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.

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.

Hematology Case Study: Presenting a Double Feature Starring Chronic Myelogenous Leukemia

One of the reasons I love working in Hematology is because when we have unexpected results they are often accompanied by visuals… and a picture is worth a thousand words! Unusual or critical results in Chemistry can be interesting, sometimes there are dilutions to perform, results to compare or puzzles to solve, I have always loved working up a good antibody or complicated multiple antibodies in Blood Bank or calculating how many units I may need to screen to find compatible ones, gram stains of unusual organisms in Microbiology can be exciting, but nothing beats some of the cells we see in Hematology! It’s always fascinating when we find unusual cells and follow up with smear reviews with our pathologists. And, being able to save these visuals in CellaVision or saving the slides for teaching, is a plus. These cases are a gift that keeps on giving! Lately I’ve had my share of “exciting” specimens, usually on a Saturday or Sunday afternoon! It never fails to get the adrenaline going when you are the first one to see a CBC with a WBC of 400,000, a differential that is over 90% blasts, rare lymphoma cells, malarial parasites, or a body fluid smear full of malignant cells. The following 2 cases are a very remarkable looking smear and a not so remarkable one, from 2 different patients with the same diagnosis.

The first patient is a 71 year old male who had a routine CBC done by his primary care physician. The blood was collected as an outpatient on a Saturday morning, and brought to our lab by a routine courier that afternoon (of course, right before change of shift!). We had one previous CBC result on this gentleman, from several years earlier, which was essentially normal. CBC result shown below:

Table 1. Case 1, CBC results. [Editor’s note: a previous version of this table noted a Hct of 231.8. The correct result is 31.8.]
Table 2. Case 1, Manual Differential results.
Image 1. Peripheral smear, Case 1, WBC 363.14.

As soon as I saw the results, I called the provider with the WBC and alerted them that I would be contacting the pathologist on call and calling back with the differential. Our pathologist confirmed blasts on the peripheral smear and requested that the sample be sent out for flow cytometry. The pathology report stated “Marked leukocytosis with left shift and <5% blasts. The presentation is suspicious for a myeloproliferative neoplasm (e.g. chronic myelogenous leukemia (CML)). Immunophenotypic studies have been ordered and will be reported separately. Clinical correlation and Hematology consult recommended.” Flow cytometry results showed left shifted maturation and FISH studies demonstrated t(9;22) BCR-ABL with 98% of positive nuclei in bone marrow. No other mutations were detected. Diagnosis: chronic myelogenous leukemia. Five days later, we had a bone marrow scheduled on a 50 year old male. A CBC done 2 weeks earlier showed a mild leukocytosis and thrombocythemia. (WBC 12.4, Hgb 17.8, Hct 52%, PLT 539). Diagnoses under consideration were possible CML, polycythemia or a myeloproliferative neoplasm (MPN). The patient’s CBC the day of the procedure is shown below.

Table 3. Case 2, CBC results.
Table 4. Case 2, Manual Differential results.

Cytogenetic analysis showed an abnormal clone characterized by the Philadelphia chromosome translocation, t(9;22). The BCR/ABL gene rearrangement was detected by FISH, with 78% of positive nuclei in bone marrow. The bone marrow was negative for other mutations. Flow cytometry analysis reported no evidence of abnormal myeloid maturation or increased blast production. There was no evidence for a lymphoproliferative disorder. Diagnosis: chronic myelogenous leukemia.

In 1959, at a time when techniques for preparing chromosomes for visualizing under the microscope were still very unsophisticated, 2 researchers in Philadelphia detected a tiny abnormality in the chromosomes of patients with CML. They noticed that part of chromosome 22 appeared to be missing. It was not until 1970, when techniques for chromosome banding became available, that this discovery was shown to be a translocation between chromosomes 22 and 9. The shortened chromosome 22 was named the Philadelphia (Ph) chromosome after the city where it was discovered.

Image 2. The Philadelphia chromosome. A piece of chromosome 9 and a piece of chromosome 22 break off and trade places (cancer.gov).

