Microbiology Case Study: A 60 Year Old Male Status-Post Orthotopic Liver Transplant with Headache and Word-Finding Difficulty

Case History

A 60 year old male with a past medical history of ulcerative colitis requiring total proctocolectomy and immunomodulatory therapy followed by an anti-Tumor Necrosis Factor α blocker for the last two years and primary sclerosing cholangitis with subsequent decompensated cirrhosis that ultimately required an orthotopic liver transplant on tacrolimus and prednisone for immunosuppression presents 17 days post-transplant with worsening headache for two weeks with associated word finding difficulty and expressive aphasia.

Laboratory and Diagnostic Findings

Brain magnetic resonance imaging demonstrated, a “Heterogeneous, partially hemorrhagic and centrally necrotic mass within the posterior left temporal lobe…infectious etiologies such as pyogenic/non-pyogenic abscesses to include fungal organisms, are highest on the differential” (Image 1). At the time of admission, his complete blood count demonstrated a leukocytosis (16.48×109 cells/L), anemia (hemoglobin of 7.8 g/dL, hematocrit of 24.8%) and a normal platelet count (367×109 cells/L). The automated differential showed 82% neutrophils, 10% lymphocytes, 6% monocytes, 1% eosinophils, and 1% basophils. A lumbar puncture was performed to obtain cerebral spinal fluid (CSF) and the analysis showed a glucose of 60 mg/dL, protein of 34 mg/dL, nucleated cell count of <1, and 6 red blood cells (completely normal CSF indices). Broad spectrum antimicrobials (Vancomycin, Piperacillin/Tazobactam, Metronidazole and Micafungin) were initiated. A 1,3-β-D-glucan test had a result of >500 pg/mL in both serum and CSF. Galactomannan, Histoplasma urine antigen, Cryptococcus antigen and other fungal testing were negative. Antifungal therapy was changed to voriconazole. Craniotomy was determined to be the best course of action and the patient was taken to surgery for debridement and pathologic evaluation.

Frozen section evaluation during the time of surgery showed granulomatous inflammation. Septate hyphae were observed on the fungal smear. Following surgery, amphotericin was added. Histologic evaluation of the tissue submitted from surgery showed pyogranulomatous inflammation with pigmented, spore-like structures present in multinucleated giant cells on hematoxylin and eosin (H&E) stain (Image 2). Grocott’s methenamine silver (GMS) stain also highlighted short segments of septate hyphae (Image 3).

Cultures from the surgical debridement grew a mould with central pigmentation (Image 4). Direct microscopic examination of the mould revealed thick-walled, oblong conidia with 3-5 cells, and uniformly pigmented hyphae (Image 5). A germ tube test showed germ tubes originating from both ends of the conidia consistent with Bipolaris species.

Image 1. T1-weighted (left) and T2-weighted (right) magnetic resonance imaging of the brain demonstrating a left temporal lobe mass.
Image 2. Hematoxylin and Eosin stained photomicrographs showing pyogranulomatous inflammation with giant cell formation and circular structures within them (left) (40x objective magnification). The right shows gold-brown pigmented structures within granulomatous inflammation (40x objective magnification).
Image 3. Grocott’s methenamine silver stain highlighting short segments of irregular septate hyphae in the brain debridement specimen (10x objective magnification).
Image 4. Mature wooly brown-black colony on potato dextrose agar.
Image 5. Photomicrograph of a lactophenol blue tape prep of the mature fungal colony. Pigmented hyphae and short 3-4 cell conidia are readily identified (40x objective magnification). This specimen also tested germ tube positive (not shown), indicating that this dematiaceous fungus is Bipolaris spp.

The patient’s mental status significantly improved following surgical debridement, 2 weeks of liposomal Amphotericin B, as well as long term treatment with voriconazole. The voriconazole was later switched to posaconazole due to concerns for fluoride toxicity. He completed a year of posaconazole with significant improvement of the abscess observed on imaging and resolution of headaches with no other visual problems. He continued to recover cognitive function with some residual difficulty with reading, comprehension and speech that eventually resolved.

Discussion

Phaeohyphomycosis refers to infections caused by dematiaceous fungi that exist in a variety of forms when seen in tissues and commonly involves skin, soft tissue and nasal sinuses.1 In rare cases, central nervous system (CNS) involvement has been reported. CNS phaeohyphomycosis is predominantly seen in immunosuppressed patients; however, cases involving immunocompetent individuals do exist.2 In one case series from Houston, Texas, five of seven cases of cerebral mycosis were caused by a dematiaceous mould.3 Interestingly, the patient presented in this case came to medical attention around the Dallas-Fort Worth area of Texas.

Cladophialophora bantiana is the most common dematiaceous fungus associated with CNS phaeohyphomycosis, but rare cases of Bipolaris species have been reported previously in literature.4-6

We report a case of CNS phaeohyphomycosis by Bipolaris species following orthotopic liver transplant with an excellent patient outcome. This case is unusual, in part, because the typical hospital course of a patient with phaeohyphomycosis is generally dismal.7 The stories of successful treatment often involve complete debridement of discrete lesions.7-8 In our case, the patient underwent surgical debridement and treatment initially with liposomal Amphotericin B and later transitioned to long term therapy with newer azole antifungals.

