Overview of Laboratory Tests for Cytomegalovirus

Introduction

Cytomegalovirus (CMV) is considered the most important pathogen in transplant recipient patients as it can cause significant morbidity and mortality. Anti-CMV treatments have proven to be effective but are not without adverse side effects. Thus, there is a strong need for sensitive and reliable tests to diagnose and monitor active CMV infection. Several testing methodologies are available in today’s clinical laboratories to evaluate a patient’s CMV status: viral culture, serology, histopathology, pp65 antigenemia, and quantitative PCR. In this post, we will review the advantages and limitations of these tests.

Viral culture

Viral culture is performed most commonly by the shell vial assay (also known as rapid culture), in which a cell line (usually human fibroblast cells) is inoculated with patient sample by centrifugation. The virus is then detected by either direct or indirect fluorescent monoclonal antibody, providing results within 1-3 days. The centrifugation step greatly improves turnaround time when compared to traditional tube cell culture technique, which may take 2-3 weeks before a result can be reported as negative.

Culturing CMV has been largely replaced by newer methodologies like quantitative PCR and CMV antigenemia. This is due to relatively weaker sensitivity for diagnosing CMV infection compared to newer tests, as well as slower turnaround time. Viral cultures of urine, oral secretions, and stool are not recommended due to poor specificity; however, for diagnosis of congenital CMV, viral culture of urine or saliva samples is an acceptable alternative if PCR is not available.

Serology

CMV serostatus is an important metric to evaluate prior to patients receiving a hematopoietic or solid organ transplant. Serologic testing is done primarily via enzyme immunoassays and indirect immunofluorescence assays. These tests check for presence of anti-CMV immunoglobulin (Ig)M and IgG to provide evidence of recent or past infection. Outside of establishing serostatus (primarily in organ donors and recipients), serologic testing for CMV is not recommended in diagnosing or monitoring active CMV infection.

CMV IgM antibodies can be detected within the first two weeks of symptom development and can be present for another 4-6 months. IgG antibodies can be detected 2-3 weeks after symptoms develop, and remain present lifelong. These antibody measurements are particularly useful in determining risk of CMV acquisition in seronegative patients (negative for IgM and IgG) at time of transplantation. IgG titers can also be measured every 2-4 weeks to assess for CMV reactivation in seropositive patients. Since CMV IgG persistently remains in circulation, testing for it has a higher specificity compared to IgM, and thus is the preferred immunoglobulin to test for in establishing serostatus. Serologic tests can be falsely positive if patients have recently received IVIG or blood products, so testing on pretransfusion samples are preferred if possible.

Histopathology

Under the microscope, cells infected with CMV can express certain viral cytopathic effects. These infected cells classically show cytoplasmic and nuclear inclusions (owl eye nuclei) with cytoplasmic and nuclear enlargement. Additionally, immunohistochemistry (IHC) can stain antibodies specifically for CMV proteins to highlight infected cells, making histologic examination quicker and improving diagnostic sensitivity.

Histopathology can be useful in identifying tissue-invasive disease, such as CMV colitis or pneumonitis. Cases in which PCR testing is negative does not necessarily exclude tissue-invasive disease; thus, the diagnosis of tissue-invasive disease relies on histologic examination (with or without IHC) or possibly viral culture. On the other hand, a negative histologic result does not exclude tissue-invasive disease, possibly due to inadequate sampling, and shows the potential for weak diagnostic sensitivity.

pp65 antigenemia

CMV antigenemia testing uses indirect immunofluorescence to identify pp65 antigen, a CMV-specific matrix protein, in peripheral blood polymorphonuclear leukocytes. Whole blood specimens are lysed and then the leukocytes are cytocentrifuged onto a glass slide. Monoclonal antibodies to pp65 are applied, followed by a secondary FITC-labeled antibody. The slide is then read using a fluorescence microscope for homogenous yellow-green polylobate nuclear staining, indicating presence of CMV antigen-positive leukocytes. Studies have suggested that a higher number of positive cells correlates with an increased risk of developing active disease. The sensitivity of antigenemia testing is higher than that of viral culture and offers a turnaround time within several hours.

This test has been utilized since the 1980s, but has seen less use recently due to the increasing popularity of quantitative PCR. Antigenemia testing is labor intensive, and requires experienced and trained personnel to interpret the results (which can be somewhat subjective). This test also must be performed on whole blood specimens within 6-8 hours of collection due to decreasing sensitivity over time, which limits transportability of specimens. Additionally, It is not recommended to be run on patients with absolute neutrophil counts below 1000/mm3, due to decreased sensitivity. Despite these limitations, CMV antigenemia testing is still considered a viable choice for diagnosing and monitoring CMV infection, especially when viral load testing is not available.

Quantitative PCR

Quantitative real-team polymerase chain reaction (PCR) is the most commonly used method to monitor patients at risk for CMV disease and response to therapy, as well as for diagnosing active CMV infection. The advantages of using a quantitative PCR assay include increased sensitivity over antigenemia testing, quick turnaround time, flexibility of using whole blood or plasma specimens for up to 3-4 days at room temperature, and the availability of an international reference standard published by the World Health Organization (WHO).

Several assays from Roche, Abbott, and Qiagen are available and FDA-approved. The targets of these assays vary, with some targeting polymerase and other targeting CMV major immediate early gene. These assays are all calibrated with the WHO international standard, which was developed in 2010 to help standardize viral load values among different labs when results are reported in international units/mL. The goal of this international standard is to decrease the interlaboratory variability of viral load, and determine the appropriate cut-offs for determining clinical CMV disease. There is still improvement to be made in this area, as variability still exists between labs.

Conclusion

There are several tests to determine the CMV status of patients. Some of these tests are suited for particular goals, such as serology for determining serostatus prior to organ transplantation, or histology and IHC to diagnose tissue-specific CMV disease. For diagnosis and monitoring of general CMV disease, the test of choice in most laboratories is quantitative PCR, which offers automated, quick and sensitive results. Antigenemia, while dated and labor intensive, is still an acceptable alternative when PCR is neither available nor cost-effective for smaller labs. Both of these testing methods are preferred over viral culture, which has poorer diagnostic sensitivity and relatively longer turnaround time.

