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COVID-19: Epidemiology, Virology, Disease Course and Testing

Gilead Sciences, Inc.Last Updated April 27, 2020

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Disclaimer

§ For up to date information on COVID-19 epidemiology, please refer to WHO, CDC, and regional authorities

§ This deck is for proactive use

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Overview

§ Epidemiology§ Virology§ Disease Course§ Diagnostic Testing

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Epidemiology

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Coronavirus Disease of 2019 (COVID-19) Timeline

1. Chen N, et al. Lancet. 2020 Feb 15;395(10223):507-513. doi: 10.1016/S0140-6736(20)30211-7. Epub 2020 Jan 30. 2. Guan W, et al. N Engl J Med. 2020 Feb 28. doi: 10.1056/NEJMoa2002032. [Epub ahead of print]. 3. The New York Times. A timeline of the coronavirus. https://www.nytimes.com/article/coronavirus-timeline.html. Published March 4, 2020. Accessed March 9, 2020. 4. Coronavirus COVID-19 Global Cases by Johns Hopkins CSSE. https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6. Accessed March 9, 2020. 5. Centers for Disease Prevention and Control (CDC). https://www.cdc.gov/coronavirus/2019-ncov/index.html (Accessed 11 March 2020) 6. WHO COVID-19 rolling update https://www.who.int/emergencies/diseases/novel-coronavirus-2019/events-as-they-happen

Dec 8, 2019

Cases of pneumonia of

unknown etiology in Wuhan, Hubei province, China

around local Huanan seafood

market

Jan 7, 2020

Novel coronavirus identified by

Chinese CDC

Apr 16, 2020

~ 2M confirmed cases

documented globally (>130,000

deaths)

Jan 13, 2020

First confirmed cases outside

China in Thailand

Jan 30, 2020

WHO declares a

global health emergency

Mar 11, 2020

WHO declares pandemic

Feb 11, 2020

ICTV announced SARS-CoV-2 as the name of the

new virus, COVID-19 as the name of the new

disease

Jan 11, 2020

First death case in China

Up-to-date Global COVID-19 Situation Available at:Johns Hopkins Coronavirus Resource Center

WHO COVID-19 DashboardICTV; International Committee on Taxonomy of Virus

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Disease Spectrum Overview

1. Jin et al, Viruses 20202. Lu et al, NEJM 20203. Cascella et al, Features, Evaluation and Treatment Coronavirus (COVID-19). StatPearls Publishing, Treasure Island, FL; 2020.4. Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-195. NIH COVID-19 Treatment Guidelines https://www.covid19treatmentguidelines.nih.gov/overview/

§ Individuals of all ages are at risk for infection and severe disease. However, the probability of fatal disease is highest inpeople aged ≥ 65 years and those living in a nursing home or long-term care facility. Other high risks population are those with underlying conditions including:5

‒ Hypertension‒ Cardiovascular disease‒ Diabetes‒ Chronic respiratory disease

Asymptomatic Mild-Moderate Severe Critical

Can be as high as

15.8% in children1, 2

Mild: mild fever, cough, muscle pain, nasal congestion and sore throat3 Severe

pneumonia with severe dyspnea,

hypoxia3,4

Respiratory failure,

septic shock, organ failure3,4

Moderate: Respiratory signs and symptoms such as cough and moderate dyspnea consistent with

moderate pneumonia3

‒ Cancer‒ Renal disease‒ Obesity

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0.2 0.2 0.2 0.4 1.33.6

8

14.8

0369

1215

0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 >80

Cas

e fa

talit

y, %

Case Fatality Rate By Age, Gender, and Comorbidities

*As of April 21, 2020, Please refer to the WHO COVID-19 Situation report for up to date information.1. China CDC Weekly Feb 17, 2020 http://weekly.chinacdc.cn/en/article/id/e53946e2-c6c4-41e9-9a9b-fea8db1a8f512. WHO COVID-19 situation report Apr 21st, 2020 https://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/

The global WHO case fatality rate for COVID-19 infected patients is estimated to be 6.8%*2 and disproportionally affects those of older age, males, and those with comorbidities

2.81.7

012345

Males Females

Cas

e fa

talit

y, %

10.57.3 6.3 6 5.6

0.90369

1215

Cardiovasculardisease

Diabetes Chronicrespiratory

disease

Hypertension Cancer None

Cas

e fa

talit

y, %

By Co-Morbid Condition

By Gender

49

0

20

40

60

Critical

Cas

e fa

talit

y, %

For Critical Cases

By AgeCase fatality rate based reports from China1:

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Regional Hospitalization, ICU Admission and Case Fatality Rates

China1, 4, 7, 9 US2, 3, 10 Europe4 Italy5 Germany8

Hospitalization, % 10.6 20.7-31.4 32 -- 17ICU Admission, %

26-32 4.9- 11.5*2.4

(ICU or respiratory support in 16 countries)

9 --

ICU Case Fatality (CF) or Mortality Rate (MR), %

49 (CF) 50 (CF) 11(Hospitalization MR in 21

Countries)26 (MR) 30 (MR)

Overall Case Fatality rate, %** 6 5.5 4.7 9 13.3 3.2

Hospitalization, ICU admission/mortality and case fatality rates vary by region, however, ICU case-fatality and mortality rates are generally high

Up-to-date Global COVID-19 Situation Available athttps://www.who.int/emergencies/diseases/novel-coronavirus-2019/situation-reports/

5. Grasselli JAMA April 6 doi:10.1001/jama.2020.53946. WHO COVID-19 situation report Apr 21st, 2020 https://www.who.int/emergencies/diseases/novel-

coronavirus-2019/situation-reports/7. CDC https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical-guidance-management-patients.html8. Germany COVID-19 Daily Situation Report of the Robert Koch Institute April 21, 20209. Wang et al, JAMA Feb 202010.Artenz et al JAMA 2020 DOI:10.1001/jama.2020.4326.

