COVID-19 and the Cytology Lab -1 · Respublica Ragusina (Dubrovnik, Croatia) A Decree of 1377 officially issued “quarantine”, the obligation for people and goods from ships arriving

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COVID-19 and the Cytology Lab (Do's & Don'ts)

Stefan E. Pambuccian, MDLoyola University Medical Center

COVID-19 Timeline

Pangolins, or scaly anteaters, 穿⼭甲critically endangered, most trafficked animal in the world

SARS‐CoV‐2 and Coronavirus Disease 2019:What We Know So Far. Pathogens 2020, 9, 231

̴800,000 cases in 199 countries and territories

̴40,000 Deaths

https://www.worldometers.info/coronavirus/

COVID‐19 CORONAVIRUS PANDEMIC

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Trends of new cases and deaths in the US and in Illinois

https://weather.com/coronavirus/l/41.8935,‐87.9395

Social distancing

Measures include:• Cancelling sports, music, cultural and even political events• Closing of gyms, schools and collegesRecommendations to • Work from home• Avoid discretionary travel• Avoid eating or drinking in bars, restaurants and food courts• Avoid social gatherings in groups of more than 10 people

Do your part by staying home

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Unprecedented?

The United States Public Health Service issued this pamphlet in October of 1918 as part of a public education campaign to slow the progress of the disease

Unprecedented?Good advice and misinformation

Masks

St. Louis Red Cross Motor Corps workers and ambulances waiting to receive influenza patients in 1918

Seattle, WA policemen wearing gauze face masks during the influenza of 1918

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Respublica Ragusina(Dubrovnik, Croatia)

A Decree of 1377 officially issued “quarantine”, the obligation for people and goods from ships arriving from plague‐affected areas to stay for 30 days on an uninhabited island, “to be purified by the sun and the wind”. This later became 40 days, “quarantine” from the Italian ”quarantina (giorni)”, or “space of 40 (days)”

In the Hippocratic tradition this is the turning point between acute and chronic disease, It also reflects the concept of spiritual purification (Jesus’ time in the wilderness).

Did the Quarantine Laws Work?Even with the new quarantine law, Ragusa continued to be hit hard by aftershock outbreaks of the plague in 1391 and 1397. As a maritime city that survived on trade, it would have been impossible to completely wall off Ragusa to disease without gutting the economy.

Impact of social distancing on mortality during the 1918 “Spanish” influenza pandemic

• 200,000 people took to the streets of Philadelphia; by the end of the week, 4,500 were dead

• In St. Louis, , two days from detecting its first cases, the city closed schools, playgrounds, libraries, courtrooms, and even churches. Work shifts were staggered and streetcar ridership was strictly limited. Public gatherings of more than 20 people were banned.

Flattening the curve

Mathematical model: In an unmitigated epidemic, we would predict approximately 510,000 deaths in GB and 2.2 million in the US, not accounting for the potential negative effects of health systems being overwhelmed on mortality. Neil M Ferguson, et al. (Imperial College COVID‐19 Response Team) Impact of non‐pharmaceutical interventions (NPIs) to reduce COVID19 mortality and healthcare demand

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Mathematical models are based on 1. Basic reproductive number (R0)

https://en.wikipedia.org/wiki/Basic_reproduction_number

R0, pronounced R nought or R zero is the expected number of cases directly generated by one case in a population where all individuals are susceptible to infectionR0 >1 indicate a propensity for the infection to spread, while R0

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Death rates according to age, sex and coexisting conditions

AGE DEATH RATE

of all cases in that age group

80+ years old 14.80%

70‐79 years old 8.00%

60‐69 years old 3.60%

50‐59 years old 1.30%

40‐49 years old 0.40%

30‐39 years old 0.20%

20‐29 years old 0.20%

10‐19 years old 0.20%

0‐9 years old no fatalities

PRE‐EXISTING CONDITION DEATH RATE

of all cases with that condition

Cardiovascular disease 10.50%

Diabetes 7.30%

Chronic respiratory disease 6.30%

Hypertension 6.00%

Cancer 5.60%

no pre‐existing conditions 0.90%

SEX DEATH RATE

of all cases of that sex

Male 2.80%

Female 1.70%

https://www.worldometers.info/coronavirus

Because both the R0 and the CFR are grossly overestimated, as asymptomatic and mildly symptomatic cases are not diagnosed and not included in the calculations, the mathematical models most likely vastly overestimate the magnitude of COVID‐19‐related deaths

Mode of transmission

• Respiratory, large droplet• Fomite• Possibly fecal‐oral• ?Airborne (small droplet)• No intrauterine transmission

Tellier et al. BMC Infectious Diseases (2019) 19:101

Viral entry (receptor)

Hoffmann, M et al. (2020). SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically-proven protease inhibitor. Cell,

The red staining represents the virus proteins.