At diagnosis, over 90% of CML cases have the t(9;22) translocation which has become a hallmark for a diagnosis of CML. However, the Ph chromosome is also detected in about 30% of adult acute B cell lymphoblastic leukemia (B-ALL), and a very small number of acute myeloid leukemias (AML) and childhood B-ALL so testing must be done for differentiation. t(9;22) is a translocation of the proto-oncogene tyrosine-protein kinase ABL1 gene on chromosome 9 and the breakpoint cluster region BCR gene on chromosome 22. The newly formed chromosome 22 with the attached piece of chromosome 9 is called the Philadelphia chromosome. The BCR-ABL oncogene is formed on the Philadelphia chromosome and the product of the Ph translocation is an abnormal fusion protein, p210, which has increased tyrosine kinase activity. This, in turn, is responsible for the unregulated proliferation of cells seen in CML. Tyrosine kinase inhibitors (TKIs) have been developed as targeted therapy for Ph+ CML.1

So, how can these 2 patients with very different CBC results both be diagnosed with CML? CML can be classified into phases of CML-chronic phase (CML-CP), accelerated phase (CML-AP), and blast crisis (CML-BP). The WHO Classification of 2017 proposed a system of cutoffs to define each phase. The phases are based mainly on the number of blasts in the blood or bone marrow. Progression from CML-CP to CML-AP is also generally recognized to correlate with an increase in BCR-ABL1 levels. Several studies have been done that discuss another phase, pre-leukemic (pre-clinical) CML. These pre-leukemic patients have the Philadelphia chromosome, the genetic hallmark of CML, without other abnormalities. They have a normal to mildly elevated WBC and are asymptomatic. In these cases, progression to CML-CP can be several months to several years. One interesting factor common in this phase, which can help in diagnosis, is the presence of an absolute basophilia (ABC) >200/mm3. This basophilia is also seen in CML-CP and often progresses with the disease.2

Results from both patients are compared below. While we may more readily recognize a new CML that presents with very high WBC, left shift, and blasts, FISH, flow and cytogenetics of both these patients indicated a diagnosis of CML. This second patient may be one that could be classified as a pre-CML. The patient is certainly fortunate to have physicians who suggested further workup so he can benefit from his early diagnosis.

Table 5. Comparison of results from 2 cases.

References

  1. Huma Amin*, Suhaib Ahmed. Characteristics of BCR–ABL gene variants in patients of chronic myeloid leukemia. Open Medicine, 2021.16:904-912.
  2. Aye, Le Le; Loghavi, Sanam; Young, Ken H et al. Preleukemic phase of chronic myelogenous leukemia: 2. morphologic and immunohistochemical characterization of 7 cases Annals of Diagnostic Pathology. April 2016 21:53-58 Language: English. DOI: 10.1016/j.anndiagpath.2015.12.004.
  3. Kuan JW, Su AT, Leong CF, Osato M, Sashida G. Systematic review of pre-clinical chronic myeloid leukaemia. Int J Hematol. 2018 Nov;108(5):465-484. doi: 10.1007/s12185-018-2528-x. Epub 2018 Sep 14. Erratum in: Int J Hematol. 2018 Nov 7;: PMID: 30218276.
  4. https://www.cancer.gov/publications/dictionaries/cancer-terms/def/philadelphia-chromosome
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.

Just A Little Too Much – Unrelated Cluster of Brucella Positive Cases in a Community Hospital

A 65 year old, patient 1, who did not have a travel history outside the United States, presented at the ED for hematuria, left upper quadrant pain, generalized weakness, and a fever of 100°F. He was admitted for suspected viral illness causing idiopathic thrombocytopenia and acute cholecystitis. However, CT scan and RUQ ultrasound upon admission was negative for cholecysitits. His AST and ALT were elevated. Blood parasite smears were ordered to rule out Babesia, which were negative. HIV and Ehrlichia antibodies were also negative. Blood cultures (BC) were also drawn on hospital day (HD) 1.

His BC became positive 3 days after incubation, with the initial Gram stain showing tiny gram negative coccobacilli (Figure 1). FilmArray Blood culture Identification panel-2 (BCID-2) returned negative. BC broth was sub-cultured to Blood, chocolate, and MacConkey agar plates and a haze of growth was observed on the BAP (Figure 2) and Choc plates after 48 hours of incubation. No growth on MAC. Our laboratory could not rule out Brucella or Burkholderia after a battery of biochemical tests (oxidase: strong positive, catalase positive, no satellite growth, nitrite positive) performed in BSL3. The isolate was then referred to the state department of health (DOH), which provided a final identification of Brucella suis. The history revealed that he ate raw beef and sushi a week before his symptoms appeared.