References

  1. Revankar SG, Sutton DA, & Rinaldi MG, (2004). Primary Central Nervous System Phaeohyphomycosis: A Review of 101 cases. CID, 38, 206-2016
  2. Filizzola MJ, Martinez F, & Rauf SJ, (2003). Phaeohyphomycosis of the central nervous system in immunocompetent hosts: report of a case and review of the literature. Int J Infec Dis, 7, 282-286
  3. Raparia K, Powell SZ, Cernoch P, Takei H, (2010). Cerebral mycosis: 7-year retrospective series in a tertiary center. Neuropathology, Jun; 30(3): 218-223.
  4. Frank T, Esquenazi Y, Nigo M, Wanger A, Portnoy B, & Shepard S, (2016). Disseminated Phaeohyphomycosis with Brain Abscess and Biliary Invasion Due to Bipolaris spp. In an Immunocompetent Patient. Annals of Clinical & Laboratory Science, 46(4). 
  5. McGinnis MR, Campbell G, Gourley WK, & Lucia HL, (1992). Phaeohyphomycosis Caused by Bipolaris spicifera, An Informative Case. Eur. J. Epidemiol, 8(3), 383-386
  6. Rosow L, Jiang JX, Deuel T, Lechpammer M, Zamani AA, Milner DA, Folkerth R, Marty FM, & Kesari S, (2011). Cerebral phaeohyphomycosis caused by Bipolaris spicifera after heart transplantation. Transpl Infect Dis, 13, 419-423.
  7. Dixon DM, Walsh TJ, Merz WG, McGinnis MR, (1989). Infections due to Xylohypha bantiana (Cladosporium trichoides). Rev Infect Dis, 11: 515-525.
  8. Gadgil N, Kupfermen M, Smitherman S, Fuller GN, Rao G, (2013). Curvularia brain abscess. J Clin Neurosci, Jan;20(1): 172-175.

-John Markantonis, DO is a second year Clinical Pathology resident at UT Southwestern in Dallas. He has interests in Medical Microbiology and Transfusion Medicine.

-Dominick Cavuoti, DO is a Professor at UT Southwestern in the Department of Pathology. He is multifaceted and splits his time as the Medical Director of the Parkland Hospital Clinical Microbiology Laboratory and Parkland Cytology attending among other administrative and educational activities.

-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.

Patient Advocacy: Introduction

“I do not really understand what pathology is,” I said during my first round of interviews at ASCP. “In fact, I have a website page in front of me that describes it and I still do not really get it. I want to be upfront about that before we go any further in my interview process,” I followed. Needless to say, I got the job, but that experience really stuck with me. As I learned more and more about pathology and laboratory medicine, I was amazed that I had not known more about it. I had been to the doctor all my life, I had received some serious diagnoses, and I thought I was pretty well-versed in what my medical care entailed.

In the last few years that I have been with ASCP I have become passionate about educating patients about the role the medical laboratory plays in patient care. Without that understanding, patients will be less empowered and less likely to advocate for themselves. Their family doctors might order tests that they do not want or not order ones they that do. They might not understand certain results, which means that they are less likely to take an active role in their care. The more we education patients and their caregivers about pathology and laboratory medicine, the higher quality health care we create. Educated patients are empowered patients and it is imperative that education includes the laboratory.

Through directing the ASCP Patient Champions program, I have been fortunate to meet incredible patients, all who have some understanding of the role the laboratory played and plays in their care. Hearing them say that without the laboratory, they would only be a memory, is incredibly powerful and humbling. The active role these patients play in their care has allowed them to be more resourceful and more hopeful. For some of them, seeing their own slides has been a cathartic experience because they could suddenly see the enemy they were fighting. Others are now educating new patients about their lab tests and taking time from their own busy schedules to volunteer at hospitals and clinics.

It can also be an inspirational experience for laboratory professionals and pathologists to hear how they impacted a patient’s life. I have personally shed many tears when interviewing patients so I can only imagine what it is like to hear from someone whose life you have impacted, let alone meet them in person. It can also really help patients to have their diagnosis be explained by someone working in the lab and to understand why their blood is drawn or why a biopsy is needed.

This new series on Lablogatory called Patient Advocacy, will explore the topic of patient advocacy from laboratory professional, pathologist, and patient perspectives. Each month, you will hear how patient interactions have impacted lives and what we can do to make more people aware of the crucial role the medical laboratory plays in patient care. You are all changing and saving lives every day. Let’s learn together how we can increase our patient advocacy to help them even more.

-Lotte Mulder, EdM, is the Senior Manager of Organizational Leadership and Patient Engagement at ASCP. She earned her Masters of Education from the Harvard Graduate School of Education in 2013, where she focused on Leadership and Group Development. After she graduated, Lotte started her own consulting company focused on establishing leadership practices in organizations, creating effective organizational structures, and interpersonal coaching. She has worked in Africa, Latin America, Asia, and the U.S. on increasing leadership skills in young adults through cultural immersion, service learning and refugee issues, and cross-cultural interpretation. She is currently working toward a PhD in Organizational Leadership.

Recognizing Disruptive Innovation in Global Health

One of the challenges of providing healthcare to patients of any type is “staying current” or “keeping up with the literature.” This can be especially challenging in the diagnostics laboratory where novel or unique approaches to a given test or test method or disease may show early promise but have no clinical utility, be too expensive, or not actually significantly change work-flow and/or patient value to justify implementation. On the other hand, sometimes a technology or test which is in development or approval can be so anticipated that clinicians and laboratorians are frustrated that it is not yet available.

In global health, there is a different problem that is encountered every day. There are technologies and tests that are approved, have documented clinical utility, and add great value to patients but they are simply not available because of supply chain, cost, administration, or geography. In such situations, the practitioners in these settings face extreme frustration—especially with stock-outs—and can become jaded and non-dependent on laboratory testing as part of care. This latter issue is a major challenge in cancer care where cancer diagnoses are required before treatment can begin; yet, in a large number of countries, access to cancer diagnostics routinely is not available. It is to that end that ASCP along with a whole host of NGO, industry, academic, and government partners are making great efforts to improve cancer care in each part of the continuum.