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Syed Z. Ali@sza_jhcyto

Clinical manifestations of COVID-19

Symptoms: • Start with a fever, chills, sweating, malaise, muscle pain,

• Followed by a dry cough, • After a week, shortness of breath, • 20% of patients requiring hospitalization

• Rarely runny nose, sneezing, or sore throat (5%)

• Rarely gastrointestinal symptoms: nausea, vomiting, diarrhea (5%)

• Rare presenting symptoms: loss of smell and loss of taste

• COVID‐19 Incubation Period:• Mean 3‐5 days• Range 2 ‐ 14 days (outliers 0 ‐ 27

days)

• Symptoms duration• mild cases: approximately 2 weeks• severe or critical disease: 3 ‐ 6 weeks• time from onset to the development

of severe disease (including hypoxia): 1 week

• Duration of viral shedding 8 to 37 days (median 20 days)

• Among patients who have died, the time from symptom onset to fatal outcome ranges from 2 ‐ 8 weeks; viral shedding continues until death

COVID-19 Differential with influenza and common cold

https://thespinoff.co.nz/science/18‐03‐2020/siouxsie‐wiles‐how‐testing‐for‐covid‐19‐works/

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How certain is the clinical diagnosis of COVID-19? There are no data on COVID-19, but extrapolating from SARS

• 14 cases presenting with progressive pulmonary symptoms with a clinical diagnosis of SARS or suspected SARS, had autopsy results:

• 8 confirmed SARS (57%)• 6 non‐SARS (pneumonia with adenovirus, Pneumocystis, Pseudomonas, S. pneumoniae, MRSA)

• Clinical differential diagnostic considerations

• ? dengue, • viral pneumonitis• nosocomial pneumonia• status asthmaticus• Sepsis, septic shock• acute myocardial infarct

Chong, P. Y., Chui, P., Ling, A. E., Franks, T. J., Tai, D. Y., Leo, Y. S., . . . Travis, W. D. (2004). Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: challenges in determining a SARS diagnosis. Arch Pathol Lab Med, 128(2), 195‐204.

Laboratory Tests

205 patients with COVID‐19 : National Institute for Viral Disease Control and Prevention, China CDC, Beijing, ChinaWang, W., Xu, Y., Gao, R., Lu, R., Han, K., Wu, G., & Tan, W. (2020). Detection of SARS‐CoV‐2 in Different Types of Clinical Specimens. JAMA. doi:10.1001/jama.2020.3786

Pathology findings in COVID-19• Mostly pulmonary: ARDS/DAD, organizing pneumonitis

• Widespread intravascular fibrin thrombi with infarction of multiple organs, suggesting the presence of disseminated intravascular coagulation.

• Hemophagocytosis can be seen

Xu, Z., Shi, L., Wang, Y., Zhang, J., Huang, L., Zhang, C., . . . Wang, F. S. (2020). Pathological findings of COVID‐19 associated with acute respiratory distress syndrome. Lancet Respir Med. doi:10.1016/S2213‐2600(20)30076‐X

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Pathology findings in COVID-19

Sufang Tian, et al Journal of Thoracic Oncology 2020

• Alveolar edema and proteinaceous exudates• Intraalveolar fibrin with early organization,

lymphoplasmacytic infiltrate, multinucleated giant cells• Pneumocyte hyperplasia and reactive changes, including

some that suggest viral inclusions

https://consultqd.clevelandclinic.org/a‐pulmonary‐pathologists‐perspective‐on‐covid‐19/