* Lower bound of range = among total; Upper bound of range with known status out of 2,449*** For Global up to date information on Case Fatality please refer to WHO

1. Jin et al. Viruses 2020 doi:10.3390/v120403722. CDC MMWR March 27, 2020/ 69 (12):343-346 https://www.cdc.gov/mmwr/volumes/69/wr/mm6912e2.htm3. Bhatraju PK. NEJM March 30 2020 DOI: 10.1056/NEJMoa20045004. European CDC Disease background of COVID-19 https://www.ecdc.europa.eu/en/2019-ncov-background-disease

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* Data Through April 20, 2020 for 32 states and Washington DC. Please refer to the APM Research Lab for updates

COVID-19 Racial Disparities Observed Among African American

Viz The Color of Coronavirus APM Research Lab https://www.apmresearchlab.org/covid/deaths-by-race

African Americans account for 11 % of total population but African Americans account for 28 % of deaths from and disproportionately impacted by COVID-19

COVID-19 Impact on African Americans as of April 20, 2020

Race/Ethnicity*1 Overall, N (%) Mortality per 100,000

All Americans 26,233 13.5

Black 7,326 (28) 23.1

Latino 3,969 (15) 9.3

White 12,680 (49) 8.6

Asians 1,147 (4) 7.9

Indigenous 83 (N/A) N/A

Other 1,028 (4) N/A

States represented

% of POPULATION % of DEATHS

44

11

14

27

6

6

32

14

31

38

15

29

14

11

79

45

45

57

36

35

6141

58

63

38

44

26

28

District of Columbia (83 deaths)

Missouri (67)

Michigan (987)

South Carolina (60)

Wisconsin (81)

Kansas (31)

Louisiana (747)

Illinois ((528)

Georgia (392)

Mississipi (107)

Arkansas (15)

Maryland (224)

New York State (2,467)

All Known Deaths (7,326)

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7. Ferguson et al, Imperial College COVID-19 Response Team, Mar 20208. Coburn et al. BMC Medicine 20099. Eur. J. Immunol. 2020. 50: 308–31610. Sanche et al, Emerging Infectious Disease 2020

Transmission Dynamics

SARS-CoV-2Incubation period1,2 • Around 5 days (range 1-14 days) Transmission routes1, 3-6 • Human-to-human through respiratory droplets (e.g coughing or sneezing)

• Transmission can still occur during asymptomatic stage• Virus particles can be detected in upper and lower respiratory tracts, as well as

fecal samples, however the infectivity is unknown• Aerosol and fomite transmission is plausible (virus can remain viable and

infectious in aerosols for hours and on surfaces up to days)• SARS-CoV-2 remained viable in aerosols for duration of the experiment (3hrs)• More stable on plastic and stainless steel than on copper and cardboard, and

viable virus was detectable ≤72 hours• Suppression requires social distancing in combination with other

interventions

1. Li et al, NEJM Jan 20202. WHO FAQ https://www.who.int/news-room/q-a-detail/q-a-coronaviruses 3. Cascella et al, https://www.ncbi.nlm.nih.gov/books/NBK554776/4. van Doremalen et al, NEJM Mar 20205. Rothe et al, NEJM, Mar 20206. Wu et al, Lancet Gastroenterol Hepatol, Mar 2020

SARS-CoV-2 is highly transmissible and each person can transmits the infection on an average 2.2-5.7 additional individuals (RO=2.2-5.7)1, 10 Virus1, 8, 9, 10 R0

SARS-CoV-2 2.2-5.7SARS-CoV 2-4MERS-CoV 1H1N1 1.4-1.6

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Four Phases of Epidemics and Response Interventions

Epidemic Phases*

Localized Transmission

Introduction or Emergence Amplification Reduced

Transmission

Response Interventions

Anticipation EarlyDetection Containment Control and

MitigationElimination or

eradication

*Not all epidemic disease necessarily go through each phaseWorld Health Organization. Managing Epidemics: Key facts about major deadly disease https://www.who.int/emergencies/diseases/managing-epidemics-interactive.pdf

Understanding the phases of epidemics and implementing response interventions can help to prevent the spread of infection and mitigate the risk of overwhelming healthcare systems

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Use of Diagnostic Testing to Assist with Management of the COVID-19 Pandemic

Cheng et al, Annals of Internal Medicine 2020 doi:10.7326/M20-1301

§ Rapid implementation of diagnostic nucleic acid amplification testing aids in case isolation and contact tracing and likely contributed to the success of a few countries controlling transmission

§ Detection of host-derived antibodies directed against SARS–CoV-2 will be crucial for surveillance, epidemic forecasting, and determination of SARS–CoV-2 immunity

Screening symptomatic patients

De-isolation Monitoring of shedding

DiagnosisScreening asymptomatic patients

Treatment initiation decisions

Monitoring contacts

Active/passive surveillance

Incubation ConvalescenceSymptomatic Illness

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Strategies Proposed After Lockdown / Shelter in Place is Lifted