In this environment, however, disruptive innovations are, in fact, much easier to recognize as forthcoming. In the early 2000’s when I was working and traveling in Malawi, our project had a landline in the hospital to call the landline at the doctor’s house for issues overnight with patients. This required 24-hour nurses to be physically in the ward, tied to the phone and the patients. Landlines were expensive to install, had a very long waiting list to be installed, and, for the most part, the majority of the population in the country had never had a phone line in their dwelling. By the mid-2000’s, our project had one or more cellphones (as did the nurses) and communications through texting were nearly constant (especially since it was less expensive than making a phone call). By 2010, cell phones were ubiquitous in Malawi (and almost everywhere else in Africa) and there was no demand for landlines. Although this is a commonly used example, consider the adoption of cellular telephones and now smartphones in the US compared with Africa. There was push back, denial, avoidance, and even refusal to use them because there was an existing, well established system of landline communication.  If you want to install cable television and internet in your home as late as 2016, you were often required to bundle with a landline. The point is that the adoption pattern was significantly different because there was a pre-existing competitor with the new technology although—clearly—the new technology was superior.

Now consider a woman of 35 years who has a breast mass on mammogram in downtown Boston today. She will likely have an imaging study with immediate ultrasound and fine needle aspiration and/or core biopsy subsequent. A pathological diagnosis will be issued within 3 to 4 business days (or sooner) which includes a histological diagnosis along with hormone receptor status and Her2 staining. She will see a clinician likely within a week for a positive cancer diagnosis and a treatment plan will be decided upon and executed. If we consider a similar woman in downtown Nairobi, Kampala, or Lagos, they may, in fact, have a similar experience because of the recent efforts globally to improve cancer awareness, diagnosis, and treatment. There may be some delays (reports may take several weeks), potential stock-outs, etc. but, in these major cities, the services might exist. They are likely, however, provided in private clinics, will cost a premium, and may or may not have any guarantees about quality.

The reality, however, is that the vast majority of women in the US or Europe who present with breast cancer do so at a very early stage because of active screening programs which include mammography. The vast majority of women in low- and middle-income countries (LMICs) present with later staged disease because of lack of screening. The latter group of women, however, often live in rural conditions and/or poverty conditions such that seeking care for a breast mass (of any size) will require them to spend time and money to travel to one of the major cities and attempt to access services. With this situation, many of these cancers are detected by the health system at a late stage where curative therapy windows have been missed.

Onto these observations let’s now overlay access to a test for a breast mass that can be performed on a fine needle aspiration biopsy and resulted in ~4 hours which will provide a diagnosis of cancer (or benign) along with prognostic features directing treatment. If we consider the woman in Boston, we may see such a test providing an incremental improvement in care because billing systems, litigation fears, compliance requirements, or accreditation standards still include routine histology and immunohistochemistry to be performed on a tissue biopsy. To some degree, the test may be rejected because it is adding a cost over the standard costs without adding value (other than speed) to the results. However, for the woman in the rural village who likely has access to a community health worker, access to such a test could mean that she starts oral therapy the same day she has the health visit without ever having to leave her village. We have now removed the journey to a clinic that can performed a biopsy, the costs associated with that travel, the time lost while traveling and waiting for a result, and removed the risk that this is not breast cancer—which would mean all the time and money were wasted. For this woman, enormous value is created for her with a test that is performed same day with immediate results.

This concept of point-of-care (POC) cancer diagnostics would arguable meet resistance in the US or European system because of competition with existing systems and other issues as mentioned previously. In an LMIC setting, as there may be no competition, such an innovation would sweep the system and become standard of care—almost regardless of cost. This last bit is very important because traditional systems for performing histology and IHC are complex, costly, and require multiple highly trained individuals to get a quality result. If that process costs $75 to $100 US dollars (to the health system) to provide and, for the individual patient, $10s to $100s of dollar for the travel, lodging, and lost wages, the cost of such a test could, in a stable, high-income country (HIC) market, fetch a hefty price. However, if such a test is priced at $25 to $50 USD (half the cost of the current system excluding the travel), the immediate replacement of the old system with this new system for the given indication must and will occur. This uptake is amplified in an LMIC when the POC test moves to the patient in a geographically distributed process. Breast cancer is an obvious target for such an approach because the tumors are easily accessible, the disease is quite common globally, and the primary therapies are very inexpensive. Could such a test have an impact in an LMICs for bone marrow-based, lung, bladder, colon, prostate, liver, kidney, or soft tissue tumors? The answer to that question lies in the availability of therapy, incidence of disease, and access to radiological equipment rather than availability of the actual POC device. That is, once you have a POC test for one cancer, creating a subsequent POC test for another cancer is a surmountable technical hurdle. But will such a test be able to have an impact because of the alignment of the other factors? It is likely that as you are reading this sentence, you have thought of a few yourself but there are certain cancers where you are likely thinking, “not possible”.

For breast cancer, two such POC approaches are coming down the pipeline. The first is the Cepheid GeneXpert Breast STRAT4 assay which measures quantitative RNA (qRNA) for ESR1, PGR, ERBB2, and MKi67. These four assays are surrogates for standard immunohistochemical staining for ER, PR, Her2, and Ki-67, respectively. In a series of published and in press feasibility and validation studies, the qRNA assay is essentially equivalent to IHC. There are nearly a dozen studies of this new testing cartridge using formalin-fixed, paraffin embedded (FFPE) tissue throughout Africa where the test is being compared to standard IHC. However, in at least one site, the test is being performed directly on FNA material. The second test is from the laboratory of Dr. Sara Sukumar at Johns Hopkins which uses a set of DNA methylation markers that can separate benign from malignant disease on FNA using only 10 markers. By combining these two approaches (benign vs. malignant followed by STRAT4 for positive tumors), a diagnosis of malignant breast disease with prognostic factors for treatment could be obtained in less than 4 hours.

Let’s jump forward to the point in time when both of these POCs are available (or, in fact, any POC for cancer is available). How would they change the approach to breast or other cancer in an LMIC? Because both tests require only an FNA of a mass and because tumors of the breast and other organs today are often late staged, community health workers could be trained to evaluate patients with masses, perform the sampling, and run the test in a remote village. Regardless of stage, starting a breast cancer patient on estrogen receptor antagonists can provide palliative relief or pre-surgical treatment. As a population down stages—which occurs as community health workers begin routine screening—the testing can triage benign and malignant disease at a fraction of the cost for both the system and the patient. Based on population epidemiology, nearly exact costs for these services can be predicted for a population and stock outs can be avoided. Corollary note: Only for those cancers for which you HAVE a POC.

