Month: May 2023

The Latest Pox, Up Close and Personal

Against the backdrop of COVID-19, the world experienced a multicounty outbreak of Mpox (formally monkeypox) beginning in May of 2022. Prior to that time, the virus was primarily known to circulate within central and west African nations causing zoonotic disease. Clinical presentations of Mpox comprise signs and symptoms including rash on the hands, feet, face or mucous membranes and patients may experience fever or an influenza-like illness.1 Historically, transmission was associated with travel to an endemic region and contact with an infected animal. Importantly, the outbreak in 2022 was associated with broad changes in Mpox epidemiology, as most infections were acquired via sexual transmission.

Pox viruses and Mpox

Pox viruses are members of the family Poxviridae, which are double stranded DNA viruses that replicate entirely in the cytoplasm of host cells. They have worldwide distribution and cause disease in humans and other animals. Infection typically manifests as the formation of lesions, skin nodules or rash. Mpox belongs to the genus Orthopoxvirus which also includes other clinically important viruses including variola virus (smallpox), vaccinia virus, and cowpox. In the context of diagnosis, differentiation between the members of the Orthopox family becomes important.

The duration of illness with Mpox is usually between 2-4 weeks, with a variable incubation time most often between 6-13 days. The Mpox rash has historically been more focused on the face and extremities,2 and will cycle through stages including encrustation, scabbing, and eventually resolution. During the 2022 outbreak, an increasing number of presentations involved the anogenital and oral regions, further highlighting the change in epidemiology. The window for transmission is currently an area of active research as new data suggests transmission can begin prior to the appearance of symptomology.3

Diagnosis – Molecular

Mpox is generally diagnosed using PCR testing from a swabbed lesion. At the onset of this emerging infectious disease, the CDC shared its algorithm and testing for Mpox with public health laboratories. The first-generation algorithm largely reflected its potential use as a tool for screening for bioterrorism agents, which included using two-tiered testing. The first test was designed to demonstrate that Orthopox DNA was present and rule out variola virus by targeting the Orthopox DNA polymerase gene found not present in Variola (E9L-NVAR). The second step was to target an Mpox-specific gene encoding the envelope protein (B6R).4 It soon was readily apparent that the only Orthopox virus in circulation was Mpox, so the CDC updated its guidance in late June 2022 to confirming diagnosis of Mpox with the single Orthopox DNA-polymerase PCR assay.

However, despite this modification to improve expediency and like the situation faced at the onset of the COVID-19 pandemic, the need for testing greatly exceeded what public health infrastructure could support. Thus, laboratories designed and validated laboratory developed tests (LDTs) to expand access to testing, thus enabling physicians to interrogate the causes of a patient’s rash more thoroughly. This flexibility was essential given rising cases numbers and relatively non-specific symptomology of Mpox. By May 2023, over 80 laboratories registered Mpox LDTs with the Food and Drug Administration,5 and commercial device manufacturers are now including it in new and forthcoming assays still in development.

Diagnosis – Histopathology

Although PCR testing is the mainstay of diagnosis, histopathologic evaluation of biopsy material from a lesion can also provide insight into the viral etiology. Mpox infected skin biopsies demonstrate similar histopathologic features of infections caused by other pox viruses. As the rash continues to evolve over time, representative histopathological changes can also be observed. Early lesions may demonstrate ballooning degeneration, acanthosis and spongiosis. More mature lesions progress to near total keratinocyte necrosis with exocytosis comprised of mixed cellular inflammatory infiltrate.6 Eosinophilic bodies may be identifiable in the cytoplasm of infected cells, commonly known as Guarnieri bodies, represent the mature virions produced in the cytoplasm of infected cells.