Despite the numerous advantages quantitative PCR has, there is still variability in viral load counts among laboratories. This is due to varying DNA extraction techniques, gene targets used by PCR, and specimen types used. There is still a lot of work to be done in standardizing testing in regards to not just CMV, but also other viral pathogens like Epstein-Barr virus, BK virus, adenovirus and HHV6. Updated standards and increased use of standardized assays will hopefully decrease this variability between labs to improve testing results and in turn, improve patient care.

References

  1. https://www.uptodate.com/contents/overview-of-diagnostic-tests-for-cytomegalovirus-infection#H104411749
  2. https://www.uptodate.com/contents/congenital-cytomegalovirus-infection-clinical-features-and-diagnosis?topicRef=8305&source=related_link#H9542666
  3. Kotton CN, Kumar D, Caliendo AM, et al. Updated international consensus guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2013;96(4):333-60.
  4. Hayden RT, Sun Y, Tang L, et al. Progress in Quantitative Viral Load Testing: Variability and Impact of the WHO Quantitative International Standards. J Clin Microbiol. 2017;55(2):423-430.
  5. Kotton CN, Kumar D, Caliendo AM, et al. The Third International Consensus Guidelines on the Management of Cytomegalovirus in Solid-organ Transplantation. Transplantation. 2018;102(6):900-931.

-David Joseph, MD is a 2nd year anatomic and clinical pathology resident at Houston Methodist Hospital in Houston, TX. He is planning on pursuing a fellowship in forensic pathology after completing residency. His interests outside of work include photography, playing bass guitar and video games, making (and eating) homemade ice cream, and biking.

Laboratory Safety and COVID-19: References You Need to Know

Three months ago, life in the laboratories in these United States carried on as usual, and no one could probably have predicted where we stand today. The COVID-19 pandemic has changed the way laboratorians work everywhere. Some staff have had hours cut because of decreased workloads, other labs worked around the clock to bring new testing on board, and others dealt with staffing shortages due to illness. It has been a wild ride, and through it all, a great many safety issues have arisen. Common lab practices are now viewed through a new lens- is it acceptable to bring hematology slides for review into a clean pathologist’s office? Can we wear surgical masks worn in the lab into the break room? There are many good questions, but some of the answers can be found using references offered from reliable sources. Not everything you read online can be believed, but here are some references that may be necessary and that provide important information.

The pandemic has created a world-wide shortage of PPE, and some have wondered what can be done as resources diminish. The CDC has some good information about calculating how long PPE can be used and how long it can last. There are good guidelines about re-use and extended use of PPE.

PPE Burn Rate Calculator:

https://www.cdc.gov/coronavirus/2019-ncov/hcp/ppe-strategy/burn-calculator.html

Strategies to Optimize the Supply of PPE and Equipment:

https://www.cdc.gov/coronavirus/2019-ncov/hcp/ppe-strategy/index.html

There are specific references regarding respirators and how they should be used.

Respiratory Protection During Outbreaks: Respirators versus Surgical Masks

Understanding the Use of Imported Non-NIOSH-Approved Respirators

Proper N95 Respirator Use for Respiratory Protection Preparedness

Some laboratory disinfectants have become more difficult to purchase. The gold standard for disinfection remains a 10% bleach solution, but there are many other options that can be used as well.

Disinfectants for Use Against SARS-CoV-2 (EPA List N):

https://www.epa.gov/pesticide-registration/list-n-disinfectants-use-against-sars-cov-2

EPA’s Registered Antimicrobial Products Effective Against Human HIV-1 and Hepatitis B Virus:

https://www.epa.gov/pesticide-registration/list-d-epas-registered-antimicrobial-products-effective-against-human-hiv-1

The CDC also offers laboratories a set of COVID-19 guidelines for performing testing, biosafety issues, waste management, and protection against aerosols. These guidelines are thorough, and they can be very helpful should safety challenges arise.

Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19):

https://www.cdc.gov/coronavirus/2019-nCoV/lab/lab-biosafety-guidelines.html

Many of these references are updated regularly, so be sure you go to go to the source when making safety policy about COVID-19 tasks.

Laboratorians are now literally on the front lines during this novel coronavirus pandemic. While many public and commercial services have been scaled back, restaurants are closing, and many people are staying or working at home, lab staff are doing their level best to keep coming to work despite the extremely unusual circumstances and hardships.

I am here to serve as well. If you have questions about how to safely navigate this national (and global) emergency while working in the lab, ask me (info@danthelabsafetyman.com). I will do my best to provide any lab safety resources you may need. Make sure the decisions you make during these days are safe, sound, and based on the most recent resources available to you.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

Microbiology Case Study: Elderly woman with Ear Pain

An elderly woman with a past medical history significant for end-stage renal disease status post deceased donor kidney transplant in 2018 (on immunosuppression), type 2 diabetes mellitus, and recurrent urinary tract infections presented to nephrology clinic with right ear pain and rash of three weeks. She was otherwise in her usual state of health. On physical exam, there were exophytic itchy papules with hemorrhagic crust and ulcerations on the ear (Image 1) and arm. A few of these papules showed central umbilication (Image 2). Erosions were also present on the upper back, face, neck, and forearms. Patient was referred to dermatology with concern for disseminated infection versus neoplasia. Complete blood count showed mildly elevated white cells. Serologies, cultures, imaging, lumbar puncture, and biopsy were performed.

Blood studies revealed a Cryptococcus antigen titer of 1:4096 along with a CSF antigen titer of 1:2048. Additionally, the CSF gram stain demonstrated yeast and cultures grew Cryptococcus neoformans. Opening pressure was normal and protein was slightly elevated to 53 mg/mL with 36 nucleated cells with a differential including 64% lymphocytes. Biopsy culture of the left cheek was positive for C. neoformans and a left forearm biopsy showed nodular aggregates of encapsulated yeasts, surrounded by relatively sparse lymphohistiocytic inflammation (Images 3-4). A CT of the chest showed innumerable pulmonary nodules concerning for infection.