How would these tests change the approach to breast cancer in an HIC? There would likely be resistance at many levels but, eventually, the relatively low cost and the increased patient value would allow the tests to replace or displace standard diagnostics. Without complete replacement, there could, at a minimum, be multimodality redundancy which increases quality. However, the tests would find purchase within the system because in some settings their cost and added value would make any other choice impossible.

For both settings, we can now add other market entrants, other tests for other cancers, and a generalize increased in cancer awareness in the community, all of which would increase demand, improve morbidity and mortality, but decrease costs. Such a situation would be highly valued by the patients and, therefore, is the most important eventuality as this disruption ensues. Recognizing forthcoming change is sometimes hard and sometimes easy; however, accepting and embracing forthcoming change in healthcare can lead to best outcomes for our patients—the central mission of ASCP.

Dr. Milner has no financial disclosures regarding this blog post and has received no fiscal or in-kind support from any entity, named or otherwise, that involves this blog post.

References

  1. Wu NC, Wong W, Ho KE, Chu VC, Rizo A, Davenport S, Kelly D, Makar R, Jassem J, Duchnowska R, Biernat W, Radecka B, Fujita T, Klein JL, Stonecypher M, Ohta S, Juhl H, Weidler JM, Bates M, Press MF. Comparison of central laboratory assessments of ER, PR, HER2, and Ki67 by IHC/FISH and the corresponding mRNAs (ESR1, PGR, ERBB2, and MKi67) by RT-qPCR on an automated, broadly deployed diagnostic platform. Breast Cancer Res Treat. 2018 Nov;172(2):327-338.
  2. Wasserman BE, Carvajal-Hausdorf DE, Ho K, Wong W, Wu N, Chu VC, Lai EW, Weidler JM, Bates M, Neumeister V, Rimm DL. High concordance of a closed-system, RT-qPCR breast cancer assay for HER2 mRNA, compared to clinically determined immunohistochemistry, fluorescence in situ hybridization, and quantitative immunofluorescence. Lab Invest. 2017 Dec;97(12):1521-1526.
  3. Downs BM, Mercado-Rodriguez C, Cimino-Mathews A, Chen C, Yuan JP, Van Den Berg E, Cope LM, Schmitt F, Tse GM, Ali SZ, Meir-Levi D, Sood R, Li J, Richardson AL,  Mosunjac MB, Rizzo M, Tulac S, Kocmond KJ, de Guzman T, Lai EW, Rhees B, Bates M, Wolff AC, Gabrielson E, Harvey SC, Umbricht CB, Visvanathan K, Fackler MJ, Sukumar S. DNA Methylation Markers for Breast Cancer Detection in the Developing  World. Clin Cancer Res. 2019 Nov 1;25(21):6357-6367.

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.

From Safety Eyes to X-Ray Vision

In the Immunohistochemical stain lab, Rory made up his special stains under the chemical fume hood. One of the reagents he used was hydrochloric acid. At the end of each month there was usually a little bit of acid that needed to be disposed of as waste. He poured the waste acid into a glass jar and labeled the jar as “waste HCl.” He then carried the jar through the door to the room next door where there was an acid storage cabinet. That was where the contracted chemical waste vendor picked up other wastes from the lab.

Lydia was working the night shift in blood bank when she was changing the waste container on the automated type and screen analyzer. She splashed some waste into her eye when pulling the container out of the analyzer. She rubbed some water from the restroom sink in her eyes and decided not to report the incident as she was already in trouble with the supervisor for her continued absences.

I often talk to Lab Safety Professionals about using their “Safety Eyes” while performing their duties. It’s a latent ability we all have and can develop with some practice. With it, one can walk into a laboratory and quickly see safety issues and even make a swift assessment of the overall safety culture. Much of what can be seen using that super-power belongs to the lab’s physical environment- that which lies on the surface and should be visible to all. But sometimes there are deeper issues, those that may be more hidden. With practice, one might easily spot incorrect use of PPE, unlabeled chemicals or trip hazards. But how do you spot those other safety issues that can be just as dangerous- or even more so? How can your Safety Eyes ability be honed into something more powerful….like X-ray vision?

In the first scenario above, you may see nothing wrong, especially if you’ve performed that process yourself for years. One week later the EPA inspector came in for a laboratory waste audit, and they cited the lab for moving waste from the point of its generation to another area which was not designated as a Central Accumulation Area (CAA). Hazardous (chemical) waste cannot be moved to another location outside the line of sight of its generation point unless that other area is treated a CAA.

In the second scenario Lydia woke up the next day because her eye began to burn. She went to the emergency room and told her story. Because she missed the window of opportunity for proper treatment of an unknown source exposure to biohazards, she had to undergo long-term treatments which involved strong medications which have unpleasant side effects. She also had to be tested regularly for Hepatitis and HIV.

Some people you may know in the lab have been performing unsafe acts for years with little or no known consequences. Have they been doing the right thing or have they been lucky? What will it take to correct those unsafe actions? A fine? An exposure or injury? Hopefully not. Sometimes the reason unsafe acts occur is that staff is unaware of the regulations or the potential consequences. Influencing others’ safety behaviors is another more subtle super-power of the Lab Safety Professional, but it can be both important and useful.

As a safety professional, make sure you develop your basic super powers- your Influence and your Safety Eyes- but also be sure to augment what you already know how to use. Learn to use some X-ray Vision. Look more deeply for those processes and actions that may have been in place for years. It is not too late to make a change and prevent an incident that was years in the making.

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.

Up in Smoke

Hello again everybody, and welcome back! Last month, I was flattered by a double feature with my post about giving a TEDx talk and Dr. Razzano interviewing me for her global health series. This month, I’d like to address a topic that’s been literally everywhere lately and is just as hard to ignore as…well, second-hand smoke. So, fasten your seatbelts, ensure your seats and tray tables are in the upright position, make sure your biases are stowed in the seat before you, and (of course) please note the no smoking sign as we take off on the topic of vaping!

