Hematology Case Study: Too Many Platelets?

Too many platelets? We know that low platelet counts can pose problems for hematology analyzers and that reporting accurate results is vital for good patient care. We learn and read a lot about thrombocytopenia and its various symptoms, causes, and treatments. But, what about thrombocytosis? What happens when there are too many platelets?

In my last blog I compared 2 cases of newly diagnosed CML. Lately I have seen so many new leukemia cases and myeloproliferative diseases that I have become fascinated with them. When I was in college and grad school (many moons ago), nomenclature, diagnoses and knowledge of these disorders were very different, so it’s been fun learning about them all over again!

Today’s case is of a 55 year old woman who was referred for a hematology consult because of a finding of increased RBC and platelet counts. White blood cells appeared normal with few reactive lymphocytes noted. The peripheral smear showed mild anisocytosis and dacrocytes. Platelets were markedly increased with large forms present. No giant platelets were noted. A bone marrow biopsy was ordered. Pre-Op diagnosis: Thrombocytosis.

Bone marrow results reported increased myeloid forms with full spectrum of maturation, erythroid elements normal in number with normoblastic maturation, and markedly increased megakaryocytes with numerous large hyperlobated forms. M:E ratio was increased. No iron was seen on iron stain. A reticulin stain showed mildly increased reticulum fibrosis (1+). Next generation sequencing studies demonstrated a JAK2 V617F mutation. BCR-ABL mutation was not detected. Diagnosis: Myeloproliferative neoplasm most consistent with Essential Thrombocythemia (ET).

Myeloproliferative neoplasms (MPN) are a group of disorders characterized by the over proliferation of WBCs, RBCs, or platelets. These can be separated into the Philadelphia chromosome (Ph) positive Chronic Myelogenous Leukemia (CML) and Ph negative neoplasms. The BCR-ABL oncogene is formed on the Ph and is responsible for the unregulated proliferation of cells seen in CML. At diagnosis over 90% of CML cases are BCR-ABL positive. (See Case Studies in Hematology: Presenting a double feature starring Chronic Myelogenous Leukemia). On the other hand, Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are the three classic Ph negative neoplasms.

Many ET patients have no symptoms at diagnosis but are found to have a high platelet count on a routine CBC. Diagnosis is based on ruling out other disease and testing for genetic mutations, which can be done from a peripheral blood sample or bone marrow. In addition to any blood tests, a bone marrow biopsy is typically recommended for differential diagnosis of MPNs. The most common mutation found in PV, ET or PMF, is in the JAK2 gene. The JAK2 V617F mutation is found in nearly all PV patients, and about 50-60% of ET and PMF patients.Othermutations found in the classic MPN group include CALR, the second most common genetic abnormality after JAK2 mutations,and MPL W515L.Normally, blood cells are only produced when the body has a need for the cells, but these genetic mutations turn a gene ‘on’, causing the unregulated production of the affected blood cell line. Until recently it was believed that a patient with PV, ET or PMF will have a mutation in only one of these genes. However, in 2018, a French group reported that CALR or MPL mutations may co-exist in a small percentage of patients with a low burden of JAK2 V617F mutation. (Accurso) Some patients are triple-negative for the JAK2MPL and CALR mutations and always have a poor prognosis.

The identification of a genetic marker in MPNs is valuable because a JAK2 mutation distinguishes PV from other disorders that may cause polycythemia. As well, a JAK2 or other mutation can distinguish ET from other causes of reactive thrombocytosis and PMF from secondary causes of myelofibrosis. In addition, most CML cases are diagnosed with a very high WBC, but occasionally patients with CML have a normal or only slightly elevated WBC with a high platelet count. Therefore, patients with suspected ET are also evaluated for CML with a test for the Philadelphia chromosome. Our patient was found to have a JAK2 V617F mutation, BCR-ABL negative and was diagnosed with ET.

ET was first recognized in the 1950’s and was termed a myeloproliferative disorder. At this time, it was not known what was causing the over proliferation of platelets. Theories were broad and ranged from ‘something environmental’ to ‘an internal defect’. Over the decades, it became more apparent that the myeloproliferative disorders were caused by internal defects in stem cells, and they were renamed MPN. In 2005, four separate research groups, using different methods, all identified the JAK2 V617F allele, which led to further understanding of PV, ET and MPN. The MPL mutation was discovered in 2006 and CALR mutations were discovered in 2013.

ET is a type of chronic leukemia and patients with ET generally have a normal life expectancy. Of the 3 BCR-ABL negative MPN, ET has the best prognosis. Treatment is often not needed, other than aspirin for prevention of blood clots. Patients are placed in risk factor groups based on risk of clots or bleeding. A patient <60 years with no JAK 2 mutation and no prior thrombosis is considered very low risk and would be simply observed or prescribed low dose aspirin. Patients <60, JAK2 V617F +, with no prior thrombosis have low risk and would be treated with aspirin, dosage dependent on any cardiac risk factors. Older patients over 60 with JAK2 wild type and no history of thrombosis may be treated with aspirin alone or with cytoreductive therapy. Lastly, the highest risk patients are those over 60, JAK2 V617F + or with prior thrombosis, and would be treated with cytoreductive therapy, such as hydroxyurea. With very high platelet counts, there is a risk of both blood clots and hemorrhage. Blood clots that develop in thrombocythemia can use up the body’s platelets and result in bleeding. For this reason, cytoreductive therapy such as hydroxyurea is recommended to reduce hemorrhage in high-risk patients with very high platelet counts over 1,000 x 103/ μL. Hydroxyurea can also be used as treatment in patients who have a mixed population of PV and ET. CALR mutated patients with ET tend to be young with a much lower thrombotic risk and do not generally require therapy. Aspirin in this group is considered overtreatment because CALR+ patients suffer more risk of bleeding with aspirin.

While there is some risk of a MPN transforming to another type, ET is the MPN least likely to transform or to progress to acute myeloid leukemia. ET also has a better prognosis than the other MPN. Even so, there is often not one clear cut entity. There can be overlap between the disorders, causing some difficulty in diagnosis and treatment decisions. For instance, a physician may have a patient, as our patient does, with a high RBC and Hgb, with thrombocytosis, and with a JAK2 mutation. Bone marrow biopsy may detect hyperlobated megakaryocytes which would indicate a diagnosis of ET; however, the physician may choose to monitor and possibly treat as PV due to the RBC counts and symptoms.

Many advances in the understanding of ET and molecular techniques for diagnosis have been made in the last 10 years. Unfortunately, many times, diagnosis is not made until after a thrombotic event. In addition, many patients with thrombocytosis are not referred for hematology consults in a timely fashion or until they too experience a thrombotic event. In 2016 WHO published a new diagnostic criterion for PV, ET and PMF. There is an effort amongst research and physician groups to ‘spread the news’ throughout the medical community to promote early detection of ET, minimize the risk of thrombotic events and improve prognosis.