Image 1. Erythematous and crusted pink plaque with ulceration on the pinna of the ear.
Image 2. Pink, domed papule on the arm with central umbilication and crust.
Image 3. Hematoxylin and eosin stain of the arm biopsy with Cryptococcus, low magnification. Note the loss of epidermis (left-hand side) and underlying foamy stroma with numerous yeasts within the dermis.
Image 4. Hematoxylin and eosin stain of Cryptococcus, high magnification. The yeasts show variable size and some demonstrate a halo of pale staining capsule. There is no significant inflammation in the background parenchyma.

Discussion

Cryptococcus is an encapsulated basidiomycetous fungus typically found in soil and pigeon droppings.1 Two species comprise the majority of Cryptococcus infections: C. neoformans and C. gatti. C. neoformans is most commonly seen in immunosuppressed patients, particularly in the setting of AIDS.2 C. gatti infections may be seen in more immunocompetent patients and appears to be more geographically restricted to the tropics and Pacific Northwest.3 C. neoformans infections can present as lung disease associated with symptoms of fever, shortness of breath, or cough and characteristically may spread to the central nervous system to cause meningitis.4 Lumbar puncture may show significantly elevated opening pressures.5 Other features of disseminated Cryptococcus infections include rash, endocarditis, ocular lesions, or multiorgan failure.6

This case is a somewhat unusual presentation of disseminated Cryptococcus infection characterized only by skin findings without clinical features of pulmonary or CNS infection. Approximately 15% of patients with disseminated infection may show cutaneous findings but primary cutaneous cryptococcosis is rare.7 Cryptococcal skin findings are quite varied, but may present similar to molluscum contagiosum, as dome shaped papules with central umbilication.7,8 On microscopy, small variably sized round yeasts without hyphae are characteristic. These yeasts may show a clear or pale staining halo representing the capsule and are highlighted well on Grocott’s Methenamine Silver or Periodic Acid-Schiff stains. Histology may demonstrate innumerable extracellular yeasts accompanied by foamy stroma and minimal inflammation or more granulomatous tissue reaction with necrosis, ulceration, and mixed inflammation. In conclusion, disseminated Cryptococcus must be considered in the context of new skin findings in an immunocompromised patient even if typical pulmonary or CNS findings are not identified.

References

  1. Sorrell TC, Ellis DH. Ecology of Cryptococcus neoformans. Rev Iberoam Micol. 1997 Jun;14(2):42-3.
  2. Bratton EW, El Husseini N, Chastain CA, Lee MS, Poole C, Sturmer T, et al. Comparison and temporal trends of three groups with cryptococcosis: HIV-infected, solid organ transplant, and HIV-negative/non-transplant. PloS One. 2012;7(8):e43582
  3. MacDougall L, Fyfe M, Romney M, Starr M, Galanis E. Risk factors for Cryptococcus gattii infection, British Columbia, Canada. Emerg Infect Dis. 2011 Feb;17(2):193-9.
  4. Sabiiti W, May RC. Mechanisms of infection by the human fungal pathogen Cryptococcus neoformans. Future microbiol. 2012 Nov;7(11):1297-313.
  5. Abassi M, Boulware DR, Rhein J. Cryptococcal Meningitis: Diagnosis and Management Update. Curr Trop Med Rep. 2015;2(2):90–99. doi:10.1007/s40475-015-0046-y
  6. Clark RA, Greer D, Atkinson W, Valainis GT, Hyslop N. Spectrum of Cryptococcus neoformans infection in 68 patients infected with human immunodeficiency virus. Rev Infect Dis. 1990 Sep-Oct;12(5):768-77.
  7. Srivastava GN, Tilak R, Yadav J, Bansal M. Cutaneous Cryptococcus: marker for disseminated infection. BMJ Case Rep. 2015;2015:bcr2015210898. Published 2015 Jul 21. doi:10.1136/bcr-2015-210898
  8. Akram SM, Koirala J. Cutaneous Cryptococcus (Cryptococcosis) [Updated 2019 Aug 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2019 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK448148/

-Dr. Stanton Miller is a second year AP/CP resident at UT Southwestern Medical Center who is interested in Dermatopathology.

-Dr. IJ Frame is a board-certified Clinical Pathologist who is completing his Medical Microbiology fellowship at UT Southwestern Medical Center.

-Dr. Dominick Cavuoti is a professor of AP and CP at UT Southwestern, specializing in infectious disease pathology, cytology and medical microbiology.

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

COVID19 and the Lessons We Learned from Prior Pandemics

With recent criticisms in the media, both foreign and domestic, on the United States’ response to COVID19 as well as accusations and summary conclusions that the United States is not a global health power house nor is it as prepared to handle COVID19 as nations around the world that are plagued by infectious challenges daily, it is important to revisit history of recent pandemics and the prior US responses to them to put the current interpretations of “failing” into perspective.

In 2003, the SARS epidemic began in China with the first possible case documented in November 2002. At the time, US relations with China were such that CDC field offices and CDC field officers, including permanent deploys and temporary lead deploys from central CDC in Atlanta, GA, were available to assist the Chinese healthcare system and government with the response to SARS. Through this effort, statements from CDC field directors such as, “This town is going to have a spike and we need 300 more beds,” was answered by the Chinese with a new hospital being built with 300 beds in less than 3 days. Such transparency, collaboration, and communication were possible at the time but relationships have diminished in recent years. During the SARS outbreak, there were 8,098 patients infected (known by positive testing) and 774 deaths (9.5%) which affected 26 countries including the US; however, the US only had 8 to 27 cases (depending on source) and no deaths. Although the first cases traced back to late 2002, the disease was not sequenced and declared until April 2003, but testing was available shortly thereafter. Control measures locally and globally with some help from testing stifled the pandemic in a matter of weeks and the threat was near zero by the end of 2003. No resurgence has occurred. From this outbreak, the US and the world learned how to deal with novel coronaviruses and how to coordinate and collaborate for future potential outbreaks. Such lessons include the need for transparent communication and direct in country collaboration, rapid move to testing distribution, and high-level knowledge of pandemics and who nations should respond.