One week after patient 1 admission, a 50 year old male (patient 2) presented to ED with 2 weeks of unexplained fever, chills, headache and occasional vomiting. Blood cultures were drawn prior to admission. Malaria screening was also performed due to his recent travel history of South Eastern Africa. Patient 3, a 36 year old female also presented with similar symptoms 2 days after patient 2 was admitted. However, Patient 3 had a recent travel to Eastern Africa. Her blood cultures were also drawn. In both patients, HIV Ag/Ab screening, malaria, Babesia, Erhlichia PCR, and Quantiferon were negative. Liver panels were also performed on both patient 2 and 3. Both ALT and AST were elevated, which is a common theme in all three patients. Both patients 2 and 3 drank unpasteurized camel milk during their trips. Similar to patient 1, the blood cultures of patient 2 and 3 became positive three days after incubation. Since the growth pattern and Gram stain characteristics of blood bottle subcultures was similar to that of patient 1, the isolates were sent to the DOH as soon as a haze of growth appeared on the media. Both isolates were identified as Brucella melitensis.

Discussion

Brucella spp. are facultative, small gram negative coccobacilli and tend to appear as clusters on the Gram stain. Brucella melitensis is the most common cause of human infection followed by B. suis and B. abortus although the latter two can be more prevalent in certain geographical regions. B. canis infection in human is rare. Brucellosis is a zoonotic disease. Most industrialized countries, with effective public health measures, have managed to control the infection. According to 2019 report by CDC, 165 cases of Brucellosis were reported in the United States. Brucella infections in certain parts of the world are particularly associated with drinking raw unpasteurized goat or camel milk.

Brucellosis symptoms appear between 1-4 weeks after exposure. Clinical presentations of Brucellosis are often nonspecific and can mimic malaria or typhoid fever in those returning from the endemic areas; therefore, Brucellosis is considered “the disease of mistakes.” One of the organ systems commonly affected in Brucellosis is liver, with the manifestation of acute liver failure and unexplained thrombocytopenia. Approximately 25 to 35 percent of Brucellosis patients have high AST and ALT associated with low platelet levels. A similar profile was observed in our patients presented here.

While person-to-person transmission is rare, Brucella is the most common laboratory acquired infection (LAI). LAI occurs via aerosolization of Brucellae upon manipulation of isolates on open benches. Manipulation includes picking colonies for rapid biochemical tests, MALDI-ToF, or susceptibility testing on an open bench. Notable LAI’s due to Brucella are reported by New York State Department of Health in 2015-2017, when more than 200 cases of laboratory workers were exposed. Because the infectious dose for Brucella is extremely low (5CFU/mL), it is considered a highly pathogenic category B Bioterrorism agent. LAI can be prevented if clinicians notify the clinical laboratory of suspicious Brucella cases when samples are sent for cultures.

Most laboratories today utilize rapid molecular blood culture panels for initial identification of positive blood cultures. Microbiology laboratories should implement a bio-alert “rule out or refer” protocol to minimize exposure when 1) rapid molecular blood culture identification multiplex panels fail to detect any organisms; 2) prolonged incubation time (blood culture bottles and subculture media) in most cases; 3) atypical Gram stain characteristics (small coccobacilli that typically tend to cluster in positive blood cultures and sometimes appear as gram variable); and 4) growth only on blood and chocolate, but not on MacConkey agar.

Identification of Brucella by MALDI-ToF or any commercial methods in sentinel laboratories is highly prohibited. Biochemical tests must be performed in a biosafety cabinet to rule out potential biothreat agents. In most cases, MALDI-ToF systems can misidentify select bioagents. One salient example was a case report by the Yale University Clinical Microbiology Laboratory, where Brucella was misidentified as Ochrobactrum anthropi by Vitek MS.

Because of a diverse range of clinical symptoms, definitive diagnosis of Brucellosis is mainly achieved by laboratory findings by means of serology or isolation of organisms in culture. Serologic diagnosis requires two serum samples – the first sample taken during the acute phase of illness and the latter should be taken 2-4 weeks. A rise in antibodies of four fold or higher is considered positive Brucellosis. The Laboratory Reference Network and Center for Disease Control (CDC) can perform Brucella microagglutination test (BMAT) for B. melitensis, B. suis, and B. abortus. American Society of Microbiology (ASM), Association of Public Health Laboratories (APHL), and Laboratory Response Network (LRN) have diagnostic protocols and guidelines for ruling out or refer potential Brucella from culture isolation.

Figure 1. Small clusters of gram negative coccobacilli from patient 1’s positive blood culture.
Figure 2. 48 hour old slow growing Brucella on Blood Agar Plate (Image courtesy: BioThreat agent bench cards for Sentinel Laboratory by Texas Department of State Health Services).