Wuhan CDC

When Near zero cases in past weeks1Downward trend within 14 days, hospitals to treat all

patients without crisis care, and testing program in place for at-risk healthcare workers4

Testing Mandatory testing before returning to work (RNA and antibody)2

Ability to set up screening/testing sites for symptomatic individuals and trace positive contacts4

Protective Masks Always3

Employers to develop policies on protective equipment4

CDC recommends wearing face coverings in public settings5

Social Distancing Risk based on app code3 Yes. Avoid groups of more than 10 people4

Building Cleaning Required disinfection2 Employers to develop policies on disinfection4

Software Apps / Health Surveys

Require “green code” on app to prove good health in order to leave (risk based social distancing)3 None

1. https://www.cnn.com/2020/04/07/asia/coronavirus-wuhan-lockdown-lifted-intl-hnk/index.html. 2. https://www.bloomberg.com/news/articles/2020-04-09/wuhan-workers-line-up-for-mass-testing-after-lockdown-lifted3. https://www.businessinsider.com/coronavirus-china-health-software-color-coded-how-it-works-2020-4. 4. https://www.whitehouse.gov/openingamerica/5. https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/diy-cloth-face-coverings.html. 6. https://www.cnn.com/2020/04/20/health/measures-coronavirus-unusual-restrictions-intl/index.html

Methods in Other Regions6

Classrooms 2.0, immunity cards/passports, weekend-only lockdowns, stay-at-home orders for vulnerable, gender-based lockdowns, delivery or pick-up only at restaurants, limited hours at banks

As regions lift shelter in place policies, various strategies will need to be implemented to limit transmission

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Epidemiology Summary

§ Cases of pneumonia first appeared in Dec 2019 in China. With rapid global spread, the WHO declared COVID-19 a pandemic in March 2020. By April 2020 there were over 2 million cases

§ While the majority of the infected people will have mild-moderate disease, asymptomatic people and those in the incubation phase may still shed and spread SARS-CoV-2 infection

§ Progression to severe disease or death disproportionately impacts older adults, people with underlying health conditions, and males.

§ There are regional differences in death rates, but generally mortality rates are high. In the US, African Americans have disproportionately higher mortality rates compared to other ethnicities

§ SARS-CoV-2 is highly transmissible, with a RO of 2.2-5.7§ Infection control starts with anticipation and preparedness, early detection, and implementation of

interventions to decrease spread§ Strategies of testing and tracking infected individuals are needed to lift shelter-in-place policies

after spreading subsides

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Virology

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Nomenclature and History of 3 Zoonotic Coronavirus Outbreaks

1. Gorbalenya et al. Nat Microbiol. 2020 Mar 2. doi: 10.1038/s41564-020-0695-z.. 2. Lu R, et al. Lancet. 2020 Feb 22;395(10224):565-574. doi: 10.1016/S0140-6736(20)30251-8. Epub 2020 Jan 30. 3. Scripps Research Institute March 17, 2020 https://www.sciencedaily.com/releases/2020/03/200317175442.htm 4. Li et al, NEJM Jan 20205. Coburn et al. BMC Medicine 20096. Mahase BMJ 2020 368:m641 doi: 10.1136/bmj.m641.

7. Wu et al Jama 2020 Feb 24. 8. Sanche et al, Emerging Infectious Disease 20209. Zhu et al, J of Med Virology 202010. WHO COVID-19 situation report Apr 21st, 2020 https://www.who.int/emergencies/diseases/novel-coronavirus-

2019/situation-reports

Nam

ing

auth

ority ICTV-CSG

WHO

Virus species1Middle East respiratory

syndrome-related coronavirus

Severe acute respiratory syndrome-related coronavirus

Virus1 MERS-CoV SARS-CoV SARS-CoV-2

Disease1 Middle East respiratory syndrome (MERS)

Severe acute respiratory syndrome (SARS)

Coronavirus disease 2019 (COVID-19)

Year2 2012 2003 2019

Probable Animal Source2,3 Camel Bats Bats

R04,5 1 2-4 2.2-5.7

Outcome2 2494 patients858 deaths

>8000 patients774 deaths Ongoing

Case Fatality Rate6,7 34% 10% 6.8%*10

SAR-CoV-2 is the third documented spillover of an animal coronavirus to humans in 2 decades that has resulted in a major epidemic

* As of April 21, 2020, Please refer to the WHO COVID-19 Situation report for up to date information.

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Coronaviruses (CoVs) and SARS-CoV-2 Structure

§ Family Coronaviridae are enveloped RNA viruses with positive-sense, single-stranded RNA genomes, 26 to 32 kilobases in length

§ CoVs are divided into four subfamilies: α, β, γ, δ§ CoVs can cause respiratory, enteric, hepatic, and

neurologic diseases and have been identified in various mammal hosts– Two human coronaviruses (HCoV-229E, HCoV-

OC43) generally cause mild disease (common cold)– Three zoonotic β-CoVs caused severe respiratory

diseases (SARS-CoV, MERS-CoV, SARS-CoV-2)– SARS-CoV-2 shares 79.5% RNA sequence identity

and 94.6% ORF1ab conserved replicase domains sequence identity with SARS-CoV

1. Gorbalenya et al. Nat Microbiol. 2020 Mar 2. doi: 10.1038/s41564-020-0695-z.. 2. Lu R, et al. Lancet. 2020 Feb 22;395(10224):565-574. doi: 10.1016/S0140-6736(20)30251-8. Epub 2020 Jan 30

SARS-CoV-2 is an enveloped RNA virus β-coronavirus most similar to SARS-CoV

SARS-CoV2 structure

3. Graham et al, Nature Reviews Microbiology 20134. Jin Y et al. Viruses. 2020 March 7

Adapted from Jin Y et al. Viruses. 2020 March 7

Spike (S)

Envelope (E)

Nucleocapsid (N)

Membrane (M)

Genome RNA

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SARS-CoV Replication and Life Cycle

Liu W, et al. Chembiochem. 2020 Mar 2;21(5):730-738. doi: 10.1002/cbic.202000047. Epub 2020 Feb 25.de Wit et al, Nature Reviews Microbiology 2016

.

