available prior to the pandemic that showed promise for addressing these concerns.^4,5 That is, in the 2020 – 2021 residency interview season, medical students are estimated to spend about 3.5 hours on an average virtual interview day instead of the 8 hour day of a traditional interview and through the elimination of travel time they may spend 7 less days on the interview trail. Thus, the cost of interviewing is also projected to be skeletonized to that of necessary professional clothing and computer hardware. Additional promising data from this small body of research suggests that 85% of virtual interviewees were satisfied with their understanding of the program and their ability to present themselves to residency programs.⁶ Furthermore, the fact that the residency program’s rank list showed no significant impact based on whether candidates interviewed virtually or in-person suggests that residency programs may feel capable of fairly assessing candidates.⁷

Beyond time and financial savings for pathology residency applicants and the assessment of candidates by residency programs and vice versa, the measurability of additional outcomes may be critical to the continuation of virtual resident interviews. In particular, there is great interest in online social events and interview day resident panels as a sufficient substitute for the naturally evolving casual conversations that occur during the dinners, lunches and tours available with on-site visits. Also, whether or not these socials combined with interviews with a small subset of faculty can accurately portray a pathology residency program’s culture. In prior surveys that compared in-person, virtual or a combined approach to interviews, candidates always favored in-person assessment when given the choice. The present circumstance will perhaps be the best attempt at an unbiased assessment of the perception of culture through virtual interviews. Last but not least, given the turbulent nature of race relations and culture in the United States over the last year combined with the ability of applicants to virtually interview without travel or financial restrictions, it will be absolutely critical to understand if virtual interviews portend to increase the spread of geographic and cultural diversity among applicants to pathology residency programs. That is, if current trends in resident recruitment can be altered from the current rate of 40 – 60% intraregional resident matriculation or whether the needs of financial and family assistance and/or intraregional familiarity are insurmountable.⁸ For if the potential for greater diversity is attainable in a significant manner that can be perpetuated into the future, it will be hard to argue for a return to the traditional format. That said, there will likely be bias in the data as an increasing number of pathology residency programs have heard the call to arms and are marching towards diversity, inclusion and equity through greater promotion, recruitment and retention efforts.⁹

In a tumultuous year that has included race relations reminiscent of the Civil Rights Era combined with a total number of worldwide pandemic deaths similar to the 1957 or 1968 influenza pandemics, medicine continues its steady progression toward improved healthcare and education for all. Following the May 2020 recommendations to implement virtual residency interviews, pathology residency programs moved expeditiously to bolster their websites, increase their social media presence, add virtual tours and prepare for the virtual interview format. Amid this tumult, the virtual interview format has already served to lessen the burdens of time and cost while also serving the practical needs of interview assessments for both medical students and residency programs. Yet, only time and methodical assessment will tell if the virtual interview format eliminates the impact of these burdens on residency decisions, allows both parties to adequately assess cultural fit and if the format and its advantages are here to stay. Regardless, it is imperative that the emphasis on diversity, inclusion and equity remains irrespective of future format.

References

The Coalition for Physician Accountability’s Work Group on Medical Students in the Class of 2021 Moving Across Institutions for Post Graduate Training Final Report and Recommendations for Medical Education Institutions of LCME-Accredited, U.S. Osteopathic, and Non-U.S. Medical School Applicants.
Pourmand, A., Lee, H., Fair, M., Maloney, K. & Caggiula, A. Feasibility and usability of tele-interview for medical residency interview. Western Journal of Emergency Medicine 19, 80–86 (2018).
Hammoud, M. M., Andrews, J. & Skochelak, S. E. Improving the Residency Application and Selection Process: An Optional Early Result Acceptance Program. JAMA – Journal of the American Medical Association 323, 503–504 (2020).
Chandler, N. M., Litz, C. N., Chang, H. L. & Danielson, P. D. Efficacy of Videoconference Interviews in the Pediatric Surgery Match. J. Surg. Educ. 76, 420–426 (2019).
Vining, C. C. et al. Virtual Surgical Fellowship Recruitment During COVID-19 and Its Implications for Resident/Fellow Recruitment in the Future. Ann. Surg. Oncol. 1 (2020). doi:10.1245/s10434-020-08623-2
Healy, W. L. & Bedair, H. Videoconference Interviews for an Adult Reconstruction Fellowship: Lessons Learned. Journal of Bone and Joint Surgery – American Volume 99, E114 (2017).
Vadi, M. G. et al. Comparison of web-based and face-to-face interviews for application to an anesthesiology training program: a pilot study. Int. J. Med. Educ. 7, 102–108 (2016).
Shappell, C. N., Farnan, J. M., McConville, J. F. & Martin, S. K. Geographic Trends for United States Allopathic Seniors Participating in the Residency Match: a Descriptive Analysis. J. Gen. Intern. Med. 34, 179–181 (2019).
Ware, A. D. et al. The “Race” Toward Diversity, Inclusion, and Equity in Pathology: The Johns Hopkins Experience. Acad. Pathol. 6, (2019).

-Josh Klonoski, MD, PhD, is a chief resident at the University of Utah, Salt Lake City, Utah, with a focus in neuroinfectious disease and global health. He has completed the first year of a neuropathology fellowship (out of sequence) and is in his final year of an anatomical and clinical pathology residency. Dr. Klonoski will return to the second neuropathology fellowship year in 2021 – 2022 and apply for a mentored clinical scientist research career development award (K08). The focus of his laboratory research is influenza and active projects include flu pneumonia, super-infections, encephalitis and oncolytic virotherapy.