Recently, the histopathological description of 20 outbreak-associated clinical cases of Mpox from Spain was reported. Epidermal necrosis and keratinocytic ballooning were commonly encountered microscopic features associated with Mpox lesions.7 Figure 1 is a skin biopsy from a patient who presented with a vesicular eruption in September with a history of mpox, syphilis and herpes simplex infection whose lesions were worsening. It similarly shows ballooning degeneration, epidermal necrosis, exocytosis of neutrophils into the epidermis, and intracytoplasmic eosinophilic inclusions (Guarnieri bodies) (Figures 2-3).

Figure 1. Histopathology of MPOX from a biopsied skin lesion (4x magnification, H&E). Intact epidermis with evidence of ballooning keratinocyte degeneration and infiltration of neutrophils.
Figure 2. Histopathology of Mpox (10x magnification, H&E). Epidermis with a cross-section of follicular infundibulum (hair follicle) is in the bottom left. The keratinocytes to the right demonstrate marked vacuolar change and small eosinophilic bodies can be observed in a background of neutrophils and necrotic keratinocytes.
Figure 3. Histopathologic findings of MPOX in a biopsy of a skin lesion (40x magnification, H&E).
High power magnification of viral inclusions, guarnieri bodies, (arrowheads) in a background of necrotic keratinocytes and neutrophilic infiltrate.

Treatment

Mpox is much milder than smallpox despite similar rash manifestations. In cases of severe Mpox infection, therapies used for smallpox have been compassionately utilized, but supportive measures are the mainstay of management of uncomplicated cases. Vaccination is now available as both a pre-exposure prophylaxis and post-exposure prophylaxis. It is important to note that the clinical effectiveness of the currently used vaccine in the United States is not known; however, early data across 32 US jurisdictions showed that among males 18-49, those who were unvaccinated had an Mpox incidence 14 times higher than similarly aged males who received at least one dose of vaccine at least 2 weeks prior.8

Conclusion

The Mpox outbreak, declared a global health emergency in July of 2022, has reinforced the need for flexibility within laboratories and industry to respond to emerging infectious diseases. The global health emergency for Mpox was declared over on May 11, 2023, but cases are still going to sporadically occur and minor outbreaks will result. The rapid development of numerous LDTs was essential to support the overwhelmed public health infrastructure, and this continued flexibility is needed to appropriately respond to future public health emergencies.

References

  1. https://www.cdc.gov/poxvirus/mpox/symptoms/index.html. Accessed April 19th, 2023.
  2. Saxena et al. J. Med. Virol. 2022;95:e27902.  DOI: 10.1002/jmv.27902
  3. https://www.cdc.gov/poxvirus/mpox/about/science-behind-transmission.html Accessed May 19th, 2023
  4. Li Y, Olson VA, Laue T, Laker MT, Damon IK. Detection of monkeypox virus with real-time PCR assays. J Clin Virol. 2006 Jul;36(3):194-203. doi: 10.1016/j.jcv.2006.03.012. Epub 2006 May 30. PMID: 16731033; PMCID: PMC9628957.
  5. https://www.fda.gov/medical-devices/emergency-situations-medical-devices/monkeypox-mpox-and-medical-devices#Laboratories. Accessed May 3, 2023.
  6. Bayer-Garner IB. Monkeypox virus: histologic, immunohistochemical and electron-microscopic findings. J Cutan Pathol. 2005 Jan;32(1):28-34. doi: 10.1111/j.0303-6987.2005.00254.x. PMID: 15660652.
  7. Rodríguez-Cuadrado FJ, Nájera L, Suárez D, Silvestre G, García-Fresnadillo D, Roustan G, Sánchez-Vázquez L, Jo M, Santonja C, Garrido-Ruiz MC, Vicente-Montaña AM, Rodríguez-Peralto JL, Requena L. Clinical, histopathologic, immunohistochemical, and electron microscopic findings in cutaneous monkeypox: A multicenter retrospective case series in Spain. J Am Acad Dermatol. 2023 Apr;88(4):856-863. doi: 10.1016/j.jaad.2022.12.027. Epub 2022 Dec 26. PMID: 36581043; PMCID: PMC9794029.
  8. https://www.cdc.gov/poxvirus/mpox/clinicians/vaccines/vaccine-considerations.html. Accessed May 3, 2023.