In 2009, the H1N1 pandemic began. The CDC activated its emergency system within 7 days of the first case, the US and the WHO declared the pandemic within 9 days of the first case, and testing was available within 14 days of the first case. The US had 60.8 million cases (confirmed positive tests) with 274,000 hospitalizations (0.5%) and 12,469 deaths (4.5% of hospitalizations, 0.02% of cases). The incidence from the disease was due to the rapid respiratory spread very similar to routine influenza but on top of a system (including hospital processes and national approaches to testing with integrated public health laboratory systems) that was prepared and able to nimbly adapt. In this case the rapid advent of testing was crucial to controlling case, getting patients on treatment, and tracking the disease. H1N1 was then subsequently included in the annual influenza vaccines.

From 2012-2014, the MERS-CoV virus, originating from and primarily endemic in the Arabian Peninsula, was a challenge for global heatlh because of the high mortality rate (30 to 40%) and the very efficient spread of the virus. All cases arising outside of the Arabian Peninsula were traced to travelers from that region. The first known cases were in April 2012 with the first recognition of the virus causing the disease in September 2012. The CDC developed a test for MERS in 2012 and subsequently an EUA from FDA was granted on June 5, 2013. The first positive cases of MERS in the US occurred in May 2014, almost 1 year after testing had been available. To date, only 2 confirmed cases of MERS have been diagnosed in the US which were traveling healthcare workers who had treated patients in Saudi Arabia.

The Ebola epidemic in West Africa from 2014-2016 had a total global case count of 27,000+ with 11,000+ deaths (46% mortality). However, in the US only 4 patients were ever diagnosed with EBOLA and 11 patients were treated for EBOLA with only 2 total deaths (18% mortality). Why was the case count so low for the US and why was the mortality nearly a 1/3rd of the overall epidemic? Immediate response from the US government to control incoming patients (the only transmission inside the US was from patients who were travelers to healthcare workers) and availability of testing prior to the outbreak (with the CDC). Nigeria was able to diagnose the first case in Lagos (a traveler from Liberia) because a scientist in Nigeria had developed a rapid EBOLA PCR six months before the outbreak occurred. Nigeria only had 8 deaths from 20 confirmed infections (40% mortality). Why did Nigeria get ahead of the game? Immediate response from government and availability of testing. The unfortunate results in Liberia, Sierra Leone, and Guinea were less about lack of response and lack of testing and mostly due to poor infrastructure for health.

The current pandemic of COVID19 started on November 17th (earliest confirmed case in China) and was a reported disease cluster from China to WHO by December 31st, 2019. The first case in the US was documented to have occurred on January 19, 2020. The FDA, in response to information from central administration and pressure from multiple entities, allowed testing for COVID19 through Emergency Use Authorization (EUA) on February 28, 2020 (more than one month after the first US case). As of April 28, 2020, the US has had 1,026,771 confirmed cases (positive testing) and 58,269 deaths (5.7% mortality) affecting all 50 states in the setting of an unprepared system (i.e., insufficient testing, insufficient pandemic planning at the national level, insufficient in country data from source countries). Data has shown in the laboratory that the SARS-CoV-2 virus shares 74 to 90% genetic homology with the original SARS virus but has a 10-fold increased affinity for binding which suggests that its natural biological virulence could be 10x that of SARS. If proper systems, testing, and planning had been in place, we can conservatively estimate that there would currently be 102,667 confirmed cases in the US and 5,827 deaths. These excess cases and excess death are, therefore, a direct result of the lack of systems, testing, and planning (52,442 excess deaths of US citizens).

There are conspiracy theorists that argue SARS-CoV-2 was created or modified from a different virus by human manipulation with a most recent endorsement of HIV Nobel Prize Laureate Luc Montagnier—statements that were almost immediately refuted by other prominent scientists. If there was a credible threat from SARS-CoV-2 when the sequence was released, that would have been an even more convincing argument that preparation was needed. But the threat of SARS-CoV-2 from just the observed medical cases and initial reports should have warranted a brisk and complete response from leadership. That such responses were delayed because of a multitude of failed responses (pandemic planning, testing, situational awareness, field deployments, etc.) can be argued from now until the next pandemic occurs. But our collective prior experience with pandemics (4 of them in 2 decades) provided plenty of evidence and case-studies for how we should have responded.

ASCP along with other organizations reached out to our membership and the community for support of a call for a National Testing Strategy resulting in tens of thousands of letters to elected representatives and a subsequent plan for a National Testing Strategy released by the US government. The CARES Act released this week includes billions for testing.

These efforts are for our membership who are the medical laboratory professionals working 12 hours shifts to provide the testing needed by their patient populations.

These efforts are for our pathologist members who are informing and controlling hospital and government responses around testing through their rapid decisions and their expertise.

These efforts are for our pathologist’s assistance at all levels who keep anatomic pathology running with our pathology trainees despite massive volume challenges.

These efforts are for our PhD members whose expertise in science, design, and evidence acquisition is rapidly leading to new testing and eventually new vaccines.

These efforts are, most importantly, for our patients, the center of all that we do, to ensure that they have access to testing and the peace of mind they need to move forward from this pandemic.

References

  1. https://www.webmd.com/lung/news/20030411/sars-timeline-of-outbreak#1
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1904415/
  3. https://en.wikipedia.org/wiki/2002%E2%80%932004_SARS_outbreak
  4. https://www.fda.gov/media/72313/download
  5. https://www.who.int/csr/sars/testing2003_04_18/en/
  6. https://www.cdc.gov/about/history/sars/timeline.htm
  7. https://www.cdc.gov/flu/pandemic-resources/2009-pandemic-timeline.html
  8. https://www.cdc.gov/flu/pandemic-resources/2009-h1n1-pandemic.html
  9. https://www.cdc.gov/coronavirus/mers/about/index.html
  10. https://www.cdc.gov/about/ebola/timeline.html
  11. https://en.wikipedia.org/wiki/Western_African_Ebola_virus_epidemic
  12. https://www.scmp.com/news/china/society/article/3074991/coronavirus-chinas-first-confirmed-covid-19-case-traced-back
  13. https://www.who.int/news-room/detail/27-04-2020-who-timeline—covid-19 https://www.nature.com/articles/s41467-020-15562-9
  14. https://www.worldometers.info/coronavirus/country/us/
  15. https://www.nejm.org/doi/full/10.1056/NEJMoa2001191
  16. https://en.wikipedia.org/wiki/COVID-19_testing
  17. http://www.xinhuanet.com/english/2020-04/21/c_138995464.htm
milner-small

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

Love in the Time of COVID-19

Hi everybody—welcome back!