References

  1. Accurso V, Santoro M, Mancuso S, et al. The Essential Thrombocythemia in 2020: What We Know and Where We Still Have to Dig Deep. Clin Med Insights Blood Disord. 2020;13:2634853520978210. Published 2020 Dec 28. doi:10.1177/2634853520978210
  2. Bose P, Verstovsek S. Updates in the management of polycythemia vera and essential thrombocythemia. Ther Adv Hematol. 2019;10:2040620719870052. Published 2019 Aug 30. doi:10.1177/2040620719870052
  3. Kilpivaara, O., Levine, R. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia 22, 1813–1817 (2008). https://doi.org/10.1038/leu.2008.229
  4. Panjwani,Laura. Management of ET, PV Requires 2 Distinct Approaches. Special Reports, Hematologic Malignancies: Polycythemia Vera, Volume 3, Issue 3. September 28, 2016
  5. https://rarediseases.org/rare-diseases/essential-thrombocythemia/
  6. https://www.mpnconnect.com/pdf/who-diagnostic-criteria-mf-pv-et.pdf
Socha-small

-Becky Socha, MS, MLS(ASCP)CMBBCM graduated from Merrimack College in N. Andover, Massachusetts with a BS in Medical Technology and completed her MS in Clinical Laboratory Sciences at the University of Massachusetts, Lowell. She has worked as a Medical Technologist for over 40 years and has taught as an adjunct faculty member at Merrimack College, UMass Lowell and Stevenson University for over 20 years.  She has worked in all areas of the clinical laboratory, but has a special interest in Hematology and Blood Banking. She currently works at Mercy Medical Center in Baltimore, Md. When she’s not busy being a mad scientist, she can be found outside riding her bicycle.

Microbiology Case Study: An Adult Male with Left Hand Pain

Case History

An adult male presented to the Emergency Department with edema and pain in his left hand. The patient stated that he was bitten by his cat 24 hours prior to admission. Bloodwork was drawn and patient was found to have mild leukocytosis (11.2 x103 cells/uL [reference range, 4-10 x103 cells/uL]) with an elevated neutrophil percentage (76.8% [reference range, 40-70%]). Debridement was performed in the operating room and purulent drainage was send to the lab for aerobic and anaerobic bacterial culture. Gram stain of the purulent drainage showed 3+ white blood cells and mixed bacteria. Mixed aerobic flora grew out in culture, but the predominate isolate was Pasteurella multocida, which grew on 5% sheep blood and Chocolate agar plates (Figure 1) as identified using MALDI-TOF MS.

Figure 1. Growth of Pasteurella multocida on Chocolate Agar after 48hrs incubation at 37°C, 5% CO2.
Figure 2. Gram stain of Pasteurella multocida showing gram negative coccobacilli.

Pasteurella multocida

Pasteurella multocida is one of the most common causes of animal bite-related bacterial infections among patients who present to the Emergency Department in the United States.1 Immunocompetent patients with P. multocida infection typically present with cellulitis at the site of physical injury. These infections usually have a rapid course that can develop within 3 – 48 hours following injury. While most commonly associated with animal bites, immunocompromised patients can acquire Pasteurella simply through animal contact. These rare cases can present as more severe infections involving the respiratory tract and invasive infections including meningitis and endocarditis.

P. multocida is a small, non-motile, facultative anaerobe, which contains an outer polysaccharide capsule. The outer polysaccharide capsule is used to further classify serogroup with most human infections caused by serogroups A and D.2 Further classification into sub-serogroups is determined by the composition of the outer lipopolysaccharide (LPS), which further separates isolates into 16 serovars.3 The bacterium stains as a Gram negative coccobacillus (Figure 2). The recommended growth medium to grow Pasteurella multocida is 5% sheep’s blood agar.4 Of note, P. multocida does not grow on MacConkey agar, unlike most other Gram negative organisms, which can aid in identification of the organism. Most strains of the bacterium test positive for oxidase, catalase, and indole.2

While P. multocida is typically susceptible to penicillin,5 due to the polymicrobial nature of animal bites, broad-spectrum anaerobic and oral flora coverage is recommended for animal bite-related infections. Often, amoxicillin-clavulanate (Augmentin) is used, which will cover the most common organisms including Pasteurella species. Antibiotics which should be avoided for therapy include cephalexin, dicloxacillin, and erythromycin as they have been shown to have inadequate activity against the bacterium.6

References

  1. Martin TCS, Abdelmalek J, Yee B, Lavergne S, Ritter M. Pasteurella multocida line infection: a case report and review of literature. BMC Infect Dis. 2018 Aug 23;18(1):420.
  2. Wilson BA, Ho M. Pasteurella multocida: from zoonosis to cellular microbiology. Clin Microbiol Rev. 2013 Jul;26(3):631-55.
  3. Harper M, Boyce JD, Adler B. The key surface components of Pasteurella multocida: capsule and lipopolysaccharide. Curr Top Microbiol Immunol. 2012;361:39-51.
  4. Lariviere S, Leblanc L, Mittal KR, Martineau GP. Comparison of isolation methods for the recovery of Bordetella bronchiseptica and Pasteurella multocida from the nasal cavities of piglets. J Clin Microbiol. 1993 Feb; 31(2):364-7.
  5. Hasan J, Hug M. Pasteurella Multocida. [Updated 2021 May 24]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK557629/
  6. Stevens DL, Bisno AL, Chambers HF, Dellinger EP, Goldstein EJ, Gorbach SL, Hirschmann JV, Kaplan SL, Montoya JG, Wade JC. Practice guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the infectious diseases society of America. Clin Infect Dis. 2014 Jul 15;59(2):147-59.

-Tristan R. Grams, is a PhD Candidate at the University of Florida in Gainesville, FL where he studies HSV-1 latency and characterizing SARS-CoV-2 antiviral agents.

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Microbiology Case Study: Fever and a Blister in a Young Female

A 25 year old female presented to the ED with a mild fever and a blister on her hand, which arose from a rat bite. She stated that she worked with different kinds of animals including rodents. The patient was discharged with clindamycin after two sets of blood cultures were drawn. Anaerobic bottles became positive 48 hours after incubation. Gram stain showed unusual gram negative bacilli (Figure 1). Rapid Multiplex PCR Blood culture identification (FilmArray BCID-2) panel was negative. Poor growth on Blood agar was observed after 48 hours of sub-culture. The organism was identified as Streptobacillus monoliformis by Bruker Biotyper MALDI-ToF.

S. monoliformis appears as pleomorphic gram negative rods, with its classic characteristics of “bulges” or swollen-rods in tangled chains and filaments (Image 1). Sodium polyanethol sulfonate (SPS) inhibits S. monoliformis growth. As aerobic blood culture bottles made by certain manufacturers contain SPS, the growth is mostly seen in anaerobic bottles devoid of SPS. In liquid broth, S. monoliformis grows as “puff balls.”

Since S. moniliformis is a fastidious organism, the Gram stain from direct smears, such as positive blood culture bottles, in the absence of growth on solid agar media provides an adequate preliminary diagnosis. In such circumstances, 16srRNA sequencing provides a definitive identification. When it grows on agar media, it takes from 2-3 days to as long as 7 days and may appear as “fried-egg” colonies.

Wild rodents as well as laboratory rats carry S. moniliformis in their upper respiratory tract. Cats or dogs preying on rodents also carry the organism. Hence, rat bite fever (RBF) typically occurs upon animal bites.

RBF is a systemic disease wherein patients present with fever, vomiting, headache, and muscle pain or joint swelling. While S. monoliformis is the only known cause of RBF reported in the United States, RBF in Asia is caused by Spirillum minus. Signs and symptoms of the RBF caused by these two organisms slightly differ in addition to the fact that S. minus cannot be cultured in vitro. Several cases of endocarditis and septic arthritis due to S. moniliformis have been reported in the United States.