-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.

-Travis Vandergriff, MD is an Associate Professor and Board-Certified Dermatopathologist and practicing Dermatologist at UT Southwestern Medical Center.

-Andrew Clark, PhD, D(ABMM) is an Assistant Professor at UT Southwestern Medical Center in the Department of Pathology, and Associate Director of the Clements University Hospital microbiology laboratory. He completed a CPEP-accredited postdoctoral fellowship in Medical and Public Health Microbiology at National Institutes of Health, and is interested in antimicrobial susceptibility and anaerobe pathophysiology.

Toxicology and Forensic Pathology: More Than a Numbers Game

I was recently reviewing new toxicology reports from my pending autopsies, and came across a report with the following results: 

Looking at this in isolation, it would be easy to assume this person died from an overdose. Even low levels of fentanyl can be dangerous to an opioid-naive individual – a level this high is rare. Then there’s the added presence of fluoro fentanyl, a fentanyl analog, which would seem to support the notion of an overdose. The problem with this assumption? This person died from blunt force trauma as a pedestrian struck by a car. He was, according to witness accounts, walking and talking right until the moment of impact. Autopsy had shown multiple blunt force injuries incompatible with life.

This situation illustrates some of the complexity of postmortem forensic toxicology. Despite methodology being nearly the same, toxicology in a forensic setting differs in many important ways from that performed in a clinical setting.

The first major difference occurs in the pre-analytical phase. The results of clinical testing may be used to alter therapy or make a diagnosis. However, forensic toxicology results are meant to be used in a court of law, meaning the chain of custody needs to be maintained. If there is no documentation of who touched the sample and when, the integrity of the specimen can be called into question and results may be impermissible.

Not all forensic toxicology is performed on deceased patients. Specimens may be taken from the living during evaluation of an alleged assault, driving under the influence, or for workplace monitoring. In autopsy specimens though, postmortem redistribution (PMR) is another pre-analytical factor to consider. After death the stomach, intestines, and liver can serve as a drug reservoir and passively transfer the drug to surrounding vasculature. Other organs can also act as reservoirs, depending on where the drug is concentrated in life. Drugs which are highly lipid-soluble and/or have a high volume of distribution will diffuse down their gradient from adipose tissue into the bloodstream – antidepressants are notorious for this, and elevated postmortem levels need to be interpreted with caution.

Autopsy specimens are also more varied in type and quality than typical clinical specimens. Vitreous fluid, bile, and liver tissue are commonly collected at autopsy, in addition to central (heart) and peripheral (femoral or subclavian) blood. Femoral blood vessels, being relatively isolated from PMR-causing drug reservoirs, are a preferred source of specimens. Decomposition or trauma can limit the types or quantity of specimens and may even alter results. After death, bacteria from the GI tract proliferate and can produce measurable levels of ethanol in the blood. Decomposition also produces beta-phenethylamine, which can trigger a ‘positive’ result for methamphetamine on ELISA-based tests.

The post-analytical phase of autopsy toxicology also poses unique challenges. Lawyers and law enforcement will sometimes ask what the ‘lethal level’ of a drug is, and they’re invariably disappointed by my response. While there are published ranges of toxicity and lethality for most drugs, these are only general guidelines. There is no absolute lethal blood level for prescription or illicit drugs. Opioid users develop tolerance, making them relatively immune to a dose which would kill an opioid-naive person. In the example of the pedestrian described above, he had a long history of heroin abuse and could therefore tolerate much higher levels than most. For stimulants like cocaine and methamphetamine, there are no documented ‘safe’ levels as any amount could act as an arrhythmic agent. To add to the complexity, most overdose deaths involve multiple substances which may have synergistic effects and interactions that are difficult to parse.