Image 1. What? I’ve been traveling a lot. There’s inspiration everywhere!

The Smoking Gun

You may have noted that in the past few weeks or months the topic of vaping has been a mainstay of nighttime news stories and front-page print articles. That’s because there’s a lot happening, and from a lot of different angles. It can be messy and confusing, especially because there’s a scientific and non-scientific debate: availability, marketing, health risk, research, and more—all happening at once. I’m going to talk a little bit about all of this, but mostly we’ll look at the medical aspect of vaping as some fantastic publications are making their way into medical journals, including our very own American Journal of Clinical Pathology (AJCP). Recently, friend, colleague, and fellow member of the ASCP Social Media Team and pulmonary pathologist at the Cleveland Clinic, Dr. Sanjay Mukhopadhyay (@smlungpathguy on Twitter) published a noteworthy article with AJCP demonstrating the histopathologic findings of vaping associated lung injury. In essence, vaping causes acute lung injury which is recognized in tissue, supporting the case that both further studies are mandated for health and safety and that vaping should currently be considered a potential critical health risk.

Image 2. About half of the official ASCP Social Media Team (#ASCPSoMeTeam!) from left to right Lab scientist and educator Aaron Odegard (@odie0222), myself (@CEKanakisMD), Dr. Sanjay Mukhopadhyay (@smlungpathguy), famous resident Dr. Adam Booth (@ALBoothMD), and Dr. Kamran Mirza (@Kmirza). If you want updates with great pathology and lab medicine stories and content—follow ALL OF THESE twitter handles!

In this paper, Dr. Mukhopadhyay, et al, tried to capture the direct tissue-related effects of vaping. EVALI, Electronic-Cigarette or Vaping use Associated Lung Injury, has received quite a bit of spotlight in the media as I mentioned. Case series featured in the New England Journal of Medicine (NEJM) highlighted patients in the Midwest with EVALI-type pulmonary disease, but the number of publications on the topic is currently scarce—let alone ones that demonstrate the actual pathophysiology in-process in those affected patients. In the AJCP paper, lung biopsies from a small number of male patients who havdrespiratory illness and concurrent histories of vaping were examined. With all other pulmonary pathology worked up and negative, their biopsies showed various patterns of acute lung injury. The NEJM cases were also worked up and found to be negative for the differentials of pulmonary disease whether infectious, inflammatory, or otherwise; adding credence to a developing body of research supporting the connection between vaping and EVALI.

Image 3. Here’s the mainstay paper I keep referencing. It’s part of a growing number of published works on the topic and part of our expanding understanding of EVALI and its health implications from both public health and pathologic/diagnostic viewpoints.

Where There’s Smoke, There’s…a Lot of Stuff, Actually

There are a ton of stories in the lay-press about vaping-related illnesses. The surveillance data from those NEJM case series and the CDC show a median age of 19 with an overwhelming 94% being hospitalized and roughly two-thirds of those requiring ICU intervention and one-third having to be placed on mechanical ventilation. Of note, 11% of these patients claimed that they vaped pure nicotine product, while 89% smoked cannabinoids/THC in their vape products. Most of them presented to medical care with oxygen saturations <89% on room air (normal O2 sats are variable by patient, but they should be above 95% in ideally healthy individuals). This is neither an endorsement or comment on the medical uses of cannabinoids or a statement on their health effects. Instead, it should be worth mentioning that not only are electronic-cigarette products a new way of smoking higher concentrations of tobacco-obtained or synthetic nicotine but also other products, which have very little data with regard to their associated health risks.

Image 4a. If you haven’t been able to read Dr. Mukhopadhyay’s paper yet, don’t worry I got you. Here are a few cases’ computed tomography (CT) scans that show clinically diagnostic evidence of pulmonary disease visible as (A) ground-glass opacity, GGO, with a pattern that mimics a peripheral eosinophilic pneumonia, (B) more GGOs with areas of consolidation, or solid-looking lung tissue, (C) lower lung GGOs and consolidation with some thickened tissue, and (D) patchy GGOs. All of these cases and more demonstrated some kind of pneumonia and lung tissue pathology but had been worked up and found negative for other causes of disease aside from their shared history of vaping.
Image 4b. Okay, this is a blog for medical laboratory professionals, right? So here’s some slides for the glass pushers! PLEASE NOTE: this is just a sample of a number of histopathologic findings published in the paper, so to see the rest go to the primary source. I’ve highlighted these images as they demonstrate the two major lung injury patterns seen in the EVALI entity: organizing pneumonia seen in (A) & (B) and diffuse alveolar damage (DAD) seen in figures (C) & (D).

Put This in Your Pipe and (please don’t) Smoke It

Okay, I mentioned cannabinoids. Now that I have your attention, I want to walk you through a unique piece of the EVALI discussion you may have seen in the media: the implication of Vitamin-E substances as a potential culprit for these lung-related injuries. The New York Times recently published a piece that cites the CDC’s consideration of Vit-E Acetate as a “a very strong culprit.” Think about it this way: the aerosol generated by vaping devices can reach very high temperatures (higher than traditional cigarettes), if a substance is inhaled at this temperature, and contains lipid-soluble-contents like Vitamin-E acetate, you’re breathing in a grease fire! Here’s an oversimplification: some studies of vaping came up with a theory that a grease fire would cause injury in the lungs similar to a pattern caused by inadvertent inhalation of mineral oil into the lungs known as “exogenous lipoid pneumonia”. However, when expert lung pathologists including Dr. Mukhopadhyay looked at lung biopsies from EVALI patients, they didn’t find even a single case of exogenous lipoid pneumonia. What does this mean? Not much at this point. It’s certainly possible that vitamin E acetate causes lung damage but not in the way mineral oil does. As the CDC materials state, this is early days if it is indeed a health epidemic (it probably is though, please stop vaping). More research is needed, as always, but you can read the NYT article and CDC primer article here.