ACE2, the cell’s receptor

Spike

Nucleus

Golgi

Type II Pneumocyte

New Virion Assembly

Virus Spike protein attaches to ACE2 on cell

surface as an entry receptor

Attachment

Entry and Uncoating

Virus enters the cells through acid-dependent membrane fusion

RdRp(+)(+)(+)

New viral genome

RNA Replication

Viral genome replication by RdRp

(+)

Release of Virus

Mature virions form at host ER/Golgi compartment

Ribosomes

RdRp

PLpro & 3CLpro

Proteases

TranslationViral translation with host ribosomes into polyproteins

(+)

ProteolysisPLpro and 3CLprocleave polyproteins into 16 non-structural and structural proteins

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Key Proteins in SARS-CoV-2 Life Cycle

Production of New Virus ParticlesReplication of RNA genome

Spike protein and Angiotensin-Converting Enzyme 2 (ACE2)

RNA-dependent RNA polymerase (RdRp) Proteinase (3CLpro) and Papain-like protease (PLpro)

Entry

Potential vaccine and drug targets include the spike protein, ACE2 receptor, RdRp, 3CLpro and Plpro*

Liu W, et al. Chembiochem. 2020 Mar 2;21(5):730-738. doi: 10.1002/cbic.202000047. Epub 2020 Feb 25.Savarino et al, Lancet Infectious Disease 2006

Ribosomes

RdRp

PLpro & 3CLpro

(+)ssRNAprotein

biosythesis Proteases

RdRp(+)(+)(+)

New viral genome

Spike protein

ACE2 Receptor

Proteins

*This is not a comprehensive list, these are some of the most obvious targets

Potential therapy:

QuinolineAntimalarial

Potential therapy:RdRp

Inhibitor

Potential therapy:

Protease Inhibitor

For internal use only

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Potential Organ Vulnerability to SARS-CoV-2 based on ACE2 Receptor Distribution

§ ACE2 is a type I membrane protein expressed in lung, heart, kidney, and intestine mainly associated with cardiovascular diseases1,2,4

– Primary function is cleavage of angiotensin (Ang) I to produce Ang-(1-9) 4

§ SARS-CoV-2 S protein binds to ACE2 receptor for entry into host cell similar to SARS-CoV1,2

§ SARS-CoV-2 S protein receptor-binding domain (RBD) binds to human ACE2 with 10–20x higher affinity than SARS-CoV 3, 4

*This analysis may not have exhausted all 2019-nCoV vulnerable organs1. Li et al, Nature 20032. Zhou P, et al. Nature. 2020 Feb 3. doi: 10.1038/s41586-020-2012-73. Zou X, et al. Front Med. 2020:Mar 12. doi: 10.1007/s11684-020-0754-0.4. Jin Y et al. Viruses. 2020 March 7

Risk Map Showing Vulnerability of Different Organs to SARS-CoV-2 Infection* 3

Affinity of SARS-CoV-2 to ACE2 receptors and distribution of these host receptors may

potentially have a role in organ vulnerability

High riskLow risk

Zou X, et al. Front Med. 2020:Mar 12.

Respiratory Tract

Esophagus

Heart

Kidney

Heart

Lung

Ileum

Bladder

Liver

Nasal mucosa

Bronchus

Stomach

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CoV Genome Replication

§ CoV replicate their genome using RNA-dependent RNA polymerase (RdRp/Nsp12)§ CoV encode a 3’→ 5’ Exoribonuclease (ExoN) which is rare for RNA viruses

– Conveys proofreading activity in nsp14 to detect and repair inappropriately incorporated or damaged bases– Allows for high fidelity (or high accuracy) with which genetic information is copied

CoV genome replication has high accuracy with potential for minimizing mutationsProcessivity factor

Polymerase core

Nsp8

Nsp7

Nsp12(RdRp)

Nsp14(ExoN and

N7-MT))Nsp10

Nsp9(ssRBP)

Nsp13(Hel/NTPase)

5’3’

3’5’

Transcription

Smith EC, et al. Annu Rev Virol. 2014;1:111–132.Minskaia et al, PNAS Feb 2006

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Virology Summary

§ SARS-CoV-2 is member of β-coronavirus and is most similar to SARS-CoV. SARS-CoV-2 is the 3rd zoonotic coronavirus to cause an epidemic outbreak in humans

§ Steps in SARS-CoV-2 lifecycle such as ACE2 receptor attachment, RNA replication by RNA-dependent RNA polymerase (RdRp), and proteolysis are potential antiviral targets

§ The binding of the SARS-CoV-2 spike protein to the ACE2 receptors and distribution of these host receptors may potentially have a role in organ vulnerability

§ Coronavirus, including SARS-CoV-2, replicate their genome using RdRp

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Disease Course

Clinical course, Management, Screening and Diagnosis

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0 10 20 30 40 50 60 70 80 90 100