References

  1. Kazak E, Akalın H, Yılmaz E, Heper Y, Mıstık R, Sınırtaş M, Özakın C, Göral G, Helvacı S. Brucellosis: a retrospective evaluation of 164 cases. Singapore Med J. 2016 Nov;57(11):624-629. doi: 10.11622/smedj.2015163. Epub 2015 Nov 13. PMID: 26768063; PMCID: PMC5331138.
  2. Ackelsberg J, Liddicoat A, Burke T, Szymczak WA, Levi MH, Ostrowsky B, Hamula C, Patel G, Kopetz V, Saverimuttu J, Sordillo EM, D’Souza D, Mitchell EA, Lowe W, Khare R, Tang YW, Bianchi AL, Egan C, Perry MJ, Hughes S, Rakeman JL, Adams E, Kharod GA, Tiller R, Saile E, Lee S, Gonzalez E, Hoppe B, Leviton IM, Hacker S, Ni KF, Orsini RL, Jhaveri S, Mazariegos I, Dingle T, Koll B, Stoddard RA, Galloway R, Hoffmaster A, Fine A, Lee E, Dentinger C, Harrison E, Layton M. BrucellaExposure Risk Events in 10 Clinical Laboratories, New York City, USA, 2015 to 2017. J Clin Microbiol. 2020 Jan 28;58(2):e01096-19. doi: 10.1128/JCM.01096-19. PMID: 31694974; PMCID: PMC6989065.
  3. Manual of Clinical Microbiology. 11th edition. 2018.
  4. Poonawala H, Marrs Conner T, Peaper DR. The Brief Case: Misidentification of Brucella melitensis as Ochrobactrum anthropi by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS). J Clin Microbiol. 2018 May 25;56(6):e00914-17. doi: 10.1128/JCM.00914-17. PMID: 29802238; PMCID: PMC5971538.
  5. https://www.cdc.gov/brucellosis/exposure/areas.html

-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: An Adult Male with HIV Presents with Shortness of Breath

Case History

An adult male presented to the Emergency Department with hemoptysis and shortness of breath. The patient was previously diagnosed with HIV-1, but was non-compliant on antiretroviral medications. At the time of admission, HIV-1 viral load was greater than 1,000,000 copies/mL and his CD4 T+ cell count was 3 cells/µL. The following microbiology tests were ordered and were negative: acid fast bacilli culture, fungal culture, and Mycobacterium tuberculosis complex PCR on sputum as well as a Legionella urinary antigen. A bronchoalveolar lavage (BAL) was obtained and sent for a laboratory-developed Pneumocystis jirovecii PCR, which was positive.

Pneumocystis jirovecii

Previously named Pneumocystis carinii, Pneumocystis jirovecii was originally thought to be a parasite. Although this organism cannot be grown in routine fungal culture, molecular analysis has revealed that Pneumocystis is a fungus. Asymptomatic colonization is common, which may play a role in transmission or in disease development when the immune system is suppressed. Colonization is more common in children than adults.

Pneumocystis jirovecii is the causative agent of Pneumocystis jirovecii pneumonia (PJP or PCP). Patients with PJP often present with cough, fevers, and dyspnea. Diffuse bilateral infiltrates are commonly observed on chest X-rays. Patients are typically treated with trimethoprim-sulfamethoxazole, which is also used for PJP prophylaxis in high risk populations. Pneumocystis can very rarely infect extrapulmonary sites, including lymph nodes, spleen, bone marrow, and liver.

Any immunosuppressed person is at risk for PJP. In particular, patients with HIV and AIDS are at a high risk for PJP, especially those with a CD4+ T cell count less than 200 cells/µL. Prior to effective antiretroviral medications and routine PJP prophylaxis in AIDS patients, PJP was one of the top causes of infections and death in those with HIV and AIDS. In the highlighted case, our patient had a CD4 T+ cell count of 3 cells/µL, which put him in the high risk category.

When PJP is suspected, a respiratory sample, either BAL or induced sputum, should be collected. The gold standard is to perform microscopy on respiratory samples using histopathology stains (Grocott-Gomori methenamine silver (GMS), hematoxylin and eosin (H&E), Papanicolaou-stained, or immunohistochemistry) and microbiology stains (calcofluor white stain). On GMS stains, Pneumocystis appears as thin-walled spheres measuring 2 – 5 microns with intracystic bodies while foamy eosinophilic exudates can be observed on the H&E stain. In the microbiology lab, fluorescein-conjugated monoclonal antibody kits are often used, which can stain the cyst and/or trophic form of Pneumocystis, depending on the kit. However, the immunofluorescent stain lacks sensitivity, especially in the non-HIV population. Molecular assays have been developed, but are not widely available or standardized. In comparison to fluorescent stains, molecular assays are highly sensitive and specific for Pneumocystis DNA, but important caveats do exist. There are no FDA-cleared Pneumocystis PCR assays, meaning that methodology, and subsequent sensitivity and specificity, varies lab to lab. While not available yet, the development and use of quantitative PJP assays have been proposed, which could offer fungal burden information and help distinguish between colonization and infection. Serology options are available, but are not specific to PJP. One serological test, (1,3)-β-D-glucan (BDG), can be used as an aid for diagnosis of PJP. BDG is estimated to be 94-96% sensitive in PJP patients. While BDG testing is non-invasive, it is positive for a variety of fungal infections including Candida spp. and Aspergillus spp. Thus, additional PJP studies are needed to support a PJP diagnosis.