Image 5. Not all that glitters is…Vitamin-E Acetate. The paper includes images of exogenous lipoid pneumonia (not from the cases studied) and endogenous lipoid pneumonia (from an EVALI case) as a comparison. Note that from a tissue standpoint, the lipid- filled macrophages on the right from an EVALI patient do not resemble the lipid-filled macrophages on the left (caused by mineral oil). Sure, there’s lipid in macrophages in the EVALI lung, but is that because a lipid is causing the damage, or because lipid from the membranes of injured cells is being cleaned up by macrophages? Lung pathologists think that the latter is more likely.

Fired Up, Ready to Go and Sending Smoke Signals

So, imagine you’re a vaper. Imagine you started because it helped you quit traditional cigarettes. That’s fantastic, good for you. You’re on the road to smoking cessation and better health! But perhaps the vaping-associated lung injury cases has made you a little defensive. Trust me I learned the hard way as I joined in the discussion earlier this month on a live-tweet pathology journal club on the AJCP article featured here. They happen under the hashtag #PathJC and lots of folks jump into the discussion from different places, institutions, time zones, and across disciplines—but its not just a bunch of pathologists analyzing an article in an academic bubble. Twitter is a public forum and that brings with it public scrutiny and commentary. As such, there were lots of lay people participating in the discussion and many individuals who held a positive opinion of electronic cigarettes. So not only did we have a very comprehensive discussion in the merits and shortcomings of published literature on the topic of EVALI, we also had to field questions and engage in non-jargon conversations with concerned (and sometimes passionate) members of the non-scientific community. Suffice it to say, it’s a tricky tightrope to walk when you’re trying to balance your anti-smoking public health crusade with some good old-fashioned medical education challenged with a sprinkle of vitriol on the most open of forums, the internet. But that’s okay! I strongly think, that in the future of medical practice, those of us in any discipline (but especially pathology and lab medicine) should lead the charge as champions of truth to connect our revered medical data to people in real terms—basically translate translational medicine.

Image 6. Why am I showing you my twitter profile picture? Easy: one of those “incendiary” comments in discussing smoking and vaping in a public forum actually included someone screen capturing my profile and accosting my “smug” pose and taste for esophageal damage in drinking hot coffee, citing poor data references for caffeine related deaths versus that of smoking. How do you deal with this? Calmly, with open honest information and, most importantly, with humility to address the barriers in communication between opposing points of view. Champions of truth, remember? But once you notice you’re talking to folks online who represent companies in the tobacco industry, ABORT MISSION, you went too far, haha! (True story, yikes!)

Once the Smoke Settles

Basically, everything’s going to be okay. There’s always a crisis or an epidemic happening that we have to address with limited data, developing knowledge, and some cohort of representative push back. That’s the nature of public health. But I’ll pull straight from the authors’ conclusion in the AJCP paper and remind you that not only is this just one, single study with very small number of cases to measure clinical outcomes, but further study is needed to support what is just beginning to be a correlation between vaping and lung injury.

TL;DR – it might seem obvious to some that hot smoke burns your lungs, but we’ve got to prove it and take steps to protect our patients everywhere.

And the good news is there are lots of us working on this. Scientists, public health officials, researchers, reporters, medical professionals, and especially pathologists are here collecting data and adding knowledge to that growing body of evidence to address this …hot topic.

Image 7. Here’s at least two of those people. Spoiler: it’s my wife and me. Here we are the recent American Public Health Association (APHA) conference in Philadelphia where the topic of vaping, smoking, and lung injury were very much in the forefront of public health research as it fits into the context of social determinants of health, medical literacy campaigns, and other concurrently related health issues like asthma and COPD.
Image 8. I actually joined that live tweet #PathJC journal club discussion from the APHA 2019 conference and was lucky enough to have, in-hand, the official EVALI clinical information release from the CDC booth in the expo floor. Check it out on my Twitter feed.

Breathe Easy

What’s past this smokescreen challenge? The same thing as always: hard work, collaboration, innovation, and paradigm shifting. If you’ve read my previous posts, you know I like to wax a bit about the future of medicine and the humanity behind our profession. Taking everything into consideration with this newest and hottest of public health concerns, our role as diagnosticians and translational representatives is as important as ever. And, if we want to ensure the recognized contributions of pathology in the wider field of medicine (and health-at-large) we should work with our colleagues in and out of the medical profession to demystify this kind of research, cleanly communicate health data to the public, and push the boundaries of personalized health and improved patient outcomes. But beware: when you address big topics like smoking, vaping, EVALI, and THC use, it can be easy to get too hot, and even burn out.

Thanks again, see you next time, and hope you had a Happy Thanksgiving!

(This is absolutely stolen from @iHeartHisto on Twitter, but enjoy a slice of pump-skin pie!)

Constantine E. Kanakis MD, MSc, MLS (ASCP)CM completed his BS at Loyola University Chicago and his MS at Rush University. He writes about experiences through medical school through the lens of a medical lab scientist with interests in hematopathology, molecular, bioethics, transfusion medicine, and graphic medicine. He is currently a 2020 AP/CP Residency Applicant and actively involved in public health and education, advocating for visibility and advancement of pathology and lab medicine. Follow him on Twitter @CEKanakisMD

Hematopathology and Molecular Diagnostics Case Study: A 63 Year Old Man with Fatigue

The following case is an interesting overlap of Hematopathology and Molecular Diagnostics, and shows the utility of sequencing to detect a cancer before biopsy could.

A 63 year old gentleman presented to a heme/onc physician with six months of intractable anasarca, fatigue, and a recent mild thrombocytopenia (Table 1). They were otherwise in healthy condition. The physician initiated a lymphoma work-up that included a bone marrow biopsy. The tests were negative for M-protein.

Table 1. Summary of symptoms and relevant abnormal labs.