COVID-19 Clinical Presentation

%

Sym

ptom

s

Fever 87.9 %Dry Cough 67.7 %

Fatigue 38.1%

Sputum Production 33.4%

Dyspnea 18.6%

Myalgia /Arthralgia 14.8%

Sore Throat 13.9%Headache 13.6%

Nausea/Vomiting 5%

Chills 11.4%

Nasal Congestion 4.9%

Diarrhea 3.7%

COVID-19 symptoms are non-specific with the most common presentation being fever, dry cough, fatigue, and sputum production

Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19) https://www.who.int/docs/default-source/coronaviruse/who-china-joint-mission-on-covid-19-final-report.pdf

Based on 55,942 laboratory confirmed cases of COVID-19 from China as of February 2020

Severe DyspneaHypoxia

Dehydration

MODERATE

MILD

Low grade feverDry cough

Myalgias and arthralgiasNasal congestion

Sore throatHeadache

High grade feverShortness of breath/supplement O2

ChillsProfound fatigue

Severe dyspneaHypoxia

Dehydration

Mild Moderate Severe/Critical

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Potential Progression of COVID-19

1. Goh et al, Annals, Academy of Medicine, Singapore 2020)2. Huang et al, Lancet 2020 3. Wang et al, JAMA Feb 2020

4. Guan et al. NEJM 20205. CDC https://www.cdc.gov/coronavirus/2019-ncov/hcp/clinical-guidance-management-patients.html6. Liu et al Journal of Infection Aprul 17, 2020 https://doi.org/10.1016/j.jinf.2020.04.008

COVID-19 patients may deteriorate one week after illness onset. Data from hospitalized patients in China, show the median hospital stay length exceeded 20 days and over 25% required ICU care

Number of Days*

**

Potential progression of COVID-19: 1-3, 5

• General timeline based on case reports from China

ARDS, Acute respiratory distress syndrome*Median time from onset of symptoms, including fever (in 98% of patients),cough (76%), myalgia or fatigue (44%), and others** ARDS and critical illness appear to develop most commonly between 1–2 weeks after the onset of symptoms

Median length hospital stay (N=99): 22 (9-46) Days for non-severe pneumonia and 25 (14-44) Days for severe pneumonia6

• Use of glucocorticoids prolonged length of hospital stay (p= 0.002)6

Among hospitalized patients, 26-32% require ICU care and develop respiratory failure3,5

Among ICU patients, ARDS is observed in 67-85%5 and septic shock observed in 13.4%4

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Radiographic Abnormalities in COVID-19

Guan W, et al. N Engl J Med. 2020 Feb 28. doi: 10.1056/NEJMoa2002032. [Epub ahead of print].Goh et al, Annals, Academy of Medicine, Singapore 2020

Non-severe (n=926) Severe (n=173)

Radiologic findings, %

Ground-glass opacity 17 30

Local patchy shadowing 26 35

Bilateral patchy shadowing 30 58

Interstitial abnormalities 3 8

Abnormalities on chest CT, %

Ground-glass opacity 56 61

Local patchy shadowing 39 55

Bilateral patchy shadowing 46 82

Interstitial abnormalities 12 26

Analysis of 1099 patients with 2019 SARS-CoV-2 reported to the National Health Commission of China between December 11, 2019 and January 29, 2020

Radiographical abnormalities may be present in patients with non-severe and severe COVID-19

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Immune and Inflammatory Lab Abnormalities in COVID-19

Prompetchara E, et al. Asian Pac J Allergy Immunol DOI 10.12932/AP-200220-0772. Wang W, et al J of Inf Dis 2020, https://doi.org/10.1093/infdis/jiaa150Qin C, et al. Clin Infect Dis. 2020 Mar 12. pii: ciaa248. doi: 10.1093/cid/ciaa248. [Epub ahead of print]Zhang B, et al. https://www.medrxiv.org/content/10.1101/2020.02.26.20028191v1.full.pdf. Guan W, et al. N Engl J Med. 2020 Feb 28. doi: 10.1056/NEJMoa2002032. [Epub ahead of print].

Case reports of patients from Wuhan, China with severe COVID-19 have been observed to have dysregulation of immune response

Common immune/inflammatory responses include ↑ neutrophils, ↓ lymphocytes, ↑ IL-6, ↑ CRPLiver transaminases (AST, ALT) are also commonly elevated in hospitalized patients (~20%)

Prompetchara et alN=99

Qin et alN=452

Wang et alN=60

Zhang et alN=82

Neutrophils ↑ ↑ na ↑Lymphocytes ↓ ↓ ↓ ↓

IL-6 ↑ ↑ ↑ ↑CRP ↑ ↑ ↑ ↑

Others↑ na

leukocytes, ESR procalcitonin, IL-2R, serum

ferritin, TNF-αESR

NLR, SII index of >500, LDH, D-dimer, cardiac troponin I, procalcitonin

↓ na monocytes, T cells, eosinophils, basophils T cells, B cells, NK cells T-cells

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Immune and Inflammatory Response to COVID-19 - Rationale

1. Prompetchara E, et al. Asian Pac J Allergy Immunol. DOI 10.12932/AP-200220-0772.2. Fu Y, et al. Virol Sin. 2020 Mar 3. doi: 10.1007/s12250-020-00207-4. [Epub ahead of print]

Aerosolized uptake of SARS-CoV-2

Infection of ACE2 expressing target cells, such as alveolar type 2 cells

Virus dampens anti-viral IFN responses

Uncontrolled viral replication

Influx of neutrophils and monocytes/macrophages

Hyperproduction of pro-inflammatory cytokines

Inflammatory response and possible “cytokine storm”

Proposed Host Immune Responses During SARS-CoV-2 Infection1

SARS-CoV-2

Alveoli

ACE2

?Type I IFN

Neutrophils

Pro-inflammatoryCytokines

Monocytes/MacrophagesAlveolarEpitheliumType II andother cells?