Because of the reasons given above, the National Association of Medical Examiners still recommends full autopsy for possible overdoses. Deciding if a death was due to overdose is more complex than just reading a toxicology report – it requires interpretation and correlation with the autopsy findings and overall investigation.   

References

D’Anna T, et al. The chain of custody in the era of modern forensics: from the classic procedures for gathering evidence for the new challenges related to digital data. Healthcare. 2023 Mar;11(5):634.

Davis GG, et al. National Association of Medical Examiners Position Paper: Recommendations for the Investigation, Diagnosis, and Certification of Deaths Related to Opioid Drugs. Acad Forensic Pathol 2013 3(1):77-81.

Pelissier-Alicot AL, et al. Mechanisms underlying postmortem redistribution of drugs: A review. J Anal Toxicol. 2003 Nov-Dec;27(8):533-44.

You Can’t Hide Those Safety Eyes!

Jamie, the manager of a large metropolitan hospital lab, has many responsibilities. She must spend most of her time in the office, on the phone, or in meetings. She does find time to come out to speak with the employees, but only for a second to check on things or maybe make a request. During a recent safety audit, Jamie received feedback that several employees were seen working in the lab without using the proper PPE. One tech was working the bench without gloves, one individual had their lab coat on but not buttoned, and one auditor noticed that no one in the lab was wearing face or eye protection. This came as a shock to Jamie, she had never noticed this before. This doesn’t necessarily mean that Jamie is a bad manager, it could be that she was so focused on daily operation issues and she failed to notice other problems.

We have all heard the term “nose blind.” It’s when a person is around a bad smell so frequently that they become oblivious to its presence, and this can actually happen with vision as well. Have you ever heard the phrase, “you can’t see the forest for the trees,” or maybe the term “snow blind?” This phenomenon occurs when someone is concentrating so hard on one problem they may miss a more serious safety issue directly in front of them. Lucky for us, we have a tool to help those safety issues stand out. We have our “Safety Eyes!”

Ok, so what exactly are Safety Eyes? Are they some kind of new eye protection device that fit directly on your eyes? Are they indestructible eyes? Not exactly. Safety Eyes is a term used to describe the ability to spot current or potential safety issues more easily. It is the ability to walk into a room and immediately scan the environment for safety issues. This ability doesn’t just magically develop, it takes time and effort to master, and once you have it, you will begin to notice issues without even trying.

There are methods you can use to develop your safety eyes. Like any other sense, it is important to practice using it frequently so that its use becomes second nature to you. Think about this in terms of a wine sommelier. A sommelier may train for several years to acclimate their nose and palate in order to detect various nuances in different types of wine. It is through experience and exposure to many different types of wine that they are able to pick up on the slightest hint of a flavor or scent. This same repeated exposure works for sharpening your Safety Eyes as well. It is probably unlikely that you have a Safety Unicorn in your lab who can pick up on potential safety issues on their first day on the job. To become better at seeing safety issues, perform periodic rounding in the department and look for specific safety issues. Start by covering one specific safety area such as PPE use, waste management or fire safety. Your ability to quickly notice issues in these areas will sharpen, and you will be able to expand your newly honed power to other areas.

By developing your Safety Eyes, you will become more aware of various types of safety issues and where they are most likely to be encountered. It is easy to become “nose blind” to safety issues in a lab where you work every day. Start by simply using a checklist to focus specifically on one new safety area and soon the issues that may have been there all along will be more easily detected. Now that you can see the forest, you can make those important changes which will improve your overall lab safety culture!

-Jason P. Nagy, PhD, MLS(ASCP)CM is a Lab Safety Coordinator for Sentara Healthcare, a hospital system with laboratories throughout Virginia and North Carolina. He is an experienced Technical Specialist with a background in biotechnology, molecular biology, clinical labs, and most recently, a focus in laboratory safety.