Although you better not have gone too far since last time, self-isolation and social distancing are still critical for us to get through this. Wash those hands, and dust off books and board games. #StrongerTogether (apart), am I right?

Yes, it’s going to be another piece on the current pandemic. It probably will continue to be so until conditions change. So, as your contributor to virology and testing these last two months, I’d like to take a minute to “zoom out” a bit and look at this pandemic in a different way.

It’s highlighting lots of things in healthcare from supply chain, to political regulatory red tape, to the mechanism of deliverables in the United States. And despite the title, I have no romantic epilogues about anything happening in Columbia at the turn of the 20th century. However, very much like the original book’s protagonist, I’d say there are a lot of us in Pathology and Laboratory Medicine alike who are champions of the scientific cause for advancement, education, and positive outcomes. And what better, more fitting of a time to celebrate this cause, than Laboratory Professionals Week 2020!

*** Never forget how vital you all are at every level. Technicians, technologists, medical laboratory scientists, administrators, directors, managers, residents, fellows, faculty, and staff all fit together in a magnificent (but often too unseen) tapestry that makes every patients’ tests results mean something so much more than numbers on a printed report. You’re all lab heroes, we’re all lab heroes. Go make sure you thank some of them this week (or anytime) and a heartfelt thank you to all of you from me as well! ***

In the mere month since I last wrote a piece for Lablogatory, so much has changed with the pandemic as well as my role in local and academic public health efforts. To name a few, I trained with the New York City Medical Reserve Corps as a public health educator early during the pandemic, I was invited to give a lecture on SARS-CoV-2 and COVID-19 testing considerations for lab professionals by the excellent pathologists who run the PathCast series, and I just recently finished a two-day series with an organization called Proceed who are sponsored by the National Center for Training, Support, and Technical Assistance (NCTSTA)—a CDC grant-funded education web series. I’ll mention a little bit about all these things (and of course link you to the material) and talk about how it all fits into what has quickly become a complicated social pandemic response.

NYC-MRC

Almost as soon as I set up shop to start my medical school clerkships and clinical training in New York City, I joined the New York City Medical Reserve Corps—a collection of volunteer healthcare first responders in the event that the city at large ever needed to mobilize every available healthcare personnel during a disaster or health emergency. *Spoilers: turn on the news* At the same time I was prepping some pre-clinical research in infectious disease healthcare and contributing to ASCP’s Choosing Wisely initiatives addressing Hepatitis C testing in vulnerable communities with some of the nation’s highest rates of infection (read the flashback primers I wrote here and here), I was attending NYC-MRC seminars and becoming a nationally certified disaster responder. There have been drills, seminars, and lectures since joining in 2018 but nothing to really contribute to while I moved through clinicals. Welp, clinicals ended, I had a few weeks off for residency interviews, then Kung Pow! Enter the defining viral pandemic of 2020! Smooth sailing lectures became phone network scrambles to see if I obtained my medical license yet since New York’s hospitals were slammed! The last meeting I remember going to was a training on disseminating appropriate information to various levels of practice/professions. I didn’t know it yet, but this became paramount knowledge for me.

Image 1. Throw back from my Instagram (@CEKanakisMD) and one of my first sessions on training to become a disaster responder for the NYC-MRC.
Image 2. The last meeting I attended, and probably the last meeting I’ll have attended while living in Manhattan. Learning some tools, tips, and tricks on effective communication during uncertain times proved invaluable for what was to come. (also from my Instagram @CEKanakisMD)

What Information Matters Most?

So what exactly did I learn? Essentially, it’s nothing groundbreaking or new, but the way you address certain topics matters more than you might realize. I once found myself in a room of mixed level healthcare providers, homeless shelter staff, local public health officials, hospital nurses, and lay people—that’s a broad range of knowledge and practice exposure. If you talk about upregulated ACE-2 receptor expression in intra-viral inflammatory response before full blown ARDS and DAD visible in lung biopsies you’ve lost half the crowd; and if you talk about epidemiology basics like reproductive number (R0), first cases found (FFX), and trace tracking, your provider audience is suddenly looking at their phones. But almost none of them know enough about laboratory testing, regulations, or quality assurance measures—so that became my target, and my bridge to connect everyone. I began collaborating with a friend and colleague Dr. Emeka Ajufo, who matched into his top-choice pain management and rehabilitation (PM&R) residency, and started creating content that connected topics like wellness, one health, and prevention while at the same time understanding deliverables and quality behind lab testing.

Image 3. We’re still in editing and sound production, but we’ve worked together before on podcasts and other content I’ve featured in previous blog posts, like this one! Check out more of Dr. Ajufo’s PM&R work here. (We may or may not be discussing the finer points of adequate nasopharyngeal swab technique here…)

PathCast

So this partnership content got noticed on Twitter (@CEKanakisMD) by the folks that run the PathCast simulcast series on Facebook and YouTube. Dr. Rifat Mannan (@mannanrifat03 on Twitter) from the University of Pennsylvania Hospital and Dr. Emilio Madrigal (@EMadrigalDO on Twitter) from Massachusetts General Hospital have been hosting and promoting a mountain of impressive faculty lectures for all kinds of topics in pathology since 2016. They host them live for viewing across an international audience, take questions, and save each video for future viewers. Their wide and comprehensive hour-a-piece lecture series is enjoyed my many and offers a free, no-hassle viewing experience. I was honored enough to be considered to give a talk on their channel, and after discussing more details with them, it appeared that there was a unique opportunity for some high-value topical information on laboratory and quality testing during this COVID-19 pandemic. If you haven’t heard of this series, you’re missing out. Please go like and subscribe to both their Facebook and YouTube platforms ASAP—you won’t be disappointed!