The notion of rat bite fever as a rare infection is likely due to the fact that 1) S. moniliformis is not a reportable disease and 2) the challenges associated with recovery and identification of this organism from culture media. Thus its occurrence maybe underestimated in the past decades. However, the advancement in technology, such as 16srRNA sequencing and MALDI-ToF, has allowed better diagnosis of RBF in recent years. S. moniliformis is generally responsive to penicillin, cephalosporins, carbapenems, aztreonam, clindamycin, clarithromycin, and tetracycline while it may have intermediate susceptibility to aminoglycoside and fluroquinolones. Since there are no definitive minimal inhibitory concentration (MIC) breakpoint guidelines for this organism, clinical laboratories do not generally perform antimicrobial susceptibility testing.

Image 1. Gram stain of Streptobacillus moniliformis from A) positive blood culture bottle and B) subculture growth on blood agar plate (Arrow showing bulging gram negative rods).

References

  1. Elliott SP. 2007. Rat bite fever and Streptobacillus moniliformis. Clin Microbiol Rev 20:13–22.
  2. Manual of Clinical Microbiology. 11th Edition. 2018
  3. https://www.cdc.gov/rat-bite-fever/index.html
  4. Gaastra W, Boot R, Ho HT, Lipman LJ. Rat bite fever. Vet Microbiol. 2009 Jan 13;133(3):211-28. doi: 10.1016/j.vetmic.2008.09.079. Epub 2008 Oct 8. PMID: 19008054.

-Phyu M. Thwe, Ph.D., D(ABMM), MLS(ASCP)CM is Microbiology Technical Director at Allina Health Laboratory in Minneapolis, MN. She completed her CPEP microbiology fellowship at the University of Texas Medical Branch in Galveston, TX. Her interest includes appropriate test utilization and extra-pulmonary tuberculosis.

Microbiology Case Study: A Male Patient with Fever, Chills, and Rigors

Case History

An 80 year old male from central Vermont with a history of coronary artery disease, hypertension, hyperlipidemia, hypothyroidism, polymyalgia rheumatica, and osteoarthritis status post bilateral knee replacements presented to the ED for fevers, chills, rigors, and fatigue complaining of a home temperature reading of 39.4C (103F). He explained that within the past week he visited a fair to enjoy petting farm animals and trying locally grown produce, meat, and dairy. In the ED he continued to complain of fever, chills, and fatigue, but did not endorse headaches, neck pain, chest pain, SOB, cough, abdominal pain, nausea, vomiting, or diarrhea. An open wound on his left arm was noted. Labs revealed mildly elevated lactic acid, hyponatremia, chronic anemia, but no leukocytosis.

Microbiology

Bacteremia was suspected, and blood cultures obtained. The next day, his blood cultures were positive. Before an extensive workup involving additional cultures and MALDI-TOF, a gram stain was performed and is shown in Image 1. Also, a preparation from a young broth culture demonstrated motility. Both findings indicate Listeria monocytogenes.

Gram positive rods seen in Gram stain from blood culture.

Discussion

Listeria monocytogenes is a gram positive rod. Features that aid in its diagnosis include a narrow zone of beta hemolysis, catalase positivity, tumbling motility, and that it is a facultative intracellular organism.  

Listeria monocytogenes has one to five peritrichous flagella and demonstrate a “tumbling motility” after incubation of a broth culture at room temperature from 8-24 hours. Listeria monocytogenes also demonstrate a “Christmas tree” or “umbrella” (shown below) pattern of motility when inoculated into a semi solid agar, or “motility medium” (Image 2). This pattern occurs because it grows best in a zone of reduced oxygen tension about 0.5cm below the surface that is also is not strictly anaerobic.1 It also has intracellular motility via polymerization of actin, referred to as “actin rockets,” and can even use this method to spread from one cell to another.

Image 2. “Christmas tree” pattern when Listeria monocytogenes is inoculated into semi-solid agar.

Listeria monocytogenes prefers colder temperatures and is often acquired through consumption of un-pasteurized refrigerated dairy products, deli meats, or produce. Pregnant individuals are more likely to acquire infection as are the young, elderly, or the immunocompromised. Listeria monocytogenes can cause meningitis, usually in newborns or the elderly. Central nervous system or bloodstream infections are treated with ampicillin and gentamycin.2

References

  1. Allerberger F. Listeria: growth, phenotypic differentiation and molecular microbiology. FEMS Immunol Med Microbiol. 2003 Apr 1;35(3):183-9. doi: 10.1016/S0928-8244(02)00447-9. PMID: 12648835. (n.d.).
  2. Gelfand, M., Thompson, J., Geeta S. (2021). Treatment and prevention of Listeria monocytogenes infection. UpToDate. Retrieved September 2, 2021, from https://www-uptodate-com.ezproxy.uvm.edu/contents/treatment-and-prevention-of-listeria-monocytogenes-infe. (n.d.).

-Joe Teague is a Pathology Student Fellow and Brianna Waller, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Microbiology Case Study: A 67 Year Old Man Develops Severe Leg Pain

Case History

A 67 year old male presented with type II diabetes, hypertension, hyperlipidemia, obstructive sleep apnea, recurrent GI bleeds, and atrial fibrillation (status post ablation and on rivaroxaban). Given the history of recurrent GI bleeds, he was taken off rivaroxaban and underwent a left atrial appendage occluder device implant procedure. Several hours later, he developed severe leg pain and loss of lower extremity pulses. CT angiogram confirmed Watchman device embolization to the abdominal aorta. The patient received emergent surgical removal of the device. In the ICU, the patient developed worsening rhabdomyolysis, anuria, hypotension, ischemic bowel disease, and died within hours. An autopsy was requested by the next of kin, which revealed an unexpected finding of a 6 cm hilar-based lung mass.

Microbiology

Premortem and postmortem cultures were not collected. Fontana-Masson stain of a section from the hilar lung tissue reveals yeast of varying size with a lighter shade in the center and a thick capsule, though the capsule does not stain and can instead be appreciated as a “halo” (Image 1). Fontana Masson stain also reveals narrow based budding (Image 2). Both findings indicate Cryptococcus neoformans.

Image. 1. Fontana-Masson stain revealing yeast of varying size with a lighter shade in the center and a thick capsule.
Image 2. Fontana-Masson stain revealing narrow based budding

Discussion

Cryptococcus neoformans is a saprophytic yeast (5-10 µm) identified best by its thick polysaccharide, antiphagocytic capsule. It can be infectious when inhaled, often from soil or avian droppings.

While most immunocompetent individuals clear the pathogen, in the immunocompromised, it can form a primary focus in the lungs and then disseminate. It is often asymptomatic when localized to the lungs but can present as a cough or dyspnea. Dissemination to the brain presents as meningitis. Cryptococcal neoformans is the most common cause of fungal meningitis.

While C. neoformans most often presents as meningitis in the immunocompromised, a retrospective case analysis found diabetes mellitus II as a newly defined independent factor contributing to morbidity and mortality. This study analyzed cryptococcal infections in patients with DMII from 1997-2015. 57% of the DMII patients did not have any other underlying disease and 69% of patients who presented with pulmonary Cryptococcus neoformans experienced a misdiagnosis and treatment delays.1

Qualities that aid in the diagnosis include urease positivity, positive latex agglutination test due to its thick polysaccharide capsule, and characteristic features on mucicarmine red, methenamine silver, India ink, and Fontana-Masson stains.2

The Fontana-Masson silver (FMS) stain is a histochemical technique that oxidizes melanin and melanin-like pigments as it reduces silver.  FMS can be used to highlight the melanin-like pigment in Cryptococcus spp., including capsule-deficient variants because this pigment is cell-wall (and not capsule) associated. FMS is a very sensitive, but not completely specific stain, for Cryptococcus spp. as other yeasts and fungi can also produce melanin and melanin-like pigments.3 Though two C. neoformans yeast close together can resemble broad-based budding, Image 2 demonstrates the narrow-based budding.

