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What Lies Ahead with COVID? We Simply Don’t Know

Recently on the local news, New York governor Kathy Hochul confidently proclaimed that we would not experience another COVID surge. Yet just two hours earlier, a quartet of experts from the Massachusetts Consortium on Pathogen Readiness (MassCPR) came to a different conclusion, on a webinar for journalists.

“Omicron took us by surprise. It is at the next level, approaching the most transmissible respiratory virus humanity has ever seen. Has it reached its natural limit? I don’t know,” said Bronwyn MacInnis, director of pathogen genomic surveillance at the Broad Institute of MIT and Harvard.

COVID-19 isn’t over.

“The fact that Omicron emerged the way it did – in stealth mode for a year before it burst onto the scene – makes us wonder what else could be out there and what features it would have. Transmission leading to minimal infection is one version of the world we may need to get used to with COVID-19, but being vigilant about immune escape and more severe disease is what we are most concerned about,” MacInnis said.

I’ll give Kathy Hochul the benefit of the doubt – perhaps she consults the databases of new viral mutations and concludes that it’s ok to downplay fears for the future. But she may be unknowingly undoing one positive outcome of the pandemic – the powerful message to the public that science is not certainty.

There’s no such thing as “scientific proof,” and there never was – that’s more an advertising phrase used to pitch a drug or a detergent. With new evidence, conclusions, always temporary, change. And SARS-CoV-2 regularly reinvents itself. No matter how much we think we know, we can’t predict the future with 100 percent accuracy – not even a governor can do that.

Yes, artificial intelligence can consult the genetic code to predict exactly how a genome can mutate, even how those mutations might assort themselves – recombine – into ever more varied viral variants. But layer on the vagaries of natural and artificial selection (like vaccines), and the crystal ball clouds.

We don’t know what lies ahead for the no-longer-novel coronavirus.

Tracking Omicron Since Thanksgiving

MassCPR has regularly updated journalists since the pandemic’s beginnings, sporadically at first, then weekly with the Thanksgiving 2021 Omicron appearance and surge. Usually four of the dozens of physicians and scientists update us briefly, and then we ask the questions that lead to the headlines in the coming days. But MassCPR’s leaders felt okay, actually almost jubilant, with ceasing at the end of February this year.

The first of the five guises of Omicron was quickly fading away. Replacing it was subvariant BA.2, which spread even faster but didn’t make us any sicker, although genetically speaking it is quite distinct.

The intermission lasted a month.

This week’s session began with the usual “weather report” from Jacob Lemieux, instructor in medicine at Harvard Medical School and infectious disease specialist at Massachusetts General Hospital.

“It’s been quiet time, but there’s also a sense that something may be brewing nationally.”

Although the numbers of COVID cases, hospitalizations, and deaths are plummeting, BA.2 is washing over the world – but it’s not been a simple “what happens in the UK happens in the US 6 weeks later.” CDC data tracker estimated the proportion of cases that are BA.2 in the US overall as 23 percent yesterday when I wrote the draft of this post. Today it’s approaching 40 percent.

Where will things go? Lemieux foresees a general transition over a month or two from BA.1 to BA.2. When that happens, he looks to current extremes to guess what might occur: The worst BA.2 outbreaks are in Brunei (part of Borneo), Hong Kong, the Philippines, and Denmark. The opposite situation is in South Africa, where BA.2 never got a strong toehold.

“Although we are in a relative low, when you look closely at the data, for the first time we are seeing a sign that we have reached the nadir and are on the upslope in a few places, including Rhode Island, Arkansas, Colorado, Maine, and Vermont. We don’t know what will happen in the coming weeks or months,” Lemieux said.

MacInnis pointed out that BA.2 cases, now the majority of infections in New England, are doubling every 6.8 days, with no increase in the number of cases or deaths. But the trend is tricky to evaluate and compare to times past because of massive changes in testing and behavioral patterns.

“Are we out of the woods? It’s too soon to know,” MacInnis said, echoing Lemieux. “There could be another curveball coming at us. Recombinant viruses in a few corners of the world are a new flavor that the scientific community needs to grapple with. Recombinant viruses open new questions.”