The bone marrow biopsy was somewhat limited, but the core contained multiple marrow elements. After a thorough review by a Hematopathologist, no evidence of dysplasia or other irregularities could be detected (Image 1). Flow cytometry detected no aberrant blast population. Cytogenetics detected 20del [16/20] and 5del [3/20]. These findings did not clearly indicate a specific diagnosis.

Image 1. 40x view of the bone marrow specimen at the initial presentation. No evidence of dysplasia was found.

As the clinical suspicion for a malignancy was high, the bone marrow specimen was sent for sequencing on a 1385-gene panel test. The test included tumor-normal matched DNA sequencing (“tumor” sample: bone marrow, normal: saliva), RNA whole transcriptome sequencing on the bone marrow, and Copy Number Variant (CNV) analysis. Tumor-normal matched sequencing helps rule out variants that are normal and present in the patient.

Somatic mutations were determined as those that were present in the “tumor” sample and not in the matched normal sample. The somatic variants found are listed below with their variant allele frequency (VAF) in parenthesis. Recall that a VAF of 40% means that a mutation is present in the heterozygous state in 80% of cells.

  • IDH2 (p.R140Q, 46%)
  • SRSF2 (p.P95T, 51%)
  • CBL (p.R499*, 47%)
  • KRAS (p.K117N, 12%)
Figure 1. View of IGV, which displays the NGS reads for IDH1 along with the variant allele highlighted in red. The color of the bars indicates the direction of the reads (forward in red and reverse in blue). This reflects the allele frequency of approximately 50%.

The mutations in these genes are commonly found in myeloid cancers including myselodysplastic syndrome. Activating mutation in IDH2 (isocitrate dehydrogenase 2) increase the production of the oncometabolite 2-HG, which alters methylation in cells taking them to an undiffereitiated state. SRSF2 (Serine And Arginine Rich Splicing Factor 2) is a part of the spliceosome complex, which regulates how sister chromatids separate from each other. Failures in the proper function of the complex creates genomic instability. CBL (Casitas B-lineage Lymphoma) is a negative regulator of multiple signaling pathways, and loss of function mutations (as seen here) lead to increased growth signals through several tyrosine kinase receptors. KRAS (Kirsten RAt Sarcoma virus) is an upstream mediator of the RAS pathway, which acquires mutations that lead to constitutive activation and sends growth signals to cells causing them to proliferate.

Furthermore the CNV analysis also found the heterozygous loss of chromosome 20 as reported in cytogenetics. CNV analysis did not detect chromosome 5 deletion, as it was below the limit of detection (20% for CNV analysis).

Figure 2. This plot shows the normalized read frequency of genes across each of the chromosomes is shown here. The drop at chromosome 20 is shown in a pale brown color on the right side of the graph. This is consistent with the cytogenetic findings. The loss of 5q isn’t seen as it is below the limit of detection of 30%.

These mutations are all individually common in MDS, but the co-occurance of each gives very strong evidence that MDS is the diagnosis (Figure 3). There have also been studies that provide prognostic implications for several of the genetic mutations present. Some mutations like SRSF2 or CBL at high VAF (>10%) indicate a poor prognosis, but mutations in IDH2 or TP53 at any frequency have not only a high chance of progression, but also a faster time to onset of disease. Another non-genetic risk factor for developing MDS is an elevated RDW, which we saw in our patient.

Figure 3. From Becker et al 2016.

All of these high-risk factors together led us to push for a diagnosis of MDS based off of molecular findings, and the patient was started on treatment with Azacitadine. Our assessment was confirmed 3 months later when, the patient’s follow up bone marrow biopsy showed significant progression with megakaryocytic and erythroid dysplasia and hyperplasia and reticulin fibrosis MF2 (Image 2). Aberrant blasts were detected (1-2%), but not elevated. This demonstrates how molecular findings predicted and predated the patient’s rapid progression to morphologic disease.

Image 2. Dysplastic, hyperplastic megakaryocytes and erythroid lineage.

In summary, multiple molecular mutations indicative of MDS were found in a symptomatic patient’s unremarkable bone marrow biopsy months before a rapid progression to MDS.

References

  1. Steensma DP, Bejar R, Jaiswal S et al. Blood 2015;126(1):9-16.
  2. Sellar RS, Jaiswal S, and Ebert BL. Predicting progression to AML. Nature Medicine 2018; 24:904-6.
  3. Abelson S, Collord G et al. Prediction of acute myeloid leukemia risk in healthy individuals. Nature 2018; 559:400-404.
  4. Desai P, Mencia-Trinchant N, Savenkov O et al. Nature Medicine 2018; 24:1015-23.
  5. Becker PM. Clonal Hematopoiesis: The Seeds of Leukemia or Innocuous Bystander? Blood.2016 13(1)

-Jeff SoRelle, MD is a Chief Resident of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX. His clinical research interests include understanding how the lab intersects with transgender healthcare and improving genetic variant interpretation.

A 66 Year Old Male with Diarrhea, Weight Loss, and Night Sweats

Case History

A 66 year old man with past medical history of recently diagnosed Clostridioides difficile colitis presented to emergency department with diarrhea, weight loss of 52 pounds in 4 months, and occasional night sweats. CT imaging revealed dilation of small bowel with thickened mucosal folds. The duodenum was subsequently biopsied to reveal diffuse intestinal lymphangiectasia containing PAS positive and Congo red negative eosinophilic material and lamina propria foamy macrophages. Laboratory investigations revealed normocytic anemia, proteinuria, and peripheral IgM kappa monoclonal gammopathy.

Biopsy Findings

Image 1. Aspirate.
Image 2. Core biopsy.
Image 3. CD138.
Image 4. Kappa ISH.
Image 5. Lambda ISH.

Bone marrow aspirate shows increased plasma cells and mast cells. H&E stained sections demonstrate a normocellular bone marrow with trilineage hematopoiesis and involvement by 35% plasma cells. By immunohistochemistry, CD138 highlights clusters of plasma cells that predominantly express kappa light chain restriction.