Th1/Th17

Plasma Cells

B cells

Major Viral Host Interaction• Delayed or suppressed Type I IFN response during initial infection• Viral replication triggers hyperinflammatory conditions• Influx of activated neutrophils and inflammatory monocytes/macrophages• Th1/Th17 is induced and specific antibodies are produced

Pneumonia Lung

Emerging Hypothesis2• Antibody-dependent enhancement (ADE) may be occurring which can

cause severe lung injury• Antiviral neutralizing antibodies cannot completely neutralize the virus.

Instead, the virus-NAb complex attaches to the Fc receptor, leading to viral endocytosis and infection of the target cells

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ARDS is an Inflammatory Mediated Response

Baric, CROI 2020 Wang D, et al. JAMA. Published online February 7, 2020.Corrected on February 20, 2020. doi:10.1001/jama.2020.1585

Mortelliti MP, et al. Am Fam Physic. 2002;65(9):1823-1830.

Direct lung injury or systemic insult (endotoxin)

↑ proinflammatory cytokines (pulmonary or circulatory)

↑ activated neutrophils(capillary → interstitial → alveolar spaces)

↑ Permeability and the subsequent influx of protein-rich fluid into the alveolar space

↑ Interleukins

↑ TNF-α ↑ Oxygen radicals and proteases

↑ Neutrophil arachidonic acid metabolites

The exact sequence of events in ARDS is unknown

From Ware LB, et al. N Engl J Med. 2000;342(18):1334-49Reprinted with permission from Massachusetts Medical Society.

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Coagulopathies

The development of coagulopathy is the most significant poor prognostic feature of COVID-191

Mechanism is unclear, but may be related to cytokine storm1

1. Thachil J, et al. J Thromb Haemost. 2020. Accepted Author Manuscript. doi:10.1111/jth.148102. Guan W, et al. N Engl J Med. 2020 Feb 28. doi: 10.1056/NEJMoa2002032. [Epub ahead of print]. 3. Tang N, et al. J Thromb Haemost. 2020 Mar 27. doi: 10.1111/jth.14817. [Epub ahead of print].

Patients with COVID-19 may be at increased risk of coagulopathies

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58 60

0

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Platelets <150,000 permm³

D-dimer ≥0.5 mg/L

%

Nonsevere disease Severe disease

More Severe Disease in Patients with Low Platelets and Elevated D-dimer2

Odds Ratio (95% CI) P-value

Prothrombin time 1.107 (1.008-1.215) 0.033

D-dimer 1.058 (1.028-1.090) <0.001

Age 1.033 (1.013-1.055) 0.002

Platelet count 0.996 (0.993-0.998) 0.001

Multivariate Correlative Factors of 28-day Mortality in Severe COVID-193

• International Society on Thrombosis and Hemostasis recommends measuring D-dimers, prothrombin time, and platelet count (decreasing order of importance) in all patients who present with COVID-19 infection1

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Potential Long-Term Sequelae for ICU Survivors

Survivors of COVID-19 who spent time on a ventilator may be at risk of long-term disability and illness. 1

§ Muscle Atrophy and Weakness1,3

– Access to rehabilitation services has been limited due to infection transmission concerns

§ Psychological Symptoms3

– Survivors may experience psychological symptoms associated with long hospital stays and isolation from social distancing (ICU delirium, PTSD, Alzheimer's like cognitive deficits, depression, lost jobs, and problems with daily activities)

§ Potential for Reduced Lung Function1,2

§ There are early reports of recovered patients experiencing reduced lung function (↓ 20-30%) and shortness of breath with exertion– Long-term follow-up is needed to understand the prevalence and duration of such changes and if chronic

lung pathology occurs after pneumonia and ARDS due to COVID-19

1. Servick K. Science Magazine (AAAS). April 8 2020. doi:10.1126/science.abc14862. SouthChina Morning Post. https://www.scmp.com/news/hong-kong/health-environment/article/3074988/coronavirus-some-recovered-patients-may-have3. https://www.washingtonpost.com/health/2020/04/03/coronavirus-survivors-recovery/

Further research and follow-up is needed to understand the long-term outcome in patients with COVID-19

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Disease Course Summary

§ Fever and cough are the most common symptoms in COVID-19 patients, while other nonspecific symptoms may also be present

§ Severe complications, which are seen in ~ 20% of the patients, generally start 7 to 10 days after the symptom onset, and over 25% of hospitalized patients may require ICU care

§ Change in immune cells and cytokine level is commonly observed in COVID-19 patients. Cytokine storm is hypothesized to lead to critical outcomes including ARDS

§ The development of coagulopathy may be the most significant poor prognostic feature of COVID-19

§ Survivors may have potential long-term sequelae. Further research is needed

For proactive use

Diagnostic Testing

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COVID-19 Testing Overview

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• Nucleic acid amplification tests (NAAT) for detection of unique RNA sequences, most commonly N, E, S and RdRP protein genes