While the India ink stain is often discussed as a popular stain for C. neoformans, it can only be performed on liquid samples (CSF, fluid samples) and cannot be performed on paraffin-embedded tissue samples. Of note, the India ink stain is a “negative stain”, resulting in the classic “halo” effect (image 3) because it is not picked up by the capsule of Cryptococcus spp. Because of this, it will miss capsule-deficient infections.4

Image 3. India Ink Stain showing “halo” effect of capsules.

Prognosis varies by the mechanism of immunosuppression. Acute mortality in in cryptococcal meningitis for HIV patients has improved dramatically with antifungals and ART, ranging from 6-16%. Poor prognostic indicators include abnormal mental status, a high yeast burden defined as CSF antigen titer > 1:1024 by latex agglutination or > 1:4000 by lateral flow assay, or a poor host response defined as CSF WBC count < 20/microL.5

Treatment of cryptococcal infections includes initial therapy with amphotericin B and flucytosine followed by long term fluconazole.6

References

  1. Boulware DR, Rolfes MA, Rajasingham R, von Hohenberg M, Qin Z, Taseera K, Schutz C, Kwizera R, Butler EK, Meintjes G, Muzoora C, Bischof JC, Meya DB. Multisite validation of cryptococcal antigen lateral flow assay and quantification by laser thermal contr. (n.d.).
  2. Li Y, Fang W, Jiang W, Hagen F, Liu J, Zhang L, Hong N, Zhu Y, Xu X, Lei X, Deng D, Xu J, Liao W, Boekhout T, Chen M, Pan W. Cryptococcosis in patients with diabetes mellitus II in mainland China: 1993-2015. Mycoses. 2017 Nov;60(11):706-713. doi: 10.1111/. (n.d.).
  3. McFadden, D., & Casadevall, A. (2001). Capsule and Melanin Synthesis in Cryptococcus neoformans. Medical Mycology, 39, 19-30.
  4. Perfect JR, Dismukes WE, Dromer F, Goldman DL, Graybill JR, Hamill RJ, Harrison TS, Larsen RA, Lortholary O, Nguyen MH, Pappas PG, Powderly WG, Singh N, Sobel JD, Sorrell TC. Clinical practice guidelines for the management of cryptococcal disease: 2010 up. (n.d.).
  5. Saag MS, Powderly WG, Cloud GA, Robinson P, Grieco MH, Sharkey PK, Thompson SE, Sugar AM, Tuazon CU, Fisher JF, et al. Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. The NIAID Mycoses Study. (n.d.).
  6. Winn, W. C., & Koneman, E. W. (2006). Koneman’s color atlas and textbook of diagnostic microbiology. Philadelphia: Lippincott Williams & Wilkins. (n.d.).

-Joe Teague is a Pathology Student Fellow and Brianna Waller, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

-Christi Wojewoda, MD, is the Director of Clinical Microbiology at the University of Vermont Medical Center and an Associate Professor at the University of Vermont.

Looking into the Pathology Mirror

Conversing with people early in their career has always been an exciting experience for me and, I hope, for those with whom I have spoken. I tend to get enthusiastic in discussing all the possibilities that lie ahead and try to keep the conversation focused on the individual in question. I try to avoid talking about my own career path unless someone specifically asks—but I keep it brief. One-on-one conversations tend to be very productive for the individual because we can delve deep into their questions, fears, concerns, hopes, and goals. Group discussions often end up being more informative for me, and I have learned a ton from listening to dynamic young people. I was recently gifted with the opportunity to lead 9 focus groups as part of a grant-funded project which included several groups with medical students and pathology residents. Although our focus was on forensic pathology, the groups were quite diverse. I would call the experience overall very positive and enlightening for all of us, but I was struck by a few observations that I felt the need to explore further on my own—so, you get to read a blog about it.

Pathology is a fascinating specialty after medical school that covers a large range of diseases and patient types, an even larger range of scopes of practice, and includes some of the lowest and highest paid jobs in the field. At the same time, the practices of pathology and medicine are evolving at an extremely rapid rate while medical knowledge is expanding exponentially. There is an entire industry based around paraphrasing the current literature for a given specialty because, even within a specialty, you can’t read every new study or follow every new development. It is this expansion that has created the demand by pathologists in the last 2 decades to be sub-specialists so that a focus on one particular area of practice will keep their expertise sharp, their diagnoses hyper-accurate, and their risk profile minimal. This expansive phenomenon in medicine in general but specifically in pathology is an excellent indicator that the field of knowledge is ripe for a disruptive innovation. It is common knowledge that the practice of anatomic pathology, for example, is based on a technique that is more than 100 years old—histology; however, what is not common knowledge is that the amount of data generated by reviewing a histology slide from, for example, a tumor, is 1/1000th or less than the data generated by performing genetic sequencing of that same tumor. Add to the mix the ability to perform transcriptional analysis, mass spectroscopy, metabolomics, lipidomics, phospholipidomics, glycobiological analysis, etc. and it becomes clear that what is contributed by an H&E pales in comparison to what we can know about a piece of tissue. There are barriers, you say? Cost, integration of information, usable outputs, or process:volume ratios? All true. But the technological ability to characterize a tumor across all these different attributes and mathematically reduce that to a multiplex assay which can perfectly classify and predict therapeutic responsiveness exists. Still don’t believe me? A collection of companies is focused on testing that has been variably called, “Universal Cancer Screening”, “Multi-cancer Screening”, and “Multi-cancer Early Detection”. These systems currently use sequencing across multiple loci to detect from 20 to 50 different cancer types. One such company can do so with stool to look for gastrointestinal cancer and is on the market today. Why am I going down this path of which many of you are already aware? Because when I was talking to a trainee recently, they told me that they originally wanted to go into forensic pathology but were talked out of it and were now considering doing GI pathology. Let’s break this down so you can understand my frustration.

GI pathology as a career is largely generating revenue through colonoscopy from screening. Yes, the field is diverse and the most complicated parts like liver, pancreas, IBD, etc. are part and parcel to the practice. But, from a C-suite perspective, the fiscal bulk of the value of the service is in biopsy reads from screening. Because of the interest in the field in the last two decades (increase in pathologists in GI) juxtaposed to the much-needed control and reduction of 88305 reimbursement (due to rampant misuse and overuse), there are a lot of GI pathologists in the United States. So many, in fact, that jobs for GI pathology are sort of hard to find. Add to the mix a product, already on the market, that can detect colon cancer in stool without screening colonoscopy and its risks, which is only the harbinger of a group of products that will arrive on the market which can do the same for many other cancers from stool, blood, etc., and one gets nervous about where GI pathology’s current revenue volume is headed. But then there is the recent recommendation that the screening age for colonoscopy be reduced to 45 (from 50). The increase in volume of biopsies from screening (if everyone was 100% compliant) would overwhelm some practices. Where is GI pathology as a specialty going? Do we have too many and should we be concerned about disruptive innovations to screening decimating revenue generating volumes? Or are we facing an overwhelming number of biopsies with the new screening guidelines? I wouldn’t dare try to predict where this is headed but there is clearly some “uncertainty” in the practice of GI pathology. And a practicing pathologist talked a resident out of forensics and into GI??