Image 4. Here’s the title card for my lecture on SARS-CoV-2 and COVID-19 testing considerations for laboratorians. It aired on Friday, April 10th at 8:00 ET and I’m proud to say that it received almost 20,000 individual views from just under 100 countries around the globe. Talk about global pandemic attention and response timing! Got an hour? Check it out here.

What’s a Good Test in a Pandemic?

Excellent question! You’ve probably already heard me talk about this before… This specific question came from discussions on social media with friend, colleague, and fellow ASCP Social Media Committee member Dr. Rodney Rhode (@RodneyRohde on Twitter). If I make it sound like I’ve been busy these last months, Dr. Rhode operates at another level: he’s publishing articles on the pandemic, running laboratory operations, is a research dean and department chair at Texas State, and is disseminating clinical information faster than I could even process it—he’s one of many pathology rockstars in our field! When we spoke before the PathCast series, we talked a bit about the problems in FDA fast-track clearance of all these new tests that would barely make the cut during non-pandemic “peace time.” The Emergency Use Authorization program allows the FDA to push forth tests available for commercial distribution with around 30 or so specimen validations that often don’t break the 60-70% sensitivity/specificity ceiling—yeah, I know. But it’s the best we’ve got and hospitals all over the country are working as hard as they can to bolster their validation studies with more specimens, better controls, modified protocols, and enhanced LDTs (laboratory developed tests) just to meet demand.

Image 5a. Here’s an updated slide from the PathCast talk. The current situation report: 43 commercially available kits, pumped out daily like an overworked approval factory, some are better than others. We’re focused more on molecular/PCR/NAAT for now for its clinical and diagnostic utility. Antibodies will become useful when we discuss “post peak curve” solutions like tracking, vaccines, and therapies like convalescent plasma. What makes a good test in a pandemic? Good laboratory practices.
Image 5b. This is the rapid serology kit that is commercially available but not FDA reviewed, at all. Courtesy of Kelly Swails. I’m not here to name or shame, but this is just one of hundreds of these tests out there. And it’s a definite challenge when those of us in Laboratory Medicine are not part of the process. So caveat emptor/buyer beware—don’t let your lab’s precious time or resources go into snake oils.

There are not enough tests, but there are also too many tests. Just before that PathCast lecture went live, I got an email from our awesome Lablogatory manager and editor, Kelly Swails (@kellyswails on Twitter) about some generic antibody testing kit that people were going nuts over. The problem was on page 6 of the manufacturers’ insert: “this test has not been reviewed or cleared by the FDA.” Well, there’s a problem—and they’re a dime a dozen. Since then, friends, colleagues, and all kinds of inquiries have come my way to ask, “is this a good test?” as people find kits available for purchase… it’s been a mess to say the least. But we laboratorians know: one of our core principles isn’t to let quantity overrule quality, especially when it comes to patient testing. That’s a non-starter.

NCTSTA

Shortly after the PathCast buzz started to settle, one of my MLS grad school classmates who now works with a local public health education and training organization in New Jersey, reached out to see if I could expand the discussion on testing to include problems with access and issues with vulnerable populations. Check and check. Go back and look at some of my posts on Zika and arbovirus work in the Caribbean and you just know I was excited to help! Proceed, Inc. has been a supportive community leader in addressing health and accessibility concerns in their local region and reaching out to form partnerships under the banner of the National Center for Training, Support, and Technical Assistance program (supported by the Centers for Disease Control and Prevention (CDC), Office of Minority Health (OMH), Administration for Children & Families (ACF), and other local entities.)

Image 6. Title card and promotional material for the Proceed/NCTSTA webinar on April 21st, at 2:00p ET with question and answer session and a following day’s meeting called “coffee house chat” where informal discussion and questions were directed at me regarding the topics discussed. It was a fantastic dual session, something I would promote and do again in the future. Unlike the previous hour lecture aimed exclusively at a pathology audience, these attendees came from mixed roles from frontline healthcare workers to government officials in public health and were located all over the US! Here’s the link to the recorded webinar, check it out here.

Who’s Vulnerable?

If you watch the news it’s individuals over 65 years of age and/or anyone with a significant related underlying condition: asthma, COPD, hypertension, etc. And, while that’s true, that’s just the tip of the vulnerability iceberg. Let’s remind ourselves for a minute about the inward and outward concepts of “social determinants of health.” When we want to label a population as vulnerable, or better put, increasingly susceptible to the negative effects of their living conditions in the setting of health care access, we have to think about all the things that contribute to a person’s health: their relationships, their stable/unstable living conditions, level of education, their income/expense ratio, possible language barriers, race/creed/color, disability, addiction, those experiencing homelessness, and concerns for their individual safety to name just a few! Inwardly, should we choose to engage these vulnerable communities we must do so with proper inclusion and a foundation of trust, communication, clarity of purpose, partnership, support, and—arguably most importantly—cultural humility. I also offered the attendees two resources as handouts which are available to you if you attend the recorded webinar as well: one COVID-19 safety factsheet directly from the CDC, and an adaptation of social determinants inventory I designed when my arbovirus team worked on Zika education in Sint Maarten. In truth, we’re all vulnerable in different ways, but when we work together to address gaps in delivery and access we end up #StrongerTogether—and that’s something our laboratory community knows a thing or two about!