Sources:

  1. Morris A, Norris KA. Colonization by Pneumocystis jirovecii and its role in disease. Clin Microbiol Rev. 2012;25(2):297-317. doi:10.1128/CMR.00013-12
  2. Bateman M, Oladele R, Kolls JK. Diagnosing Pneumocystis jirovecii pneumonia: A review of current methods and novel approaches. Med Mycol. 2020;58(8):1015-1028. doi:10.1093/mmy/myaa024
  3. Miller JM, Binnicker MJ, Campbell S, et al. A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. 2018;67(6):e1-e94. doi:10.1093/cid/ciy381
  4. Zhang SX, Babady NE, Hanson KE, et al. Recognition of Diagnostic Gaps for Laboratory Diagnosis of Fungal Diseases: Expert Opinion from the Fungal Diagnostics Laboratories Consortium (FDLC). J Clin Microbiol. 2021;59(7):e0178420. doi:10.1128/JCM.01784-20
  5. Onishi A, Sugiyama D, Kogata Y, et al. Diagnostic accuracy of serum 1,3-β-D-glucan for pneumocystis jiroveci pneumonia, invasive candidiasis, and invasive aspergillosis: systematic review and meta-analysis. J Clin Microbiol. 2012;50(1):7-15. doi:10.1128/JCM.05267-11
  6. Theel ES, Doern CD. β-D-glucan testing is important for diagnosis of invasive fungal infections. J Clin Microbiol. 2013;51(11):3478-3483. doi:10.1128/JCM.01737-13
  7. Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev. 2011;24(2):247-280. doi:10.1128/CMR.00053-10

-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: 70 Year Old Man with a History of Papillary Urothelial Carcinoma

Case Description

A 70 year old male with a past medical history of hypertension and non-invasive multifocal high grade papillary urothelial carcinoma was being followed closely for recurrence and underwent magnetic resonance imaging (MRI) of the abdomen and pelvis. The report described a 2.6 x 3.9 x 5.2 cm lobulated cystic mass involving the left psoas muscle. Furthermore, there was encasement of the left common iliac artery and less involvement of the left common iliac vein (Image 1). Further evaluation of the lesion was pursued to determine the etiology. An important aspect of this case to consider is the patient’s prior cancer treatment regimen, which included intravesical Bacille Calmette-Guerin (BCG) for 5/6 cycles. The final BCG treatment was held because the patient developed “BCG-osis” comprised of chest pain, rigors, chills and hypotension. Given the only pathology to date on the patient was non-invasive papillary carcinoma (even though it is high grade), the oncology group did not think the psoas muscle lesion was a metastasis. Fine needle aspiration (FNA) was pursued and the CT-guided aspiration demonstrated “abundant histiocytes and acute inflammation with necrotic debris…Acid fast organisms identified on AFB (acid fast bacilli) stain…Negative for malignant cells” (Image 2). Microbiology cultures were obtained at the time of FNA. The AFB smear showed 1+ AFB. AFB grew in culture and reacted with the Mycobacterium tuberculosis complex probe. The patient’s interferon gamma response assay (IGRA) was negative the year prior. Antimicrobial susceptibilities (AST) using the Mycobacterial Growth Indicator Tube system with single drug concentrations revealed susceptibility to isoniazid, rifampin, and ethambutol with resistance to pyrazinamide. Per protocol, the isolate was sent to a reference laboratory for identification which returned as BCG. During the interim, before AST was available, the patient was referred to our Infectious Diseases outpatient clinic where he was started on R(ifampin)-I(soniazid)-P(yrazinamide)-E(thambutol) therapy and then followed up his care with the county health department.  

Image 1. Magnetic resonance imaging of the pelvis demonstrating an enhancing multilobulated cystic mass overlying the left psoas muscle Top: transverse plane (orange arrow). Bottom: sagittal plane (orange arrow).
Image 2. Photomicrographs of the drained fluid from the psoas muscle abscess.
Top) hematoxylin and eosin photomicrograph demonstrating abundant neutrophils and necrotic debris (10x objective).
Bottom) AFB stain demonstrating multiple AFB (see arrows) within the necrotic cellular debris (50x oil immersion objective).