Pathology findings in COVID-19 in cynomolgus macaques

(A) Two foci of pulmonary consolidation in the left lower lung lobe (arrowheads). (B) Area of pneumonia (HE, 2X).(C) Edema fluid in alveolar lumina (HE, 40X)(D) Neutrophils, as well as erythrocytes, fibrin, and cell debris, in an alveolar lumen flooded by edema fluid (HE, 100X)(E) Mononuclear cells, either type II pneumocytes or alveolar macrophages, in an alveolar lumen flooded by edema fluid (HE, 100X). (F) Syncytium in an alveolar lumen (HE, 100X objective). Inset: Syncytium expresses keratin, indicating epithelial cell origin (IHC for pankeratin 10 AE1/AE3, 100X)(G) SARS‐CoV‐2 antigen expression is colocalized with areas of diffuse alveolar damage (IHC for SARS‐CoV‐nucleocapsid, 20X)(H) Type I (flat) and type II (cuboidal) pneumocytes in affected lung tissue express SARS‐CoV‐2 antigen (IHC, 40X). (I) Epithelial cells of mucous glands in nasal cavity expresses SARS‐CoV‐2 antigen (IHC, 40X).

Barry Rockx et al Comparative Pathogenesis Of COVID‐19, MERS And SARS In A Non‐Human Primate Modeldoi: https://doi.org/10.1101/2020.03.17.995639

Pathology findings in SARS: Reactive and multinucleated pneumocytes

Atypical pneumocytes in patients with SARS. (A)Multi‐nucleated giant pneumocytes with

irregularly distributed nuclei (H&E, x640). (B) (B) A giant atypical pneumocyte with

prominent eosinophilic nucleoli (H&E, x640).

G M‐K Tse Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS) J Clin Pathol 2004;57:260–265

CDC/Dr. Sherif ZakiSyncytial giant cell of epithelial (derivation)

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Pathology findings in SARS: virus localization

• SARS CoV RNA could be found in 100% of lung tissues, 73% of bowel samples, 69% of lymph nodes, 53% of spleens, 41% of livers, and 38% of kidneys

• By IHC, the virus was found in the epithelial cells of the trachea, bronchi, and bronchioles, and in pneumocytes, both intact and degenerated, desquamated or forming syncytial giant cells.

• They were also present in the lymphocytes in the epithelial cells of the intestinal mucosa and distal renal tubules, and the neurons of the brain

Tony Mazzulli, et al Severe Acute Respiratory Syndrome Overview With an Emphasis on the Toronto Experience Arch Pathol Lab Med. 2004;128:1346–1350Gu J, Gong E, Zhang B, et al. Multiple organ infection and the pathogenesis of SARS. J Exp Med. 2005;202: 415‐424

How the SARS-CoV-2 outbreak will evolve and (hopefully) end

Francesco, C., Pettke, A., Michele, B., Fabio, P., & Helleday, T. (2020). Managing COVID‐19 in the oncology clinic and avoiding the distraction effect. Ann Oncol.


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What are the implications for the cytology laboratory? • How can the cytology laboratory be useful in the diagnosis of COVID‐19?

• How can the cytology laboratory continue to function during social distancing?

• How can we continue our educational role in the middle of this pandemic?

• These are all good questions to which I don’t claim to have the answers, but we can, maybe, work them out together

Polls

Stefan Pambuccian@pambuccian

Feb 21, 2020

How useful is respiratory tract cytology (sputum, tracheal aspirates, BAL fluid) in the diagnosis of COVID-19?

• (A) Cytospin from COVID‐19 BAL revealed many clusters of activated plasma cells (Papanicolaou, 40x).

• (B) Alveolar macrophage intranuclear cytopathic inclusion in SARS‐CoV‐2 infection (Papanicolaou, 100x, courtesy of Dr Francesca Bono, MD).