Image 7a. Some selected slides from the Proceed/NCTSTA webinar on testing and vulnerability. (51) addresses that “tip of the vulnerability iceberg” that goes beyond physical susceptibility to viral infection, (52) clearly demonstrates that the number of confirmed cases in the 5 boroughs of New York City—the hardest hit location in the United States right now—correlates not only to income/expense ratios, but also people of color, and especially those individuals who can’t leave their work because of the “essential” nature of the service industry. (53) This tense situation between paycheck-to-paycheck workers and depending on employment for insurance is point proven if you look at unemployment claims going back to the 60’s! Notice the dramatic spike of 3.3 million claims this year because of the pandemic! That’s more than any oil embargo, dot-com burst, housing bubble, or recession we’ve ever seen! Finally, in (54) I introduce what I think is a “new” type of vulnerable population: individuals who don’t “buy-in” to the science and medical literacy of the current situation. That’s a whole other blog post folks…
Image 7b. Here it is, my COVID-19 Assessment and Preparedness Inventory Toolkit which incorporates data from FEMA, the CDC, the WHO, previous literature on inciting behavioral changes, and evidence-based best practices for addressing the most vulnerable populations.

The Tipping Point

The $64,000 two-part question: are things getting better or worse, and when will things go back to normal? My 64¢ answer: we don’t really know yet because there’s not enough active current data. The best estimates have case-peaks in places like Manhattan reaching a sort-of plateau as non-emergent hospitalizations, intubations, and COVID-19 cases slow down—but don’t mistake that for a full-on stop. Social distancing and quarantine initiatives in places like New York, Chicago, and other cities are the most effective NPI (non-pharmaceutical intervention) we’ve got. And that’s saying a lot. We have data that suggests previous pandemics had second waves as soldiers came back from war during the 1918 Flu pandemic in the states, so we’ve got to be careful and mindful of what we’re up against. But it’s getting easier and easier to become listless and bored of Netflix and stress-baking. People are getting legitimate cabin fever, although I’d rather we all had that than another, more topical viral illness these days. We’ve got a ways to go with all our frontline work, our NPI distancing, and stratified testing/tracking measures and we have to keep at it, otherwise we’ll undo all the progress we’ve made. And, that “new” vulnerable population, with motivations most certainly rooted in fear and stress, demanding to “reopen” the country since COVID is, after all, a hoax: don’t underestimate their power to tip the scales and send us back. The marriage of policy and politics is a patchy one at best, but efforts from professional advocacy societies like the ASCP are making strides, pushing both at the local and federal levels to demand active and appropriate responses to address proper COVID testing. But things don’t have to be so contentious.

Image 8. “Operation Gridlock”: re-open protestors in Denver, CO come face-to-face with frontline workers like this nurse in the middle of this tenuous mass protest and battle between politics and science. Scenes like this are happening all over the country like in Ohio, Michigan, and more. (Image: NBC news)
Image 9. ASCP has been the definitive forefront in leading the national charge to address our federal response to proper testing guidelines, strategy, and support. Read more about it here.

Fear and Loathing vs. Love in the Time of COVID?

So where does this leave us now? You’ve listened to my litany of testing complexities and considerations for preserving quality of healthcare delivery to all types of patients and you know where I stand on having a passion for preserving the importance and integrity of our professional role as leaders in this field. Do we give in to frustration or keep fighting this pandemic in more, creative ways? The answer, to me, is obvious. We move forward, as always. But most especially, this lab week should be something different because it not only highlights our work as traditionally “behind the scene,” but underscores our critical importance to the delicate house of cards balanced between clinical healthcare, decision making, public health, and public opinion. It’s not only our job to make sure the tests are good (even during pandemics) but that we represent a consistent and reliable message of evidence-based truth for patients and clinicians to rely on—like we always do.

Happy Lab Week 2020. Stay safe, wash your hands, and remember social distancing doesn’t just mean staying at home. It also means integrating compassion into a new routine, and caring for neighbors, colleagues, and friends in new profound ways.

See you all next time!

Image 10. Lab week 2020, ASCP’s Fellowship of the Lab, One Team to Diagnose Them All


-Constantine E. Kanakis MD, MSc, MLS (ASCP)CM is a new first year resident physician in the Pathology and Laboratory Medicine Department at Loyola University Medical Center in Chicago with interests in hematopathology, transfusion medicine, bioethics, public health, and graphic medicine. His posts focus on the broader issues important to the practice of clinical laboratory medicine and their applications to global/public health, outreach/education, and advancing medical science. He is actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Watch his TEDx talk entitled “Unrecognizable Medicine” and follow him on Twitter @CEKanakisMD.

COVID-19 Laboratory Safety: The Wrong Conclusion?

In the classic Barry Manilow song, Copa Cabana, the singer admonishes his audience at the end not to fall in love. We are told to avoid love because some guy named Rico might exterminate our loved one and we will end up a drunken barfly like Lola in did in the song. It’s a pretty funny conclusion when you think about it. If you were the victim in the story though, you might draw the same conclusion, however flawed. As the COVID-19 pandemic continues, laboratorians are seeing people draw incorrect conclusions as well, and some of those assumptions are leading to some potentially unsafe practices in some laboratories.

It’s easy to understand and even to forgive any misconceptions. The COVID-19 pandemic is unprecedented in our lifetime. The virus is new, it has spread very quickly, and there are unknowns about its make-up and mode of transmission. The situation has changed rapidly, and agencies like the CDC have had to change safety recommendations almost daily at times. Couple that with a media that by its nature uses fear and drama to ensure continued viewership, and people everywhere are going to make some fast decisions, and not all of them will be good.

One example of a change in guidance came from the CDC. At first, they were very careful not to give out any more information for lab safety than what was posted on their COVID-19 web page. When asked if typical lab specimens (blood and urine) from COVID-19 patients could be transported via a pneumatic tube system, they said that all specimens should be hand-carried to the lab. Since many patients were asymptomatic and many more could not be tested because of kit shortages, that would basically mean that all specimens in all hospitals would be carried to the lab. Before hospitals could react or even find the information, the CDC changed its recommendations. Only respiratory specimens from these patients should be carried directly to the lab (https://www.cdc.gov/csels/dls/locs/2020/transport_recommendations_for_covid-19_specimens.html), blood and urine can safely be transported. Given what is known about viruses and other pathogens in blood, walking all specimens to the lab doesn’t sound like a necessary guideline, does it? But this judgement in guidance simply illustrates what can happen when a situation changes rapidly.