Discussion

BCG is an attenuated strain of Mycobacterium bovis that has, historically, been used as a vaccine to Mycobacterium tuberculosis (MTB) most often used in areas of endemicity. M. bovis is a member of the MTB complex so hybridization probes used in clinical laboratories can distinguish MTB complex from Mycobacterium kansasii or Mycobacterium avium complex but cannot distinguish the MTB complex members from one another. Reference laboratories have molecular techniques including PCR or sequencing that can separate the differing members of the MTB complex. Another traditional distinguishing characteristic between M. bovis (including BCG) and MTB is susceptibility to pyrazinamide. MTB carries a pyrazinamidase which is required to activate the antibiotic; M. bovis does not, so it is intrinsically resistant to pyrazinamide. A laboratorian or clinician should be cognizant of this when a culture result returns as MTB complex that is susceptible to rifampin, ethambutol and isoniazid but is resistant to pyrazinamide alone. Furthermore, IGRAs were designed, in part, to distinguish those who have been vaccinated with BCG versus those exposed to MTB or wild type M. bovis who are latently infected. IGRAs will be negative in those exposed to BCG but would be reactive with either MTB exposure or M. bovis (non-BCG strains).

This case describes an uncommon complication of BCG immunomodulator therapy as a treatment of superficial papillary urothelial carcinoma. BCG’s use as a therapeutic intervention for malignancy is unique. It is postulated that the instillation of the organism stimulates the host’s immune response which can cause inflammation and sloughing of the bladder lining (urothelial cells), which effectively removes foci of superficial pre-cancerous in situ lesions or other intact foci of superficial urothelial carcinoma. The typical course of treatment is 6 cycles of BCG. Local inflammation resulting in cystitis is the most common complication experienced in 27-95% of patients.1 However, approximately 19% of patients undergoing BCG therapy experience severe enough complications to prematurely terminate BCG therapy, according to one study by the European Organization for Research and Treatment of Cancer.2 The patient described developed systemic symptoms during the course of his BCG therapy which prompted his physicians to terminate it. Although far less common than local genitourinary symptoms, mycotic aneurysms can occur in an estimated 0.7-1.4% of cases.1 Psoas abscesses are thought to arise from mycotic aneurysm leak.3

References

  1. Liu Y, Lu J, Huang Y, Ma L. Clinical spectrum of complications induced by intravesical immunotherapy of bacillus Calmette-Guerin for bladder cancer. Journal of Oncology. 2019. DOI: 10.1155/2019/6230409
  2. Sylvester, R, Brausi M, Kirkels W, et al. Long-term efficacy results of EORTC genito-urinary group randomized phase 3 study 30911 comparing intravesical instillations of epirubicin, bacillus Calmette-Guerin, and bacillus Calmette-Guerin plus isoniazid in patients with intermediate- and high-risk stage Ta T1 urothelial carcinoma of the bladder. European Urology. 2010. 57:5; 766-773.
  3. Leo E, Molinari A, Rossi G, et al. Mycotic abdominal aneurysms due to Mycobacterium bovis after intravesical bacillus Calmette-Guerin therapy. Annals of Vascular Surgery. 2015. 29:6;1381.e1-1318.e6.

-Dominick Cavuoti is a Professor at UT Southwestern Medical Center who specializes in Cytopathology, Infectious Disease pathology and is a medical director of the Microbiology laboratory at Parkland Health and Hospital System.

-Kelley Carrick is a Professor at UT Southwestern Medical Center who specializes in Cytopathology and Gynecologic pathology. She is the Chief of Cytopathology at Parkland Health and Hospital System.

-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.

Hematology Case Study: Unusual Lymphocytes Seen in an Apparently Healthy Young Adult

A healthy 30 year old woman visited her primary care physician concerned about a rash with questionable infection on her hands. The physician prescribed an antibiotic for infection and ordered a CBC. From the results below, it can be seen that the patient had a pancytopenia and a relative lymphocytosis.