Amy Barczak, assistant professor of medicine at Harvard Medical School and infectious disease specialist at Mass General and the Ragon Institute of MGH, MIT, and Harvard, agreed with MacInnis and Lemieux, but apparently not New York’s governor, that “new variants will continue to emerge and drive new surges.” And that will bring a slew of new unknowns:

• Transmissibility
• Resistance to vaccines and treatments
• Duration of immunity from vaccines or infection

Omicron was a Surprise and a Wake-Up Call

SARS-CoV-2 surprised in ways other than hiked transmissibility.

“As more people got immunity, it wasn’t surprising that we started to see viruses become better at transmitting to people who were vaccinated or survived. What is surprising is how fast this happened and how creative the virus was in coming up with ways to infect people who had immunity, and how much the spike protein tolerated lots of mutations. That opens the question that if SARS-COV-2 could tolerate so many mutations in Omicron, could it tolerate more? In other places? A completely different set of 30 to 50 mutations?” asked Lemieux. He recently attended a workshop at the NIH where 20 molecular virologists concluded that no one knows just what this virus is capable of.

“Omicron was not a predictable step on an evolutionary path that anyone saw coming” said MacInnis.

When it comes to COVID, I’m afraid the only certainty we have is the unrelenting uncertainty.

Lemieux calls for Congress to pick up the ball on the stalled funding for preparedness and response. “If you’re coming out of a curve on a road and can go faster, that doesn’t mean you stop looking at the road. You must prepare for the possibility there may be a curve coming. One thing we know about this virus is that there are curves coming.”

It seems as if we are moving towards a compromise lifestyle in which most people return to some version of normalcy, while those of us at higher risk of infection and serious disease avoid exposure. I’m relieved to know that while our governor seems certain that the worst is over, the experts are still carefully scrutinizing the changeling pathogen.

Discussion
  1. On February 26, 2022, George Gao of the Beijing China National CDC published the preprint

    1) ‘Surveillance of SARS-CoV-2 in the environment and animal samples of the Huanan Seafood Market’, by George Gao, William Liu, and many others, Chinese Academy of Sciences, National Institute for Viral Disease Control and Prevention, and others https://assets.researchsquare.com/files/rs-1370392/v1_covered.pdf?c=1645813311

    which contains the results of 33 environment samples collected January 1 or 12, 2020, from the Huanan Seafood Market (HSM) in Table 1 of the article, and results of samples collected on later dates. On the same day Michael Worobey and Christian Andersen study group released their preprint article below, that contained 33 positive environment samples from the HSM in their Table S2.

    (2) ‘The Huanan market was the epicenter of SARS-CoV-2 emergence’, by https://zenodo.org/record/6299116#.Yh_ErJhMFdh, by Michael Worobey, Joshua Levy, Pekar, and others

    Worobey and Anderson probably consulted with Dr. Gao, to get their list of environmental samples of the HSM.
    Dr. Gao’s Table 1 lists the Huanan Seafood Market positive environment samples for Covid-19, and he observed, “live viruses were isolated from samples F13 (wall) , F54 (ground), and B5 (ground), which were the only three samples with Ct values <30 (cycle threshhold) ….. samples F13 (wall) and F54 (ground) were from the stalls with confirmed patients ……..The genome sequences of two environmental samples, F13 (wall) and F54 (ground), were found to be highly identical to the reference strain HCoV/Wuhan/IVDC-HB-01 (WH01, sequence identity of 99.993%) and completely identical to the human stain Wuhan-Hu-1 (GenBank: NC_045512) …….Commercial products of swabs and virus preservation solution were used for the sampling (Disposable Virus Sampling Tube, V5-S-25, Shen Zhen Zi Jian Biotechnology Co., Ltd., Shenzhen, China). For environmental samples, sampling swabs were applied to smear the floors, walls or surfaces of objects and then preserved them in virus preservation solution.”
    Dr. Gao says F13 environmental sample (wall) is highly identical to WH01 (Wuhan person #1- NC_045512) isolate. WH01 isolate was collected December 26, 2019 (according to patient information table in the article, ‘Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding’, by Roujian Lu, Xiang Zhao, and others, Beijing National CDC, and others.

    Dr. Gao also indicated F13 (wall sample) had a Ct value of 23.85, and F54 sample (ground) a Ct value of 25.8 in his Table 1. Cycle threshold means the number of cycles required for the fluorescent signal to cross the threshold. Ct levels are inversely proportional to the amount of target nucleic acid in the sample, according to Igho Onakpoyo article ‘Sars-Cov-2 and Role of Fomite Transmission’. Wuhan person # 1 had a Ct value 23.967 according to the article ‘A new coronavirus associated with human respiratory disease in China’, Fan Wu, Su Zhao, and others, extended data figure 4. For a person aged 41 admitted December 26, 2019. F13 wall sample and Wuhan person #1 cycle threshold almost match and five days difference in the sampling.