Let’s contrast this with forensic pathology so my point is clear. There are currently only about 500 FPs in the United States and there is a need—to meet minimum requirements for coverage—of 1200 FPS. That’s a difference of 700 FPs, all of which must be board certified pathologists. There are more than 50 current open full-time positions for FPs that are funded (i.e., actively recruiting to hire today) that were identified on the most common sites for these listings. Seven of these programs offer tuition repayment for FPs from $100,000 to $250,000. Outside of those seven programs, there are three federal programs that specifically offer loan repayment for FPs and a fourth for which they are also eligible. Doing the math, basically, anyone wanting to practice forensic pathology likely qualifies for a loan repayment program (hint: that’s not true for the majority of pathology jobs). Although the average salary for an FP is often reported as ~$110,000 (about half of the average salary for a pathologist according to publicly available data), the current open positions I mentioned have an average of $240,612 (with a range of $175,000 to $350,000). The work of forensic pathologists includes death scene investigation, varying levels of postmortem examination (e.g., chart review, external examination, complete autopsy, etc.), medicolegal reporting including court appearances, participation in public health investigations, participation with local government, etc. This role is vital to the functioning of society and is required by law to be performed. Stated another way, we will always need FP (and we desperately need them now!). It is very difficult to imagine a disruptive innovation or even a transformative innovation that will replace this role in the next several decades. That same can’t be said for other parts of pathology (see my GI example above). And yet, we struggle to find FPs. Why?

Certainly not the only reason but a valid and real reason that we struggle is the presence of microaggressions in the medical community. These are common for pathology in general but can be extremely harsh and rampant for forensics (even coming from other pathologists!). The real example I have given you of the resident selecting GI after being talked out of forensics is a true story. And, more importantly, it was reiterated by nearly every medical student and resident (and fellow) with whom we talked about their experiences. Considers these statements (which are direct quotes):

“You’re too smart to do pathology.”

“Why would you waste your brain on forensics?”

“You’re too good with people and patients to be a pathologist.”

“Forensics is a dead specialty (pardon the pun)”.

Excuse me?? Are you kidding? It’s not that these microaggressions are inappropriate because they are damaging to a young person’s passions and interests. It is that these microaggressions, which are heard repeatedly, are simply wrong. Pathology, if nothing else, is a data and knowledge heavy specialty where we spend most of our time thinking, solving problems, and receiving, processing, interpreting, and synthesizing data into a useful answer on which a clinician can act. And we don’t do it one patient at a time. We produce literally thousands if not tens of thousands of tests results per day in an average laboratory. Forensics requires highly intelligent, detail-oriented individuals who can not only synthesize an entire patient’s life and death into a succinct story—but they have to defend their opinion in court. Every day! I’d like you to ask your primary care doctor if every decision he/she made for each of their patients in one day they would be comfortable defending in court. Every decision! It requires a special person who is not only amazing with data and knowledge but extremely talented at interacting with people—many of which are trying to prove you wrong. Moreover, few medical specialties call upon the physician to routinely deal with families at the lowest point in their lives in every single encounter. A person that is good with people and patients is exactly the person that can become a successful forensic pathologist—one that provides meaningful care when care is most needed. And lastly, forensics is thriving as a job market (as I described). And yet, our “mentors” who train our medical students and pathology residents continue to provide microaggressions (or outright rebuke) for those brave, brilliant individuals who would choose forensics as a career. Considering the state of the field and the perks of the practice at the moment, forensics seems like a pretty smart choice today. But stepping back from this rhetoric to a 10,000 foot view—because, remember, this is me thinking through a problem and forcing you to read about it—the overall observation I have is that the field of pathology (internally) needs to understand where it is going, what its scope of practice will look like tomorrow, 5 years, and 10 years from now, and, more importantly, what the needs of our patient community are (alive or dead). Without a global view of the total need in pathology, how can we possibly have meaningful conversations with individuals early in their career that both enhance their passion and meet the needs of the community of practice?

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.

Microbiology Case Study: A 28 Year Old Woman with Fever and Rash

Case History

A 28 year old female presented to the emergency department (ED) with fevers, chills, and rash. A maculopapular rash began two days prior to her presentation starting at her shins, then spreading to her abdomen, chest, and arms. On presentation she had left knee and right elbow pain and had pain with walking. The patient denied neck pain, headache, blurry vision. Her past medical history is significant for bacterial meningitis between the ages of 10-12. She does not recall the causative pathogen but does recall that her mother had to quarantine for a period of time. On physical exam, the patient was in no distress, had a low-grade fever (38.2 °C) with normal heart rate and blood pressure. Her left knee and right knee were swollen, warm, and painful to the touch with limited range of motion. She was found to have round, erythematous papules and plaques, some with central purpura primarily involving both legs as well as the back and arms (Figure 1).

Initial labs showed a WBC of 11.24/L, Hgb 10.2 g/dL, platelets of 172/L. Her acute inflammatory marker was elevated (CRP 24.7 mg/dL) and her sed rate was 30 mm/hr. One of two blood cultures on admission grew Neisseria meningitides. She was initially started on vancomycin and piperacillin-tazobactam and later transitioned to ceftriaxone to complete a 7 day course. Further blood cultures remained sterile. Dermatology was consulted for her skin lesions and a biopsy of a thigh lesion revealed leukocytoclastic vasculitis. Orthopedic surgery was consulted given her left knee and right elbow swelling. X-ray revealed trace effusions in both joints. A tap of both joints was unsuccessful. The patient developed acute renal insufficiency secondary to acute tubular necrosis during her hospitalization, which improved prior to discharge. Her fevers resolved and the joint swelling and pain improved prior to discharge. She had off and on headaches during her hospitalization but denied neck pain/stiffness and never required a lumbar puncture. She was discharged on prophylactic amoxicillin.

Two months after discharge she was seen in immunology clinic at which time she was tested for humoral and complement immune deficiencies. Her total complement (CH50) was found to be low at 15 U/mL while C3 and C4 were normal, suggesting a terminal complement deficiency. Her humoral immunity panel was normal.