Table 1. CBC results.
Table 2. Automated differential results

A manual differential was performed on CellaVision and the presence of large, clefted lymphocytes with immature features was noted. A request for pathology review was sent to the pathologist. The pathologist’s review stated “ Atypical lymphocytosis, specimen to be submitted for flow cytometry. Report to follow. Occasional atypical lymphocytes with immature features also noted. Lymphocyte population is predominantly mature”

The peripheral blood sample was sent out for immunophenotyping by flow cytometry and FISH studies. Flow cytology reported “precursor B-cell population expressing CD19, CD10, HLA-DR, and CD34 is identified. Percent of abnormal cells, 30%. These findings are consistent with precursor B-lymphoblastic leukemia.” While we tend to associate a leukemia diagnosis with a high white blood cell count, and the presence of blasts, this patient was unusual in that she did not have a high WBC or blasts seen on the peripheral smear. Pancytopenia in ALL has been noted in literature. A study of new onset pancytopenia in adults showed that the majority of cases were acute myeloid leukemia, but ALL and other lymphomas also caused pancytopenia3. Another study noted that “pancytopenia followed by a period of spontaneous recovery may precede the diagnosis of acute lymphoblastic leukemia.”1 While the pathologist did not identify blasts on this differential, and cells were predominately mature, WBC was very low, and our analyzer did flag “?blasts/abnormal lymphs” and reflexed the manual differential.

Image 1. Clefted lymphocytes seen on peripheral smear.
Image 2. Clefted lymphocytes on CellaVision.
Table 3. FISH report.

Leukemia is a broad term that includes a number of different chronic and acute diagnoses. Chronic and acute forms are further broken down into myeloid and lymphoid and then into subtypes. The French-American-British (FAB) classification of acute leukemias was devised in the 1970’s and 1980’s and was based on cytochemical staining and morphology of cells. These tests were performed manually and relied on what the cells look like under the microscope. The series of stains were used to differentiate myeloblasts from lymphoblasts. I’m old enough that I remember learning about these stains when they were being developed and thinking how amazing they were!

We’ve come a long way since the early 1980’s! Although the FAB diagnostic criteria are not entirely forgotten, the World Health Association (WHO) classification, first published in 2001, has largely replaced the FAB classification. The newest guidelines for Acute Lymphoblastic Leukemias (ALL) were published by WHO in 2016. These new guidelines supplement morphology and cytochemical staining with newer testing which can now identify and distinguish B cell and T cell ALL. In making a diagnosis, peripheral blood and/or bone marrow aspirate samples are subject to flow cytometry immunophenotyping and chromosome testing such as cytogenics or fluorescence in situ hybridization(FISH). Molecular tests can also be done to look for specific gene changes in the leukemia cells. The WHO classification has become preferred because these new tests can give more information that is important for treatment. Prognosis for ALL depends on patient age, WBC counts at diagnosis and these specific test results which tell us which subtype of ALL is present. The presence and identification of chromosomal alterations is important for diagnosis and therapy decisions. Identifying chromosomal alterations can also lead to better risk classification which is significant because of the knowledge that, while rearrangements tend to have poorer outcomes, some rearrangements actually offer a better prognosis. With the future era of individualized, targeted therapy for leukemia, combining conventional cytogenics with molecular and FISH methods will greatly enhance the accuracy of information and provide patients with more specific and customized treatment options.

While ALL is the most common childhood leukemia, it is not as commonly seen in adults. B cell ALL is more common than T cell ALL in all ages, and accounts for about 90% of ALL cases in children and about 75% of ALL cases in adults. Cure rates in children exceed 90% but in adults varies with age and depending on chromosomal alterations. Most B cell ALL subtypes with chromosome translocations tend to have a poorer outcome than those without translocations. As well, younger adults, <50 years old, have better prognosis than older adults.

This patient did not have a BCR/ABL rearrangement or MLL gene locus 11q23 translocation, which both carry poorer prognoses, but she also did not have a translocation between chromosome 12 and 21 or more than 50 chromosomes, both of which offer more favorable prognoses. This young woman therefore would be in an average risk category and appears to have been diagnosed very early in the course of her disease. We have not seen any further workup, as the patient is being treated at another facility. We wish her well in her leukemia treatments.

References

  1. Hasle H, Heim S, Schroeder H, et al. Transient pancytopenia preceding acute lymphoblastic leukemia (pre-ALL). Leukemia. 1995 Apr;9(4):605-608.
  2. Iacobucci I, Mullighan CG. Genetic Basis of Acute Lymphoblastic Leukemia. J Clin Oncol. 2017 Mar 20;35(9):975-983. doi: 10.1200/JCO.2016.70.7836. Epub 2017 Feb 13. PMID: 28297628; PMCID: PMC5455679
  3. Bone Marrow evaluation in new onset pancytopenia. Human Pathology. Vol 44, Issue 6. June 2013
  4. Hematology: Basic Principles and Practice, 7th Edition. Ronald Hoffman, Edward J. Benz, et al. 2018 Elsevier
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.