    Wuhan person #1 who worked in seafood at HSM, bronchoalveolar lavage fluid (BALF) for late December 2019, “the viral load in the BALF sample was estimated by qPCR to be 3.95 × 108 copies per ml”. —- in the article, ‘A new coronavirus associated with human respiratory disease in China’, Fan Wu, Su Zhao, Bin Yu, and others, Shanghai Public Health Center and others. They named the isolate MN908947.

    In February 2022, Jonathan Pekar calculated tMRCA of lineage B Covid-19, as being mid-December 2019. (last time when all coronavirus alleles were still in their original source.)
    “We inferred the median tMRCA of lineage B to be 13 December (95% HPD: 29 November to 23 December) and the median tMRCA of lineage A to be 25 December (95% HPD: 17 December to 30 December) (Fig. 6A), under the unconstrained model…… our results indicate that lineage B was introduced into humans no earlier than November 2019, and lineage A cross-species transmission likely occurred within days to weeks of the first event.” —-in the article, ‘SARS-CoV-2 emergence very likely resulted from at least two zoonotic events’, by Jonathan Pekar, Andrew Magee, and other, University California San Diego, and others Time to most recent ancestor possibly mid-December 2019, according to Pekar. Wuhan person # 1 showed signs of virus on December 20, 2019 according to Roujan Lu study referenced above. Wuhan person # 1 had high viral titers December 26, 2019. Mid-December 2019 is a common time period for the two different studies. Others had signs of Covid-19 before then. How did that live F13 Covid-19 sample get on the wall in the HSM, possibly associated with Wuhan person #1 (according to Gao), is the purpose of this comment. According to the WHO, Covid-19 transmission occurs either by droplet transmission, airborne transmission, or direct contact.

    “Droplet transmission occurs when a person is in in close contact (within 1 m) with someone who has respiratory symptoms (e.g., coughing or sneezing) and is therefore at risk of having his/her mucosae (mouth and nose) or conjunctiva (eyes) exposed to potentially infective respiratory droplets. Transmission may also occur through fomites in the immediate environment around the infected person. Therefore, transmission of the COVID-19 virus can occur by direct contact with infected people and indirect contact with surfaces in the immediate environment or with objects used on the infected person (e.g., stethoscope or thermometer).

    Airborne transmission is different from droplet transmission as it refers to the presence of microbes within droplet nuclei, which are generally considered to be particles <5μm in diameter, can remain in the air for long periods of time and be transmitted to others over distances greater than 1 m.”———- in the article, ‘Modes of transmission of virus causing COVID-19: implications for IPC precaution recommendations’, by the World Health Organization, https://apps.who.int/iris/handle/10665/331601

    An Egyptian study group observed.

    “The aerosol particles can be divided into (1) ultrafine particles (less than 0.1micrometer-.000001 m), (2) fine particles (0.1-2 micrometers), and (3) coarse particles (greater than 2 micrometers). The size of PM in aerosols plays a pivotal role in determining their deposition site and mechanism at the respiratory system. The mechanisms of respiratory deposition are classified based on particles sizes into different mechanisms, namely diffusion, sedimentation, impaction, and interception. Particles with sizes smaller than 0.5 micrometers are deposited in the alveoli by diffusion mechanism…..Sedimentation is another deposition mechanism, which plays a significant role in the setting out particles of aerodynamic size between 1-5 micrometers in the smaller airways of bronchioles and alveoli where the airflow is low. Sedimentation is governed by gravitational forces, particle velocity and aerodynamic size…….Particles with sizes greater than 5 micrometers m are deposited at the bronchial regions by impaction. Impaction is highly dependent on aerodynamic diameter and mass. “ in the article, ‘Inhaled nano- and microparticles for drug delivery’, by Ibrahim El-Sherbiny, Nancy El-Baz, Magdi Yacoub

    Proper sizing the particles in the F13 wall sample is very important in determining how that Covid-19 got on that HSM wall, eg, sedimentation, impaction, etc..