• (C‐D) Groups of polyclonal CD138‐positive plasma cells (cell block, Hematoxylin & Eosin, 20x; CD138 staining, 40x)

Marco Giani, Davide Seminati, Alberto Lucchini, Giuseppe Foti, and Fabio Pagni Exuberant plasmocytosis in bronchoalveolar lavage of the first patient requiring Extracorporeal Membrane Oxygenation for SARS‐CoV‐2 in Europe, prepublished Journal of Thoracic Oncology, 2020

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Reactive bronchoepithelial cells with nonspecific viral like nuclear and cytoplasmic changes in a background of severe acute inflammation. This is from bronchoalveolar lavage of a patient with #ARDS who tested positive for #COVID19. Ben Coulter, MD @Ben_WolfPath


Bronchoalveolar lavage (BAL) from a COVID‐19 patient. Increased lymphocytes, some with plasmacytoid morphology. Is the cell pointed with arrow lymphoid or type2 pneumocyte? Similar type cells reported with H1N1 BAL Amir Behdad @AmirBehdadMD

Paola Faverio et al Influenza A/H1N1 Severe Pneumonia: Novel Morphocytological Findings in Bronchoalveolar Lavage. Interdisciplinary Perspectives on Infectious Diseases Volume 2014, Article ID 470825

How useful was respiratory tract cytology (sputum, tracheal aspirates, BAL fluid) in the diagnosis of SARS?

G M‐K Tse Sputum cytology of patients with severe acute respiratory syndrome (SARS) J Clin Pathol 2004;57:256–259

Morphological changes of macrophages, including (A) cytoplasmic foaminess, (B) distinct vacuoles, (C) multinucleation, and (D) ground glass appearance of the nucleus

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Cytology has limited utility in the management of patients with known or suspected COVID-19Cytology may be useful in two circumstances:

• 1. The patient is infected with the SARS‐CoV‐2, but a superimposed viral (CMV), fungal (aspergillus) or bacterial infection is suspected or cannot be ruled out

• Some of these infectious organisms can be diagnosed cytologically before definitive culture or microbiology test results are available

• Bacterial infections show numerous neutrophils rather than the numerous macrophages, few lymphocytes and plasma cells typically seen in coronavirus‐related pneumonia

• 2. The clinicians are uncertain if this really is COVID‐19, either because of lack of testing or because of a complex presentation in which other conditions cannot be excluded.

• These may include some conditions in which cytology may be helpful, albeit not characteristic, like status asthmaticus (eos), nosocomial pneumonia, sepsis (PMNs), cardiac failure (hemosiderin‐laden macrophages)

Should cytology of respiratory samples be performed routinely? • Because respiratory samples may be collected for viral testing, BAL being the best specimen for viral detection, specimen aliquots could be routinely sent to the cytology laboratory

• We believe that routine cytology testing on such specimens is not warranted and is currently not supported by the available evidence

• Cytology may be performed selectively, based on the clinical judgement that valuable information to guide clinical management can be obtained from this test

• Ultimately, this should be discussed with the clinicians, and the limited utility of cytology should be weighted against the risks posed to the laboratory personnel


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How can the cytology laboratory continue to function during social distancing?1. Use general safe laboratory practices• Train all personnel in the use of the protective equipment

• Limit access to the laboratory

• Frequent handwashing, and wearing personal protective equipment (PPE)

• Avoid touching mucosal membranes of the eyes, nose and mouth

• Decontaminate all work surfaces multiple times per day, using EPA approved disinfectant solutions, wipes, towelettes or sprays. In addition to benches, this includes computer keyboards, phones, and frequently touched areas of microscopes.

Laboratory measures

1. Use universal precautions when dealing with any cytology specimen

2. Special precautions should be taken handling specimens the preparation of which involves steps that can lead to aerosol formation

• All technical procedures should be performed in a way that minimizes the generation of aerosols and droplets.

• Preparatory steps that may generate aerosols or droplets include expelling aspirates from the needle or syringe, smearing the aspirated material, and potentially air‐drying or heat drying the smears, in which pathologists, trainees or cytotechnologists may be involved during rapid on site evaluation (ROSE)

ROSE?

• Clinical judgement should be used to determine if ROSE is absolutely necessary for the success of the biopsy procedure

• If performed, appropriate personal protective equipment (PPE) including gloves, laboratory coat/gown and goggles or face shields for eye protection and respiratory protection using a properly fit‐tested filter respirator (N‐95)

• .

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FNAs?

• Suspend the activity of a pathologist‐run FNA Clinic for the duration of the pandemic

• Consider performing FNAs only on a case‐by‐case basis, weighing the risks and benefits of the procedure for each patient.