• Reverse Transcription-PCR(RT-PCR)*• Isothermal Amplification

• Serological antibody testing• ELISA • Rapid Diagnostic Tests

• Rapid antigen test (TBD)

Testing Methods

WHO Laboratory testing for 2019 novel coronavirus https://www.who.int/publications-detail/laboratory-testing-for-2019-novel-coronavirus-in-suspected-human-cases-20200117

• Upper Respiratory tract• Nasal swab• Oropharyngeal swab or aspirate

• Lower Respiratory tract• Sputum• Endotracheal Aspirate• Bronchoalveolar Lavage

• Other: Stool, whole blood, urine, autopsy

Specimen Collection Sites

*WHO Recommended

Results may vary depending on assay used, specimen collection site, specimen quality, timing in illness, mutationsWHO: Additional research is needed to understand• Optimal timing and type of clinical material to sample• Relationship between viral concentration and disease severity• Duration of viral shedding, and relation to clinical picture (e.g. shedding persists despite clinical improvement)

Limitations of Testing

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Viral Load TestingViral RNA Levels Vary by Specimen Location2,3

1. Wang W, et al. JAMA. 2020 Mar 11. doi: 10.1001/jama.2020.3786. [Epub ahead of print]. 2. Huang Y, et al. Am J Respir Crit Care Med. Published April 15, 2020 as 10.1164/rccm.202003-0572LE. 3. Zou L, et al. N Engl J Med. 2020 Mar 19;382(12):1177-1179. doi: 10.1056/NEJMc2001737. Epub 2020 Feb 19.

Higher viral loads detected in sputum, compared to nose and throat2

Asymptomatic patients can still have positive swab3

Detection Results by Specimen1

93

7263

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Bronchoalveolarlavage fluid

Sputum Nasal swab Fibro-bronchoscopebrush biopsy

Pharyngeal swab Feces Blood

Posi

tive

test

resu

lt, %

Specimens from multiple sites of 205 patients with COVID-19 showed lower respiratory tract

samples most often testing positive1

Sputum/ETA Nasal swab Throat swab

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25

30

35

40

**

Ct V

alue

Viral detection rates are higher in the lower respiratory tract compared to the upper respiratory tract

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Duration of Viral Shedding

Higher viral load detected early after symptom onset, with peak concentrations before Day 102

Viral load levels can remain elevated beyond 28 days after onset of symptoms (69% patients with >28 days)3

1. Tan W, et al. medRxiv 2020.03.24.20042382; doi: https://doi.org/10.1101/2020.03.24.20042382.2. Zou L, et al. N Engl J Med. 2020 Mar 19;382(12):1177-1179. doi: 10.1056/NEJMc2001737. Epub 2020 Feb 19. 3. Huang Y, et al. Am J Respir Crit Care Med. Published April 15, 2020 as 10.1164/rccm.202003-0572LE.

Median Duration of RNA Shedding1

12

19 18

0

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NP Sputum Stool

Day

s

Median duration of viral shedding varies by location and is >30 days in 21% of patients1

Timing of Viral Shedding2,3

Viral shedding duration varies by collection site and viral load detection may continue for > 28 days in a substantial portion of patients

0 7 14 21 28 35 42 48 56Days Since Onset of Symptoms

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alue

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Aggregated Ct Values Sputum/ETANasal swabThroat swab

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Viral Load and Disease Severity

Higher initial and peak viral load in patients with severe disease, but did not reach significance

1. Liu Y, et al. Lancet Infect Dis. 2020 Published Online March 19, 2020. https://doi.org/10.1016/S1473-3099(20)30232-2.2. Wang To KK, et al. Lancet Infect Dis. 2020 Mar 23. pii: S1473-3099(20)30196-1. doi: 10.1016/S1473-3099(20)30196-1. [Epub ahead of print].

Initial and Peak Viral Load According to Severity of Disease2

5.11 5.296.17

6.91

0

5

10

Initial Viral Load Peak Viral Load

Log 1

0co

pies

/mL

Mild disease (n=13) Severe disease (n=10)

Viral Dynamics in Patients with Mild and Severe Disease1

Patients with severe COVID-19 tend to have a high viral load and a long virus-shedding period

p=0.56 p=0.52

Additional research is needed to understand the relationship between viral load and disease severity

Mild, n=21Severe, n=10

-10

-5

0

5

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25

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ΔCT

Day of onset

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Recurrence vs. Prolonged Nucleic Acid Conversion

N Two negative RT-PCR tests results after initial illness

Timing of newly positive RT-PCR test Clinical symptoms

Lan1 4 Yes 5 - 13 days • All asymptomatic

Jiang2 6 Yes 8 - 15 days• 1 significant clinical symptoms• 1 mild cough• 4 asymptomatic

Chen3 1 Yes 3 days • Asymptomatic

1. Lan L, et al. JAMA. 2020 Feb 27. doi: 10.1001/jama.2020.2783. [Epub ahead of print]. 2. Jiang DM, et al. J Infect. 2020 Apr 11. pii: S0163-4453(20)30152-3. doi: 10.1016/j.jinf.2020.03.024. [Epub ahead of print].3. Chen D, et al. Int J Infect Dis. 2020 Mar 5;93:297-299. doi: 10.1016/j.ijid.2020.03.003. [Epub ahead of print]. 4. Ling Z, et al. http://rs.yiigle.com/yufabiao/1183297.htm. 5. CDC. https://www.cdc.gov/coronavirus/2019-ncov/hcp/faq.html.