    Physics behind how sample F13 may have gotten on the HSM wall —–

    Airborne transmission, also known as aerosols. F13 sample was collected by Gao from a vertical wall. Was there direct contact to contaminate the wall? Or perhaps, horizontal trajectory of droplets or aerosols being influenced by air flow, heat, rate of evaporation and other factors. A recent University of Missouri study concluded,

    “horizontal velocity of respiratory droplets depends strongly on human activity, age, and ambient environment. The trajectory of the exhaled respiratory droplets is affected by both the expired air flows profile and surrounding air flow patterns. Existing studies treated the exhaled air as a turbulent round jet , and the turbulent flow will enhance the heat and mass transfer between the droplet and the surrounding air. Therefore, respiratory droplets will likely evaporate faster than the simulated results in this study, and a larger fraction of respiratory droplets and viruses may remain airborne for a longer period of time…. airborne droplets can travel a horizontal distance of 2.64 m after 10 s, and 3.95 m after 30 s”,—–'Modeling the load of SARS-CoV-2 virus in human expelled particles during coughing and speaking’, by Yang Wang and others, University of Missouri

    Droplets and airborne transmissions should eventually drop to the ground (a horizontal surface) because of evaporation, unless air flow or direct contact caused the virions or particles to stick to the vertical HSM wall. An University College London study observed,

    “The adhesion mechanism of SARS-CoV-2 on environmental surfaces has yet to be adequately delineated, but it has been predicted that it is primarily driven by electrostatic attractions (e.g. pH, isoelectric point ( pI) and ionic strength), then hydrophobic effects, and minorly non-covalent bonds (e.g. van der Waals forces) which could all govern the binding of the S protein to solid surfaces.” — in the article, ‘Surface interactions and viability of Coronaviruses’, by Mehmet Onur Aydogdu, Esra Altun and others

    Was there some electrostatic attraction or hydrophobic effects between the Covid-19 source, and the part of the HSM wall where the F13 sample was collected? The section of Aydogdu’s above article, ‘SARS-CoV-2 adsorption mechanism on different inanimate surfaces’, is well worth reading. Did the density, inertia drag, and size of the particles play an intricate part in the Covid-19 collected from the HSM wall? In 1986, Paul Baron of the NIOSH observed,

    ‘The aerodynamic diameter (of a particle) is defined as the equivalent of the settling velocity diameter. For particles being measured 0.8 to 20 μm in diameter the particle settling velocity is sufficiently small that the viscous drag predominates…for larger particles the viscous and inertia drag are significant as indicated by the size of the particle ‘s Reynolds number in Table 1. …The Weber number is used to characterize breakup of droplets when they are in motion relative to air, is the ratio of the drag force to the surface tension force for a particle in motion.” —–in the article, ‘Calibration and Use of Aerodynamic Particle Sizer’, by Paul Baron, National Institute for Occupational Safety and Health, 1986

    The aerodynamic diameter of the (possible Covid-19) particles depends of the density of the particle in still air, according to Baron. Viscous, thick sticky consistency, between solid and liquid. Could F13 sample on the wall of HSM been a result of drag related and density of aerosoled or droplet particles? Baron continued,

    “When a droplet moves through the air at a high velocity, the drag force exerted by the air can be described by the equation ….”

    Viscous and inertia drag in conjunction with particle surface tension can create distorted particles, oblate spheroids, according to Baron.

    Recently, many studies have tested surfaces for Covid-19 but reported results in concentration of gene copies per μL, eg, Joshua Santarpia and Danielle Rivera and others in their article, ‘Aerosol and surface contamination…’. Molar concentration per unit volume. That has important medical and autopsy purposes.

    But determining and analyzing surface particles μm size (micrometer-.000001 m) and pleomorphism seems more practical regarding forensics, origin of the coronavirus, determining particles density, their Weber and Reynolds numbers, Womersley number associated with oscillatory flow capturing the importance of unsteady acceleration through the ratio of the oscillation frequency to viscous effects and perhaps trace those F13 sample particles possible source of origin.

    The Covid-19 virons themselves ——-

    Dr. Gao properly sized the Covid-19 virions themselves using transmission electron microscopy.

    “Negative-stained virus particles were generally spherical, pleomorphic and 60-140 nm (nanometers) in diameter.” (0.000000001 m).

    Verified by Dr. Na Zhu in February 2020. Pleomorphic means the occurrence of more than one distinct forms of a natural object, eg, oblong, star shaped, etc.