Discussion

Meningococcemia without meningitis occurs in 20-30% of patients with invasive meningococcal disease.1 This patient showed evidence of invasive disease with a maculopapular rash, joint involvement, and renal injury. However, her disease never reached the level of septicemia or disseminated intravascular coagulation (DIC) likely because she presented early enough in the course of her infection. A prior history of bacterial meningitidis raised suspicion for a terminal complement deficiency that was later confirmed by a low CH50. Patients with terminal compliment (C5-C9) deficiency are 1,400-10,000x more likely to develop meningococcal disease and 40-50% of these individuals can have recurrent infection.2 Deficiencies in other components of the complement pathway such as C3 and C4, especially in association with systemic lupus erythematous (SLE), and properdin (a promoter of the alternative complement pathway) can lead to invasive meningococcal disease.3 Patients with anatomic or functional asplenia and patients on eculizumab therapy are also at increased risk.4

Neisseria meningitidis is a gram negative diplococcus and is an obligate human pathogen that is also a common human commensal found in the nasopharynx of 3-25% of the population. Meningococcal disease most commonly manifests as meningitis (40-65%) and meningococcemia (20-30%) followed by pneumonia (10%), septic arthritis (2%), and chronic meningococcemia.1 The major virulence factor associated with meningococcal disease is a capsular polysaccharide, which are classified into 13 serogroups (A-L, W-135, X, Y, Z).5 Serogroups A, B, C, W-135, X, and Y are associated with invasive disease and prevalence of each serogroup is geographically varied. Diagnosis is by visualizing gram-negative diplococci on gram-stain (see Figure) from CSF or other sterile body fluid and by culture of these sterile body fluids. It is worth noting that intravenous antibiotics can sterilize meningococci in the CSF within 3-4 hours after administration.6 Transmission is by large respiratory droplets and direct contact from those with carriage or infection. Disease, especially among high-risk patients with complement deficiency, is preventable with the meningococcal conjugate vaccine. Chemoprophylaxis with rifampin, ceftriaxone, ciprofloxacin, or azithromycin is recommended for close contacts regardless of vaccination status.

Figure 1: Maculopapular rash with central purpura of both legs.

References

  1. Stephens, D., Neisseria meningitidis. 9th ed. Principles and Practices of Infectious Diseases, ed. J. Bennett. Vol. 2. 2015.
  2. Ram, S., L.A. Lewis, and P.A. Rice, Infections of people with complement deficiencies and patients who have undergone splenectomy. Clin Microbiol Rev, 2010. 23(4): p. 740-80.
  3. Fijen CA, K.E., te Bulte MT, Daha MR, Dankert J, Assessment of complement deficiency in patients with meningococcal disease in The Netherlands. Clin Infect Dis, 1999. 28(1): p. 98-105.
  4. Lebel, E., et al., Post-eculizumab meningococcaemia in vaccinated patients. Clin Microbiol Infect, 2018. 24(1): p. 89-90.
  5. Chang, Q., Y.L. Tzeng, and D.S. Stephens, Meningococcal disease: changes in epidemiology and prevention. Clin Epidemiol, 2012. 4: p. 237-45.
  6. Crosswell, J.M., W.R. Nicholson, and D.R. Lennon, Rapid sterilisation of cerebrospinal fluid in meningococcal meningitis: Implications for treatment duration. J Paediatr Child Health, 2006. 42(4): p. 170-3.

-Denver Niles is the Medical Microbiology fellow at UT Southwestern Medical Center. Prior to his Medical Microbiology fellowship, he completed pediatric infectious disease training at Baylor College of Medicine/Texas Children’s Hospital.

-Dominick Cavuoti is a professor of Pathology at UT Southwestern Medical Center who specializes in Medical Microbiology, ID Pathology and Cytology.

-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.

Microbiology Case Study: A Female in her 60s with Retro-orbital Headaches

Case History

The patient was a previously healthy female who presented with a five day history of retro-orbital headaches, lightheadedness, and intermittent falls. Her presentation was consistent with meningitis and further studies were pursued. Head computed tomography (CT), CT angiogram of the head and neck, brain magnetic resonance imaging (MRI), and electroencephalogram (EEG) were unremarkable. Analysis of the cerebrospinal fluid (CSF) demonstrated an elevated white blood cell count (605 white blood cells/µL) of which 88% were lymphocytes, 9% were monocytes, and 3% were neutrophils. CSF glucose was slightly decreased at 33 mg/dL and protein was elevated at 81 mg/dL. Additional tests requested on the CSF included herpes simplex virus (HSV), varicella zoster virus (VZV), West Nile virus (WNV), and Epstein-Barr virus (EBV). The CSF was positive for HSV-2 and negative for HSV-1, VZV, and EBV by PCR. WNV IgG and IgM were negative. Of note, the patient had two episodes of viral meningitis in the past of unknown etiology. The patient received a one week course of valacyclovir and was discharged. Per the patient, she continues to have fluctuating headaches and occasional lightheadedness. Follow-up imaging has been unremarkable.

Figure1. Results of the HSV-1 and HSV-2 PCR. HSV-2 (green) and internal control (purple) amplified. HSV-1 (red) was not detected.

Discussion

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are enveloped, double stranded DNA viruses that are members of the Herpesviridae family. They are common viruses that cause cold sores or fever blisters. HSV is a lifelong infection, and latent infection can cause reactivation. While both HSV-1 and HSV-2 can affect any area, HSV-1 is typically associated with non-genital sites whereas HSV-2 typically causes genital infections. In addition to herpetic gingivostomatitis, herpes labialis, and herpes genitalis, other associated clinical conditions include encephalitis, meningitis, keratitis, esophagitis, neonatal herpes, and disseminated primary infection. Most cases of HSV encephalitis have been linked to HSV-1 while HSV meningitis is typically caused by HSV-2. As seen in our patient, HSV-2 has been implicated in recurrent, aseptic, and self-limiting meningitis, also known as Mollaret meningitis.1 There are no specific treatment guidelines for HSV-2 meningitis with the main therapeutic strategy being symptom management. The utilization of acyclovir to manage uncomplicated HSV-2 management is controversial and there is no current consensus.2

Clinically, patients with meningitis typically present with acute onset of fever, headache, and neck stiffness. Other associated symptoms include malaise, rash, nausea, vomiting, sore throat, lymphadenopathy, and genitourinary symptoms. In order to differentiate between the infectious etiologies (i.e. viral, bacterial, tuberculous, or fungal) that cause meningitis, a lumbar puncture may be performed. For viral meningitis, CSF will usually show an elevated white count with predominantly mononuclear cells. The CSF:serum glucose ratio and protein levels are often elevated. The most common CSF viral pathogens in the non-immunosuppressed population are enteroviruses, HSV-1, HSV-2, and VZV, which can all be detected by real time polymerase chain reaction (RT-PCR) technology Molecular methods are faster, more sensitive, and more widely available that viral culture.3 Antibody tests are not recommended for HSV as ~70% of adults will be positive for HSV-1 and ~20-50% of adults will be positive for HSV-2.4

Given the broad range of infectious etiologies that can cause meningitis, there has been interest in the development of a multiplex molecular test. Currently, the FilmArray meningitis/encephalitis panel is the only one that has received FDA clearance. This panel includes 14 bacterial, fungal, and viral targets, including HSV-1 and HSV-2. However, this panel should be used cautiously as several studies have shown a high proportion of false negatives in the detection of HSV-1, HSV-2, and Crytococcus neoformans/gattii. It has been suggested that for HSV-1 and HSV-2, the multiplex panel does not work as well if the viral load is near the limit of detection of the assay or if the patient is having a reactivation of HSV. If there is a high clinical suspicion, particularly in neonates and immunosuppressed patients, an assay for detection of only HSV-1 and HSV-2 should be performed.5

References

  1. Koelle DM and Corey L. (2008) Herpes simplex: insights on pathogenesis and possible vaccines. Annual Review of Medicine, 59: 381-395.
  2. Bamberger DM. (2010) Diagnosis, initial management, and prevention of meningitis. American Family Physician, 82: 1491-1498.
  3. Logan SAE and MacMahon E. (2008) Viral meningitis. The BMJ, 336: 36-40.
  4. Schiffer JT, Corey L. (2020) Herpes simplex virus. Bennett’s Principles and Practice of Infectious Diseases, 9th edition.
  5. Tansarli GS and Chapin KC. (2020) Diagnostic test accuracy of BioFire FilmArray meningitis/encephalitis panel: a systematic review and meta-analysis. Clinical Microbiology and Infection, 26: 281-290.