China Experience4

• Recommends continued isolation and observation for 2 weeks after discharge

• Recommends close observation to prevent secondary infection

CDC5

• The CDC does not yet know if people who recover from COVID-19 can be re-infected

• Patients with MERS-CoV are unlikely to be re-infected shortly after they recover

Recommendations to Prevent Reinfection

Positive RT-PCR after recovery may be related to prolonged nucleic acid conversion rather than reinfection

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Serologic Antibody Testing

1. Yuen K-S, et al. Cell & Bioscience. 2020: 10(40). https://cellandbioscience.biomedcentral.com/articles/10.1186/s13578-020-00404-4. 2.Guo L, et al. Clin Infect Dis. 2020 Mar 21. pii: ciaa310. doi: 10.1093/cid/ciaa310. [Epub ahead of print]. 3. Xiang F, et al. Clinical Infectious Diseases. 2020 Apr 19. https://doi.org/10.1093/cid/ciaa461. 4. Zhao J, et al. Clin Infect Dis. 2020 Mar 28. pii: ciaa344. doi: 10.1093/cid/ciaa344. [Epub ahead of print]

§ Serologic antibody testing can be used during an outbreak and retrospective assessment

– During an outbreak antibody testing can be combined with PCR to increase detection rate

§ Serologic antibody tests detect IgM and IgG antibodies against SARS-CoV-2 proteins

§ Time to seroconversion1-4

– IgM and IgA: 3-24 days– IgG: 10-24 days

§ Sensitivity of Ab testing is higher than RNA test after Day 84

§ Two forms of serologic antibody testing: ELISA (quantitative), Rapid Antibody Tests (qualitative)

Positive Detection Rate of Cases Using PCR Method vs PCR plus IgM ELISA assay2

52

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020406080

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PCR PCR+IgM ELISA

Posi

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dete

ctio

n, %

Serologic assays can be used for surveillance during an outbreak and for retrospective epidemiologic studies

6754 46

19

54

80

29

7394

020406080

100

Days 1-7 Days 8-14 Days 15-39

Sen

sitiv

ity, %

RNA IgG IgM

Performance of RNA and Antibody Tests Over Time4

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Current Limitations of Antibody Testing

1. https://www.fda.gov/medical-devices/letters-health-care-providers/important-information-use-serological-antibody-tests-covid-19-letter-health-care-providers2. http://s2027422842.t.en25.com/e/es?s=2027422842&e=324732&elqTrackId=376c7bc788024cd5a73d955f2e3dcbdc&elq=7d247b3948774291aa3d15f7fe64afbf&elqaid=12176&elqat=1

§ Some firms are falsely claiming that their serological tests are FDA approved or authorized, or falsely claiming that these tests can diagnose COVID-191

§ FDA is not aware of an antibody test that has been validated for diagnosis of SARS-CoV-2 infection2

RECOMMENDATION: The FDA recommends health care providers:

§ Continue to use serological (antibody) tests, as appropriate, and be aware of their limitations2

§ Do not use serological (antibody) tests as the sole basis to diagnose COVID-19 but instead as information about whether a person may have been exposed2

§ Be aware that not all marketed serological tests have been evaluated by the FDA2

COVID-19 immunity is not yet fully understood and currently FDA authorized serological testing is only available under the Emergency Use Authorization (EUA) program. Providers should refer to the FDA EUA

page for more information

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COVID-19 Diagnostic Testing Summary

§ RT-PCR to detect virus nucleic acid is the current standard testing for COVID-19 diagnosis§ Viral detection rates are higher in the lower respiratory tract compared to the upper respiratory

tract§ Viral load normally peaks before Day 10 of disease onset; and can remain elevated >28 days§ Viral shedding duration varies by collection site and can be prolonged§ Additional research is needed to determine the relationship between viral load and disease

severity§ Positive PCR test can occur days to weeks after two negative RT-PCR tests in some patients.

Further research needed to understand recurrence vs prolonged nucleic acid conversion§ Serologic assays can be used for surveillance during an outbreak and for retrospective

epidemiologic studies. Median time to seroconversion can vary, 3-24 days days for IgM and 10-24 days for IgG

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Overall Summary Slide

§ Since the first cases reported in Wuhan over 2 million people globally have confirmed cases of COVID-19, with ongoing increase spread in Europe and North America

§ There may be regional differences on hospitalization and mortality rates, but ICU mortality is generally high

§ Preparedness and anticipation of infection by screening and implementing plans to stop transmission will be important for eradicating the disease

§ SARS-CoV-2 is the 3rd zoonotic coronavirus to cause an epidemic outbreak in humans. SARS-CoV-2 and SARS-CoVare highly conserved and share similar key proteins which include the spike protein, RdRp, 3CLpro and Plpro

§ Symptoms of COVID-19 can be mild, but may progress to needing hospitalization with about a quarter developing severe disease

§ There is a hyper-inflammatory response due to the infection which may contribute to progression of the disease to ARDS, a complication seen in severe COVID-19

§ Lower respiratory tract specimen may yield better detection of the virus compared to upper respiratory tract samples

§ Prolonged viral shedding and variations in viral shedding by collection site have been observed

§ Further research is needed on viral dynamics to understand the relationship between viral load and disease severity and to accurately differentiate between prolonged nucleic acid conversion and reinfection

§ Antibody testing can assist in epidemiologic assessment of an outbreak. Time to seroconversion may occur as early as 3 days, but may be delayed for as long as 24 days