    “Electron micrographs of negative-stained 2019-nCoV particles were generally spherical with some pleomorphism (Fig. 3). Diameter varied from about 60 to 140 nm” (0.000000001 m) ——in the article, ‘A Novel Coronavirus from Patients with Pneumonia in China, 2019’, by Na Zhu, Dingyu Zhang, and others.

    A Zhejiang and Tsinghua Universities study in 2020 elaborated on the pleomorphism nature of Covid-19 virions.

    “SARS-CoV-2 virions (ID: ZJU_5) were collected on January 22, 2020 from a patient with severe symptoms and were propagated in Vero cells……Intact and unconcentrated virions were directly visualized from the supernatant by cryo-EM, showing ellipsoidal and spherical enveloped particles (Figure S1A), consistent with the observation of virions concentrated by ultracentrifugation through a sucrose cushion (Figure S1B). We modeled 2,294 virions as ellipsoids by meshing their lipid envelopes, measuring average diameters of 64.8 plus or minus 11.8 nm, 85.9 plus or minus 9.4 nm, and 96.6 plus or minus 11.8 nm (average – SD) for the short, medium, and long axis of the envelope, respectively…. Our observations of the structures and landscape of the intact SARS-CoV-2 are consistent with two other cryo-ET studies that were published at the same time (Turo_nova´ et al., 2020; Ke et al., 2020)”. —–in the article ‘Molecular Architecture of the SARS-CoV-2 Virus’, by Hangping Yao, Yutong Song, and many others, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou , Zhejiang Province, Tsinghua University, Beijing, and many others

    The study group visualized the Covid-19 virons using cryo-EM in their unconcentrated form/nature as ellipsoids and spherical. They then observed the Covid-19 virons as concentrated, using ‘ultracentrifugation through a sucrose cushion’, with the similar observational results. Their figure 4E shows the ellipsoidal Covid-19 virons as being oblong in nature. Certainly not the result of nebulization. Supplemental figure Figure S1 B shows spherical and oblong Covid-19 virons together. The study group also shows the inner packing of ribonucleoproteins in the Covid-19 virons. But they don’t show or size the possible surface particles that Covid-19 virions possibly existed in, or were transmitted in.

    A Sharchar-Berman study group observed a few years ago regarding ellipsoidal particles.

    “Whether natural or anthropogenic (e.g. biomass burning), the majority of inhalable airborne particles are intrinsically non-spherical (Kleinstreuer and Feng, 2013)…..For micron-sized airborne particles in the absence of electrostatic forces, transport dynamics are hence foremost governed by viscous drag, aerodynamic lift and gravity (in the negative y-direction;”, —–in the article ‘Transport of ellipsoid fibers in oscillatory shear flows: Implications for aerosol deposition in deep airways’, by Lihi Shachar-Bermana and others.

    Ellipsodal Covid-19 virons may be a product of nature, or other means. Particle dynamics study is necessary. In July 2021, Vincent Munster of the NIAID observed regarding the possible larger particles housing Covid-19 virons,

    “demonstration of true aerosol transmission of SARS-CoV-2 should only include particles less than 5 μm (micrometer=.000001 m), over longer distances and in the absence of any other potential transmission routes such as fomite or direct contact….Epidemiological data suggests that the principal mode of infection with SARS-CoV-2 is via airborne transmission”——-in the article, ‘Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS-CoV-2’, by Vincent Munster, Julia Port and others, NIAID Montana

    Munster did not address methods for determining surface particle sizes for Covid-19 environment samples. Only methods for collecting aerosol size particles. Those particles were measured using a Model 3321 aerodynamic particle sizer spectrometer from TSI.

    Methods for Collecting and Determining Surface Samples Particles Sizes —

    Similarly in 2020, the World Health Organization did not address collecting direct transmission environment surface samples, other than to say,

    “Environmental samples need to be taken using a swab with a synthetic tip and a plastic shaft (2,3,9-12). The swab specimen collection vials should contain 1-3ml of viral transport medium (e.g. protein stabilizer, antibiotics and buffer solution) including neutralizing buffer to counteract the effects of any residual disinfectant (e.g. Tween 80)………sequencing of environmental samples may be challenging and may need to be discussed with laboratories with coronavirus sequencing expertise.”— in the article, ‘Surface sampling of coronavirus disease (COVID-19): a practical “how to” protocol for health care and public health professionals’, (No. WHO/2019-nCoV/Environment_protocol/2020.1). https://apps.who.int/iris/bitstream/handle/10665/331058/WHO-2019-nCoV-Environment_protocol-2020.1-eng.pdf?sequence=1&isAllowed=y