-Melissa Tjota, MD, PhD is a Molecular Genetic Pathology fellow at the University of Chicago Medicine and NorthShore University HealthSystem. She completed her MD/PhD (Immunology) and AP/CP residency at the University of Chicago.

-Paige M.K. Larkin, PhD, D(ABMM), M(ASCP)CM is the Director of Molecular Microbiology and Associate Director of Clinical Microbiology at NorthShore University HealthSystem in Evanston, IL. Her interests include mycology, mycobacteriology, point-of-care testing, and molecular diagnostics, especially next generation sequencing.

Microbiology Case Study: A Middle Aged Man with a Non-Healing Ulcer

Case History

A middle-aged man with osteogenesis imperfecta, poorly controlled HIV, and hepatitis C presented for outpatient management of an infected non-pressure ulcer on his ankle. The patient had no history of recent travel and was current on vaccinations. He was afebrile but reported increasing difficulty with ambulation due to pain from the lesion. Per the patient, the wound began as an itchy “bug bite” two weeks prior, which he had scratched, causing skin breakage. The ulcer became progressively larger with corresponding increases in pain, warmth and swelling. Specimens from the ulcer were collected for culture, and the patient started empiric doxycycline in addition to prophylactic amoxicillin/clavulanate he was already taking for management of a pre-existing ulcer on the opposite foot. The non-healing wound progressed to the size of a nickel with worsening pain. Due to these symptoms and the associated microbiological data, the patient was instructed to present to the emergency department where he was admitted for additional evaluation. Upon admission, the ulcer exhibited no surrounding erythema, but a slight exudate and pitting edema was noted (Image 1). An X-ray of the ankle was obtained which revealed soft tissue swelling, but no fracture or crepitus.

Image 1. Photograph of the ulcerated lesion when seen at hospital admission.

Microbiology

Specimens of the ulcerated lesion were submitted to the microbiology laboratory for routine bacterial culture. No growth was observed on MacConkey agar plates, while two beta-hemolytic morphotypes and one non-hemolytic morphotype were observed on blood agar. The two beta-hemolytic species were identified as Streptococcus dysgalactiae and Arcanobacterium haemolyticum by MALDI-TOF MS. The third, non-hemolytic organism was a catalase-positive, gram positive coryneform rod (Image 2A and B), and was identified by MALDI-TOF MS as Corynebacterium diphtheriae. This identification was subsequently confirmed by both a commercial reference laboratory and the US Centers for Disease Control and Prevention.

Image 2. Growth of the isolate of C. diphtheriae on a blood agar plate and associated Gram stain.  A) Non-hemolytic colonies observed after 24 hours incubation.  B) gram positive coryneform rods of C. diphtheriae.

Discussion

Corynebacterium diphtheriae is the etiological agent of diphtheria, a toxin-mediated disease classically associated with respiratory and cutaneous infections. C. diphtheriae is infrequently encountered in the United States due to a robust national vaccination program but remains endemic in other parts of the world. Respiratory manifestations include pharyngitis with dysphagia, lymphadenitis (often described as a “bull-neck” appearance), and the development of a characteristic white/grey pharyngeal pseudomembrane which can cause airway obstruction. The pathogenesis of C. diphtheriae is mediated by diphtheria toxin which inhibits host cell protein synthesis leading to cell death. Diphtheria toxin can also cause cardiac, nephrotic, and neurological sequalae due to dissemination. The diphtheria toxin gene (tox) is encoded on a bacteriophage which lysogenizes into the bacterial chromosome and is expressed in response to low iron concentrations. While C. diphtheriae is most frequently associated with diphtheria toxin production, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis can also express the toxin if infected with the bacteriophage.

Cutaneous C. diphtheriae infections manifest initially as a vesicle, eventually developing into a painful ulcerative lesion that may or may not have a pseudomembrane. These infections can be caused by either fully toxigenic strains, non-toxigenic strains lacking the tox gene, or non-toxigenic toxin gene bearing (NTTB) strains.1 NTTB strains are genotypically positive for the presence of the tox gene on the lysogenized phage, but do not express functional diphtheria toxin. This can be due to 1) mutation of truncation of the tox gene coding sequence, 2) promotor mutations, or 3) alterations in proteins regulating tox expression. NTTB strains are important from an epidemiological perspective in that they serve as an environmental reservoir for tox gene-harboring phage which could convert circulating non-toxigenic C. diphtheriae into toxin-producing organisms.1-3 This phenotype also presents additional diagnostic challenges as toxin gene expression must therefore be confirmed by more laborious phenotypic methods instead of genotypically (i.e. by PCR).

Recovery of C. diphtheriae in the routine setting is challenging as the organism morphologically resembles other coryneform rods usually representative of flora in cutaneous and respiratory specimens. While selective and differential medias are available (i.e. Cystine Tellurite Blood Agar or Tinsdale medium), they are not used routinely due to low incidence. As both toxigenic and non-toxigenic strains of C. diphtheriae circulate, this isolate was referred to the CDC’s Pertussis and Diphtheria laboratory for additional typing and toxin analysis. The organism was determined to belong to the mitis biotype and was positive for the tox gene by PCR. Toxin gene expression was then evaluated by the Elek Immunodiffusion test (Image 3). In this classical method, a filter paper strip saturated with anti-toxin is placed perpendicular to control and test strains of the organism on non-selective media. If the organism expresses diphtheria toxin, the toxin and the antisera form a complex and precipitate from solution. This phenomenon is visualized as precipitin lines in the agar after 24 hours of incubation at 37°C.4 Despite tox gene PCR-positivity, the Elek Immunodiffusion test revealed that this patient’s isolate did not express diphtheria toxin. Thus, this C. diphtheriae isolate was a representative example of a NTTB strain.

Image 3. Representation of the Elek Immunodiffusion assay for the detection of diphtheria toxin.  Filter paper soaked with antitoxin placed perpendicular to test and control isolates.  The presence of precipitin lines indicates diphtheria toxin gene expression.  Figure adapted from (3).

In contrast to respiratory presentations which have declined due to vaccination, cutaneous infections with C. diphtheriae have become more frequently recognized. Unlike respiratory diphtheria, cutaneous diphtheria was not reportable to the National Notifiable Diseases Surveillance System until the clinical criteria changed in 2019. Since that time, toxigenic isolates recovered from either respiratory or cutaneous sources are reportable.5 Additionally, the incorporation of MALDI-TOF MS into routine workflow has facilitated identification of the organism when isolated clinically and likely increased reporting.

Although immunization protects against clinical diphtheria, it does not prevent colonization by non-toxigenic C. diphtheriae including NTTB strains.2,3 Non-toxigenic C. diphtheriae causing cutaneous infections are often recovered with other pyogenic organisms including Staphylococcus aureus, beta-hemolytic streptococci, and A. haemolyticum6 as was observed in this case. Risk factors for cutaneous infections include a compromised immune system, eczema, travel to endemic regions, intravenous drug use, homelessness/unsanitary living conditions, and alcoholism.3,7  It is unclear how this patient was exposed, although he did have risk factors including immunosuppression and those associated with his osteogenesis imperfecta. The patient was started on erythromycin and ampicillin/sulbactam for subsequent management and has continued to improve when seen at follow-up. Subsequent cultures of the wound have remained negative for C. diphtheriae.