The Great Mimicker

I’ve witnessed that radiologists are often hesitant to perform a core biopsy on a spleen due to its vascularity, so when I attend spleen FNAs, I rarely push. Even when the oncology team requests both a core biopsy and FNA for a hematology workup, I will acquire as many passes as possible from an FNA to work up cytomorphology and flow cytometry before risking a core-induced hemorrhagic complication. When I was called to attend an ultrasound-guided spleen biopsy, I went in knowing two things: the patient has both splenic and brain lesions, and I was going to make the most of what I was given. When I arrived in ultrasound, the radiologist informed me that the patient had polycythemia vera (PV), which would explain the splenomegaly, but not the brain lesions. The patient, a 65 year old male, received the diagnosis in 2009 and was managed with phlebotomies for six years until a rising platelet and white blood cell count required an intervention of hydroxyurea. Within 18 months, the patient developed a PE and dizziness and began therapeutic anticoagulation. At the same time, the patient’s “metastatic lesions” were identified on imaging. The first state of business is finding out if his PV had progressed into myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). There was something… off… about this case though. It didn’t feel like a heme case (not that I prefer non-heme cases… well, okay, I’m a cytotech, so I kind of do). The brain lesions concerned me, and I didn’t know what to infer.

Let’s progress to the FNA. Here are the air-dried, Diff-Quik smears that changed everything.

Images 1-2: Splenic mass, fine needle asperation, DQ-stained smears.

What non-heme surprise is THIS?! Are these histiocytes? There’s no way, those nucleoli are aggressive! Look at the chromocenters! The variation in nuclear size! What epithelial beauty is this? Is it epithelial?! Is that a fibrovascular core? It can’t be sarcomatoid, could it? Twenty inner monologue questions later, “okay, it’s adequate,” I said to the radiologist. But wait… should I do it? Should I press for a core biopsy? The radiologist asked me if I needed anything else for the diagnosis. Perhaps she saw my puzzled expression, compelling her to tell me that she felt the imaging looked weird – she thought it wasn’t a heme case either. At least I’m not alone here. I took a deep breath, expecting the worst and hoping for the best, and I asked the radiologist to collect a core biopsy. She checked the ultrasound Doppler for excessive vascularity, and much to my surprise, she agreed to perform the core. After collecting more FNAs for my cell block and dropping the core biopsies off in surgical pathology, I showed the case to my attending pathologist. He also agreed that we don’t need to triage it for heme. He asked if the patient had any significant history other than PV, splenic lesions, and brain mets, and I told him nothing was reported in his chart. I checked the chart again for good measure while he was running through his differential diagnoses of lung, GI, prostate, etc., and saw the patient was scheduled to see dermatology later that week. I mentioned to that to my attending, and he suggested it could be a melanoma.

The following morning, I examined the pap-stained slide and began to second guess myself. Was I wrong? Could it be a heme case? Hodgkin’s wasn’t in the differential, and these cells look so much more aggressive than Reed-Sternberg cells. At least we have a core biopsy either way. We could run immunos on the cell block and save the cores for molecular. I screened the pap-stained slides a little longer, focusing on the macronucleoli, the owl-eye and eccentric nuclei, the poorly-differentiated nature of this… MELANOMA. YES! So, the cells might not contain any melanin pigment, but my attending’s inkling was exactly right. Waiting for my cell block to arrive, I listed melanoma as my primary diagnosis with a differential of lung or prostate cancer.

Images 3-4: Splenic mass, fine needle aspiration, Pap-stained smear.

The cell block confirmed my non-heme diagnosis and kept my differentials at bay. The attending pathologist ordered an immunohistochemistry profile of S100, HMB-45, and Melan A, as well as AE1/AE3. The first three immunostains (prior to our adoption of SOX-10) confirmed a diagnosis of metastatic malignant melanoma. Soon thereafter, the patient’s primary lesion was identified on his back, and he was treated with radiation and immunotherapy. Unfortunately, the metastases were not responding to the immunotherapy, and a few days after a clinical trial was offered, the patient passed away.

Images 5-8: Splenic mass, fine needle aspiration. 5 and 6, cell block, H&E; 7 and 8, Melan A+.

Melanoma is known as the great mimicker, especially in amelanotic form, and it should always be in the back of your mind as a differential diagnosis. Lack of melanin pigment and a large cherry red macronucleoli leads us to favor lung, prostate, or serous adenocarcinoma), renal cell carcinoma, hepatocellular carcinoma, Hodgkin’s lymphoma, or even an epithelioid sarcoma. This case highlights the need to remember that metastatic melanoma is always a possibility, even when you do not have a primary site or previous clinical history of the disease.

-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.