PPE: Strict adherence to full PPE is essential to avoid infection

VoxCoronavirus: Cytokine storm syndrome treatments for Covid-19

First autopsy results provide new pathology information on COVID-19

N95 masks (respirators)• National Institute for Occupational Safety and Health (NIOSH)

• N for Not resistant to oil. Used when oil particulates are not present

• 95 means that the minimum efficiency level of the filter is 95%

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“It should be noted that the surgical masks are primarily designed to protect the environment from the wearer, whereas the respirators are supposed to protect the wearer from the environment”.

• Two N95 filtering face piece respirators and 2 different models of surgical masks were evaluated

• As anticipated, the tested surgical masks showed a much higher particle penetration because they are known to be less efficient than the N95 respirators.

• The 2 surgical masks, which originated from the same manufacturer, showed tremendously different penetration levels of the MS2 virions: 20.5% and 84.5%, respectively

• N95 respirators will protect their wearers properly against particles of 300 nm and larger

Am J Infect Control 2006;34:51‐7

Hand washing

683 persons were randomized to • hand hygiene with soap and water (257 persons),

• hand hygiene with alcohol‐based hand rub (202 persons),

• control (224 persons). • The intervention period (16 months) included the 2009 influenza pandemic

• 6.7% reduction of infection episodes (especially respiratory infections) in the soap‐and water arm (p = 0.04) but not in the hand rubbing with alcohol‐based disinfectant arm

Mitka, M. (2009). Hand washing, a key anti‐flu strategy, often neglected by health care workers. JAMA, 302(17), 1850‐1851.

Savolainen-Kopra, C., Haapakoski, J., Peltola, P. A., Ziegler, T., Korpela, T., Anttila, P., . . . Hovi, T. (2012). Hand washing with soap and water together with behavioural recommendations prevents infections in common work environment: an open cluster-randomized trial. Trials, 1

“Avoid touching your face”• In subways in the US and Brazil, individuals touched common surfaces and their mouth/nose mucosa at a rate of 3.3 ‐ 3.6 touches per hour, indicating that the opportunities for hand recontamination in public settings occur at a much higher rate than any viable hand‐washing frequency.

Alonso, W. J., Nascimento, F. C., Shapiro, J., & Schuck‐Paim, C. (2013). Facing ubiquitous viruses: when hand washing is not enough. Clin Infect Dis, 56(4), 617.

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How can the cytology laboratory continue to function during social distancing? 2. Communicate• Provide up to date information regarding the most recent developments in our knowledge about the disease

• Provide candid information regarding all aspects of the personnel's job

• Provide emotional support and an opportunity to discuss any personal and family concerns

• Communicate effectively the risk to laboratory personnel and trainees, without overly reassuring them, and acknowledging that a lot is unknown about this infection.

How can the cytology laboratory continue to function during social distancing? 3. Continue teaching and training

• Cancel educational sessions involving the physical gathering of over 10 people, such as lectures, Grand Rounds presentations, journal clubs, etc.

• Limit face to face activities such as lectures or sign‐out session in a multiheaded microscope

• Consider moving to sequential viewing of slides rather than "double‐head" scoping or review

• Use online lectures • Use unknown sessions using digitized slides

• Make sure that trainees have access to the technologies used even from home

• Encourage and facilitate self‐directed learning

• Identify "teachable moments" in your daily work and share them with the trainees

• Give brief "teaching points" summaries for more unusual or difficult cases

• Give mini‐assignments; consider giving mini‐quizzes

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How can the cytology laboratory continue to function during social distancing? 3. Continue teaching and training • Consider setting up discussion groups to maintain a sense of community

• Encourage questions and be accessible to provide answers

• Give regular and meaningful feedback; be as specific as possible

• Ask for informal feedback and try to make any changes suggested

• Use the best tools or platforms available for online teaching, reevaluating them frequently

• Keep up with developments in online teaching and adopt best practices for distance learning

• Keep in touch with your colleagues and adopt the techniques that work

• Consider providing an online "office hour", and to be available to answer trainees’ questions during this time

• Check on the mental and physical well‐being of your trainees, be available to discuss their concerns and be supportive

Thanks

Questions?

Güliz Akdaş Barkan @barkanga

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Documents

COVID-19 and the Cytology Lab -1 · Respublica Ragusina (Dubrovnik, Croatia) A Decree of 1377 officially issued “quarantine”, the obligation for people and goods from ships arriving