  1. Zakikhany K, Neal S, Efstratiou A. 2014. Emergence and molecular characterisation of non-toxigenic tox gene-bearing Corynebacterium diphtheriae biovar mitis in the United Kingdom, 2003–2012. Eurosurveillance 19:20819.
  2. Doyle CJ, Mazins A, Graham RMA, Fang N-X, Smith HV, Jennison AV. 2017. Sequence Analysis of Toxin Gene-Bearing Corynebacterium diphtheriae Strains, Australia. Emerging infectious diseases 23:105-107.
  3. Sharma NC, Efstratiou A, Mokrousov I, Mutreja A, Das B, Ramamurthy T. 2019. Diphtheria. Nature Reviews Disease Primers 5:81.
  4. Kates O, Starr K, Bourassa L, Burnham C-AD. 2020. The Brief Case: Nontoxigenic Corynebacterium diphtheriae in a Nonhealing Wound. Journal of Clinical Microbiology 58:e00506-00520.
  5. United States Centers for Disease Control and Prevention. 2019. Diphtheria (Corynebacterium diphtheriae) 2019 Case Definition – National Notifiable Diseases Surveillance System. https://ndc.services.cdc.gov/case-definitions/diphtheria-2019/. Accessed August 1st, 2021.
  6. Lowe CF, Bernard KA, Romney MG. 2011. Cutaneous Diphtheria in the Urban Poor Population of Vancouver, British Columbia, Canada: a 10-Year Review. Journal of Clinical Microbiology 49:2664-2666.
  7. Gubler J, Huber-Schneider C, Gruner E, Altwegg M. 1998. An Outbreak of Nontoxigenic Corynebacterium diphtheriae Infection: Single Bacterial Clone Causing Invasive Infection Among Swiss Drug Users. Clinical Infectious Diseases 27:1295-1298.

-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.

By the Book

One of my favorite parts of being a cytotechnologist is the delight of having cytology students rotate through our institution as a practicum site. The pandemic caused a clinical rotation hiatus for the safety of both our staff and students, but thanks to widespread healthcare vaccination, we were able to bring in some fresh minds to experience the variety of interesting cases we enjoy every day. I think what I love most about having students here is reminiscing of when I was in their shoes seven years ago. I remember going into my rotations using nothing but morphologic criteria I memorized from lecture and labs. My clinicals served as a rude awakening that we rarely see any textbook perfect cases. Cancer is like a shape-shifter – one melanoma looks entirely different than another. Two lung squamous cell carcinomas from the right upper lobes from two different patients could look entirely different. The unique variation within and between cancer types is what makes this field so beautifully fascinating. The first time a cytotechnology student shows me a case, tells me their thoughts, works through the criteria, and lists the differentials, I look up and say, “nothing is quite by the book.” How often we fall into a routine of relying on criteria, closing our minds to certain diagnoses because it doesn’t quite look like the clinical impression. When the pathologic and clinical impressions divide, more diagnostic tests are performed, CPT codes fill our billing tab, and we start to panic. “It’s supposed to be adenocarcinoma, so why doesn’t it look like adenocarcinoma?!?

A few weeks ago, the lab received a left pleural fluid from a patient who presented with a history of small cell cervical cancer. I remember learning about this in my first semester of grad school – how rare a finding of small cell carcinoma is, accounting for less than 5% of cervical cancers. It essentially mimics small cell carcinoma of the lung and other neuroendocrine carcinomas, where you should be able to identify the telltale salt-and-pepper chromatin, nuclear molding, scant cytoplasm, loosely cohesive or isolated, necrosis, usually an absence of nucleoli, a high proliferation index with mitotic figures, etc. It’s an aggressive disease to say the least, just like its lung counterpart. When this cancer metastasizes, it takes its same characteristics with it, spreading rapidly without care.


The first step in processing a fluid is to prepare a fresh, air-dried, Diff-Quik-stained cytospin to triage the specimen and decide whether the specimen should be processed routinely or hand-prepped and stained with overtly positive fluids to prevent cross-contamination. There was one cluster identified on the Diff-Quik preparation, but compared to the background of mesothelial and inflammatory cells, the tumor content was insufficient to push it up to hand-processing. The bluish cytoplasm caught my attention as a feature of neuroendocrine tumors AND lymphomas, but the nuclear molding had me favoring neuroendocrine.

Image 1. Pleural fluid, left. DQ-stained cytospin.

That afternoon, I examined the pap-stained smears and SurePath liquid-based preparation, identifying similar cells of interest. However, despite the presence of nuclear molding and scant cytoplasm, the nuclei presented with prominent nucleoli. An interesting feature, to say the least.

Images 2-5. Pleural fluid, left. 2-3, Pap-stained smears (2, lightened to highlight nucleoli); 4-5, Pap-stained SurePath liquid-based preparation.

The following morning, I screened the cell block slides and came across molded groups of cells (appearing as a garden aerial view). Still the prominent nucleoli baffled me, and I thought, “Why doesn’t this look like a classic small cell carcinoma? They clinical history even included known lung mets from the patient’s small cell cervical cancer!”

Images 6 and 7: pleural fluid, left. 6, H&E cell block section 100X; 7, H&E cell block section 400X.

When I sent the case for review by the pathologist, I wrote up a diagnosis of Positive for Malignant Cells; Carcinoma, small cell? Recommend correlation with IHC.” My attending was just as intrigued. She ordered a thorough panel of immunohistochemistry stains based on the morphologic findings.

Images 8-11. Pleural fluid, left. 8, synaptophysin+; 9, CD56+; 10, TTF-1+; 11, BerEP4+.

The tumor cells are positive for synaptophysin, CD56, TTF-1, and BerEP4, focally positive for CK7 and chromogranin (not shown), and negative for calretinin, PAX-8, and p40 (also not shown). The findings support the diagnosis of metastatic high grade carcinoma with neuroendocrine differentiation.

While the stains support a diagnosis of small cell carcinoma, the morphologic diagnosis was mildly questionable. I went back to the patient’s record to see what we may have missed in the clinical history. It turns out the patient initially presented with Stage IB2 HPV+, moderately-differentiated cervical adenocarcinoma in 2020. After completing brachytherapy and one cycle of chemotherapy, but could not tolerate additional treatments due to leukopenia and elevated LFTs. Shortly thereafter the patient complained of abdominal pain and a liver mass and bulky lymphadenopathy were identified on imaging. An FNA of a supraclavicular lymph node confirmed not only metastasis of the patient’s cervical cancer, but discovered a small cell/neuroendocrine transformation. And this is why proper documentation of clinical history is so important to pathologists and laboratory professionals. In one of my earlier posts, I preached that cancer doesn’t discriminate; so why should we? Keeping an open mind is paramount to both succeeding in and enjoying the field of cytopathology. If it looks like a duck, and it walks like a duck, it might actually have transformed into a goose.

-Taryn Waraksa, MS, SCT(ASCP)CM, CT(IAC), has worked as a cytotechnologist at Fox Chase Cancer Center, in Philadelphia, Pennsylvania, since earning her master’s degree from Thomas Jefferson University in 2014. She is an ASCP board-certified Specialist in Cytotechnology with an additional certification by the International Academy of Cytology (IAC). She is also a 2020 ASCP 40 Under Forty Honoree.