    The WHO suggested using ‘swabs with synthetic tips’ to collect the environmental sample and ‘discuss with laboratories with coronavirus sequencing expertise’. If the WHO wants to standardize a method to collect and size Covid-19 surface particles, a Harvard University study group had a good technique and collected μm size (micrometer) particles as follows,

    “We used custom-designed Harvard Micro-Environmental Cascade Impactors (Demokritou et al., 2002) to collect simultaneous samples in three distinct size fractions: fine (less than or equal 2.5 μm-micrometer aerodynamic diameter), coarse (2.5–10 μm), and large (greater than or equal 10 μm) . The first impactor stage used a polyurethane foam (PUF) substrate to collect large particles. The second impactor stage used a smaller PUF substrate to collect particles in the coarse size range. The last stage collected fine particles on a 37-mm diameter glass-fiber filter We confirmed our hypothesis, finding positive samples in the fine size fraction and in a pattern indicating location-specific size distributions. A key finding is that the size distribution of particle-associated SAR-CoV-2 was related to the location where the particles were collected.”—— in the article, ‘Levels and particle size distribution of airborne SARS-CoV-2 at a healthcare facility in Kuwait’, by Rebecca A. Stern, Ali Al-Hemoud, and others

    If that Harvard Micro-Environmental Cascade Impactor can be used to collect particles from the HSM environment surface samples, then maybe it can be used to help size environment sample F13 (wall) and F54 (ground) particles. Would the key would be collecting samples on the polyurethane foam (PUF) substrate, native to the Impactor? What if no large (greater than or equal 10 μm – micrometer ) particles were found in the F13 wall sample? What if only nanometer sized Covid-19 virions are found, not housed in any particles whatsoever?

    A Singapore study group collected environment surface samples using Puritan EnviroMax Plus pre-moistened macrofoam sterile swabs, and seeming integrated the contents of those swabs into a NIOSH air sampler, to analyze particles greater than 4 μm in diameter.

    “Surface samples were collected with Puritan EnviroMax Plus pre-moistened macrofoam sterile swabs (25-88060) ……Particles collected with the NIOSH sampler are distributed into three size fractions. Particles greater than 4 μm in diameter are collected in a 15mL centrifuge tube, particles 1–4 μm in diameter are collected in a 1.5 mL centrifuge tube, and particles less than 1 μm (micrometer) in diameter are collected in a self-assembled filter cassette containing a 37-mm diameter, PTFE filter with 3 μm pores”, —-in the article ‘Detection of air and surface contamination by SARS-CoV-2 in hospital rooms of infected patients’, by Po Ying Chia, Kristen Coleman, and others, National Centre for Infectious Diseases, Singapore, Singapore, Duke NUS Medical School, and others

    Seemingly, the Singapore group integrated Puritan surface collection swabs into NIOSH air sampler, to collect particles greater than 4 μm in diameter, utilizing a two-stage bioaerosol cyclone samplers provided by the National Institute for Occupational Safety and Health (NIOSH). Their methods section is fuzzy regarding this. Hopefully Gao, the WHO and Atlanta CDC can consider standardizing the Singapore’s or Rebecca Stern’s technique in collecting and sizing surface environmental particles, for documenting and tracing the origin of possible future outbrakes. If the HSM was not aerosoled, droplet transmitted, nebulized, maybe the direct contact (large particle) source was present at the location.

    Lastly, digital drop PCR has been used to size Covid-19 aerosols into micrometre particles. Why not use digital drop PCR to size particles collected from surface environmental samples? In the article, ‘Aerodynamic analysis of SARS-CoV-2 in two Wuhan hospitals’, by Yuan Liu, Zhi Ning, and others, used digital drop PCR to “size Covid-19 aerosols into submicrometre region (dp between 0.25 and 1.0 μm-micrometers) and the other in supermicrometre region (dp greater than 2.5 μm) aerodynamic particles into….. total of three size-segregated aerosol samples was collected using a miniature cascade impactor (Sioutas Impactor, SKC) that separated aerosols into five ranges (greater than 2.5 μm, 1.0–2.5 μm, 0.50–1.0 μm and 0.25–0.50 μm on 25-mm filter substrates, and 0–0.25 μm on 37-mm filters) at a flow rate of 9.0 l min−1”.