FISH and Mutation Analysis

FISH demonstrated loss of chromosome 11 and gain of chromosome 15, which was consistent with plasma cell dyscrasia. MYD88 mutation analysis did not detect the mutation.

Diagnosis

The findings of the patient’s normocytic anemia, IgM monoclonal gammopathy, and intestinal lymphangectasia with an associated plasma cell dyscrasia involving the bone marrow favor a lymphoplasmacytic lymphoma/Waldenström macroglobulinemia.

Discussion

Waldenstrom macroglobulinemia (WM) is a malignant B-cell lymphoproliferative disorder characterized by lymphoplasmacytic infiltration of the bone marrow and peripheral IgM monoclonal gammopathy.1 It is rare with an overall incidence of 3 per million persons per year, accounting for 1-2% of hematologic cancers.1 It occurs predominantly in Caucasian males, with a median age of 63-68 years old at diagnosis.1-3

Patient may be asymptomatic for years and require observation or experience a broad spectrum of signs and symptoms. These symptoms may be attributable to the tumor infiltration of the bone marrow and lymphoid tissues, IgM circulating in the blood, and IgM depositing into tissues. The most common clinical presentation of WM is fatigue and nonspecific constitutional symptoms, such as fever, night sweats, and weight loss, due to normochromic, normocytic anemia. 20-30% of patients may exhibit lymphadenopathy and hepatosplenomegaly due to infiltration of peripheral tissues. High concentration of IgM in the circulation may lead to hyperviscosity, resulting in oronasal bleeding, gingival bleeding, blurred vision due to retinal hemorrhages, and neurological symptoms, including headache, ataxia, light-headedness, dizziness, and rarely, stroke.2-3 The gastrointestinal manifestations are rare; however, IgM monoclonal protein may deposit into the lamina propria of the GI tract, causing diarrhea, steatorrhea, and GI bleeding.4 Other IgM-related manifestations include cold agglutinin hemolytic anemia, cryoglobulin, and amyloid deposition in tissues.3

Diagnosis of WM includes evidence of IgM monoclonal gammopathy and at least 10% of bone marrow infiltration by lymphoplasmacytic cells.5 Monoclonal gammopathy can be detected by the monoclonal spike, or M-spike, on serum protein electrophoresis.3 Serum immunofixation may be performed to identify the type of monoclonal protein and the type of light chain involved.3 In terms of immunophenotype, neoplastic cells express surface IgM, cytoplasmic Igs, CD38, CD79a, and pan B-cell markers (CD19, CD20, and CD22). CD10 and CD23 are absent. Expression of CD5 occurs in approximately 5-20% of cases.6 Recent studies have reported two most common somatic mutations in WM, which are MYD88 L265P mutations (90-95% of cases) and CXCR4 (30–40% of cases).7 Absence of these mutations, however, do not completely exclude the diagnosis of WM.

The International Staging System for WM identifies five factors associated with adverse prognosis, including age older than 65, hemoglobin < 11.5g/dL, platelet count < 100K/μL, beta-2-microglobulin > 3mg/L, and monoclonal IgM concentration > 7g/L.3 Patients younger than the age of 65 years with 0 or 1 of these factors are in the low-risk category with a median survival of 12 years.3 In contrast, patients with 2 or more risk factors are in the intermediate- and high-risk categories and have a median survival of almost 4 years. 3

Management of WM depends on the patient’s clinical manifestations.Furthermore, patients with minimal symptoms should be managed with rituximab, whereas patients with severe symptoms related to WM should receive more aggressive treatment, including dexamethasone, rituximab and cyclophosphamide. Hyperviscosity syndrome is an oncologic emergency that requires removal of excess IgM from the circulation via plasmapheresis.8

References

  1. Neparidze N, Dhodapkar MV. Waldenstrom’s Macroglobulinemia: Recent advances in biology and therapy. Clin Adv Hematol Onco. 2009 Oct;7(10): 677-690.
  2. Leleu X, Roccaro AM, Moreau AS, Dupire S, Robu D, et al. Waldenstrom Macroglobulinemia. Cancer Lett. 2008 Oct;270(1):095-107.
  3. Tran T. Waldenstrom’s macroglobulinemia: a review of laboratory findings and clinical aspects. Laboratory Medicine. 2013 May;44(2):e19-e21.
  4. Kantamaneni V, Gurram K, Khehra R, Koneru G, Kulkarni A. Distal illeal ulcers as gastrointestinal manifestation of Waldenstrom Macroglbulinemia. 2019 Apr; 6(4):pe00058.
  5. Grunenberg A, Buske C. Monoclonal IgM gammopathy and Waldenstrom’s macroglobulinemia. Dtsch Arztebl Int. 2017 Nov;114(44):745-751.
  6. Bhawna S, Butola KS, Kumar Y. A diagnostic dilemma: Waldenstrom’s macroglobulinemia/plasma cell leukemia. Case Rep Pathol. 2012;2012:271407.
  7. Varettoni M, Zibellini S, Defrancesco I, Ferretti VV, Rizzo E, et all. Pattern of somatic mutations in patients with Waldenstrom macroglobulinemia or IgM monoclonal gammopathy of undetermined significance.
  8. Oza A, Rajkumar SV. Waldenstrom macroglobulinemia: prognosis and management. Blood Cancer Journal. 2015;5:e394.

-Jasmine Saleh, MD MPH is a pathology resident at Loyola University Medical Center with an interest in dermatopathology and hematopathology. Follow Dr. Saleh on Twitter @JasmineSaleh.

–Kamran M. Mirza, MD, PhD, MLS(ASCP)CM is an Assistant Professor of Pathology and Laboratory Medicine, Medical Education and Applied Health Sciences at Loyola University Chicago Stritch School of Medicine and Parkinson School for Health Sciences and Public Health. A past top 5 honoree in ASCP’s Forty Under 40, Dr. Mirza was named to The Pathologist’s Power List of 2018 and placed #5 in the #PathPower List 2019. Follow him on twitter @kmirza.