With the push for the need for COVID-19 testing, news has come out about specimen collection kits and new and faster testing platforms. Not everything that has been reported has been factual. Every night we see numbers of cases and mortality rates reported in the media. Mathematically, one cannot determine a mortality rate without knowing the total number of patients’ cases. If there aren’t enough test swabs and reagents, and if we don’t test those who are asymptomatic (estimated to be about 25% of cases), how can we calculate any kind of accurate rate? Some laboratories have been pushed to bring on testing, and with little experience with brand new instruments, there has been work performed in some locations without adequate PPE or other safety measures. Look at your testing platform. Review the steps to see if there are any potential aerosol-creating steps. Are the correct PPE or engineering controls in place? Now more than ever it is vital to adhere to lab safety regulations while paying attention to the latest COVID-19 safety guidance. If you feel there is work being performed without adequate safety protection, escalate the situation as soon as possible. There will be no one to run teste if all of the laboratory staff becomes sick.

In many locations, hospitals have responded to the shortage of N95 respirators and surgical masks by developing methods to decontaminate and reuse them. While that is an innovative idea, make sure the system in place has physical and biological checks in place to ensure the reprocessing is effective. Spot test disinfected N95 respirators by performing fit-testing with them as batches are reprocessed. Use a biological indicator to ensure the COVID-19 virus particles have been eradicated. Do not reprocess N95 respirators used for protection against tuberculosis unless it is certain the disinfection process is known to effectively destroy TB bacteria.

Sometimes we come to the wrong conclusion, and sometimes mistakes are made, even in the world of science. But we are human, and we have to come to terms with that or we might end up alone in a disco like the aforementioned Lola. That’s why it’s important that we stop for a moment and pause, especially when an event occurs rapidly and transforms the very environment where we work. Take the time to ensure safety measures are in place for everyone involved. If something doesn’t make sense, question it. Examine it, and work with your staff as a team to make sure we can do what we are called to do- to safely perform lab work in order to provide quality results for the patients we serve.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

COVID-19 Q&A: Laboratory Safety

The evolving COVID-19 pandemic has changed the world already- the way we educate, the way we conduct business, and way we socialize (or don’t). In the lab setting, there are many practices and procedures that are now being questioned. Sometimes the answer changes quickly, sometimes there is national guidance to assist with a particular issue, and sometimes your own facility or organization makes the decisions for you. As a lab safety consultant, I have received many lab safety questions about handling certain situations in these times, and I want to share some of those because others may be wondering the same thing.

Q: Should nurses or other staff come into the laboratory wearing surgical masks, even if they are taking care of COVID-19 patients?

A: Yes, it is acceptable and safe for nurses and other staff to enter the lab wearing surgical masks (unless a company policy prohibits it). Remember, if someone else wears a surgical mask, that protects you, NOT them. Even if the employee is taking care of a COVID-19 patient, science tells us that virus particles are not falling off their mask. It is generally safe for people to enter the department wearing a surgical mask.

Q: Should nurses or other staff come into the department wearing gloves?

A: No, it is generally not acceptable for others to enter (or exit) the lab wearing gloves. Door handles should be considered clean. If specimens are being delivered, the outside of the bag/container being carried is considered clean (even if a COVID-19 specimen is inside). Post signage to remind non-lab staff to remove gloves, and notify people immediately if you see them using gloves to enter or exit the lab.

Q: My lab co-worker seems to be sick. Can I tell them to go home or self-quarantine?

A: No. As an employee you do not have the right to approach a co-worker or dictate to them how to proceed. If you have concerns, escalate them to the manager. If you’re the manager, contact your employee health department. That is the department that can best determine who needs to be sent home for isolation. Remember, there are many other reasons people cough, sneeze, or have a runny nose. For instance, allergy season is in full swing. Harassing a co-worker about their health would be considered a violation of Human Resources policy.

Q: How does laboratory staff maintain an appropriate social distance from each other while working?

A: The answer to this really depends on your lab set-up and processes. Is it possible for staff in smaller labs to change work patterns in order to better maintain a distance from co-workers? Try a different receiving location so that those from outside the department can drop off specimens from a distance, like placing a cart near the lab door. Some facilities require masking of all staff, and if that is the case, then social distancing while at work may not be an issue. Be sure to limit staff in break rooms as well, and make sure areas are disinfected regularly.

Q: Should autopsies be performed during this pandemic?

A: Some pathologists have suspended autopsies at this time. While it is possible to perform autopsies on COVID-19 patients safely using the appropriate PPE, many deem the use of such PPE to be unnecessary when so many others need it.

Q: Can PPE be disinfected and re-used safely?

A: Some locations have moved to reprocessing N95 respirators, surgical masks and isolation gowns. Make sure there are appropriate quality and safety checks being performed on this equipment before it is re-used. While some methods can disinfect items used for COVID-19 patients (i.e. UV light treatment, hydrogen peroxide mist, etc.), they have not been approved for the disinfection of tuberculosis. Therefore, if PPE has been used to protect staff from TB, it should not be reprocessed with the other PPE used for COVID-19 patients.

Q: Histology and cytology slides from fresh (unfixed) samples are made in the lab space and moved to offices for reading. Can these slides from potential COVID-19 patients be safely read in clean office areas?

A: This is a valid question at all times, not just during this pandemic, and the answer depends on the process used for making the slides. It is known that xylenes or solutions containing 60% ethanol or more easily inactivate COVID-19 and other pathogens. Slides made with these chemicals can safely be transferred to office areas for reading. Fresh tissue/sample slides using other methods (i.e. methanol fixation) should be read in the lab space only. Check your reagents in your slide stains to see if they eradicate pathogens. If not, check to see if it is possible to fix the slides in stronger ethanol solutions before staining.

These are just some of the many questions people are asking. There are references which can help to provide some of the answers (CDC.gov, WHO.int, OSHA.gov, etc.), but if you need more lab safety information, please feel free to send me questions at info@danthelabsafetyman.com. I will provide the most current safety guidance for your issue. This is a rapidly-changing situation, and information comes from many sources, Make sure you do your best to validate what you hear, and continue to use Standard Precautions and good behaviors to keep you and your laboratory staff safe from COVID-19 and other harmful pathogens.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.