    A different impactor than the one used by the Harvard study group. Why not try using a similar technique regarding sizing environmental samples particle sizes? And standardize that method. Other surface collection of virus particles articles worth reading are,
    ‘Contact transmission of SARS-CoV-2 on fomite surfaces: surface survival and risk reduction’, Abhimanyu Tharayil, and others, Ghandi University, India and others, encyclopedia on virus surface factors, nothing on method of collection

    ‘Sustainability of Coronavirus on Different Surfaces’, Rajiv Suman, and others, very little on method used

    ‘ddPCR: a more accurate tool for SARS-CoV-2 detection in low viral load Specimens’, Tao Suoa, Xinjin Liub, and others used digital drop PCR to report copies/reaction

    ‘Stability of SARS-CoV-2 and other coronaviruses in the environment and on common touch surfaces and the influence of climatic conditions: A review’, by Hamada Aboubakr, Tamer Sharafeldin, and Sagar Goyal1, encyclopedia type of article environmental surfaces, very little on coronavirus surface collection methodology

    Nebulization:

    In 2020, the NIAID, Tulane University, Fort Dietrich study group used nebulizers to generate Covid-19 particles, and the Aerodynamic Particle sizer to size the particles generated.

    “The 3-jet (C3), 6-jet Collison (C6), or Aerogen Solo (AS) nebulizers were used for generation of viral aerosols ( MERS-CoV and SARS-CoV to SARS-CoV-2 )…… Aerosol particle size was determined using an Aerodynamic Particle Sizer solely or with the addition of a diluter (TSi, Shoreview, MN, USA). scanning electron microscopy was used to size virions” — ‘Persistence of Severe Acute Respiratory Syndrome Coronavirus 2 in Aerosol Suspensions’, by Alyssa Fears and Robert Garry-Tulane, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland, USA (M. Lackemeyer, J.K. Bohannon, R. Johnson) , US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, USA (A. Nalca, A. Totura, D. Dyer, B. Kearney), and others, Emerging Infectious Diseases • http://www.cdc.gov/eid • Vol. 26, No. 9, September 2020

    In 2020, the NIAID and Atlanta CDC generated Covid-19 aerosol particles using a Collison nebulizer.

    “Aerosols (<5 μm) containing SARS-CoV-2 (105.25 50% tissue-culture infectious dose [TCID50] per milliliter) or SARS-CoV-1 (106.75-7.00 TCID50 per milliliter) were generated with the use of a three-jet Collison nebulizer and fed into a Goldberg drum to create an aerosolized environment. The inoculum resulted in cycle-threshold values between 20 and 22, similar to those observed in samples obtained from the upper and lower respiratory tract in humans.”— in the article ‘Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1’, by Neeltje van Doremalen, Vincent Munster (National Institute of Allergy and Infectious Diseases-Hamilton, MT), Azaibi Tamin, Jennifer Harcourt, Natalie Thornburg, Susan Gerber,Centers for Disease Control and Prevention Atlanta, Georgia, N Engl J Med 382;16, April 16, 2020

    Vincent Munster of the NIAID used a standard spray bottle containing chemicals to help generate droplets and aerosols from hamsters inoculated with Covid-19.

    “particle size next validated the caging design using an aerodynamic particle sizer to analyze the aerodynamic size of particles (dynamic range from <0.5-20 μm) traversing from donor to sentinel cage. Droplets and aerosols were generated in the donor cage (20% (v/v) glycerol solution, sprayed with a standard spray bottle “, in the article, ‘Increased aerosol transmission for B.1.1.7 (alpha variant) over lineage A variant of SARS CoV-2’, by Julia Port, Claude Kwe Yinda, Victoria Avanzato, Jonathan Schulz, Myndi Holbrook, Neeltje van Doremalen, Carl Shaia, Robert Fischer, Vincent Munster, NIAID Montana

    Nebulization the hardway. As if they have nothing else to do at the NIAID in Montana other than inoculate hamsters with Covid-19. The article, ‘Utility of Three Nebulizers in Investigating the Infectivity of Airborne Viruses’, by Sadegh Niazi,a Lisa K. Philp,b Kirsten Spann, Graham Johnson, Queensland University of Technology — worth reading

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