Microbiology Case Study: An 88 Year Old Male with Headache

Case History

An 88 year old male presents with fever, nausea, and headache. The patient reported a diffuse headache accompanied by malaise, fatigue, and nausea without vomiting. He denied confusion, irritability, or a personal and family history of headaches. According to the patient, he frequently attends cookout parties and enjoys fruits, salads, wine, and cheese. Temperature is 38.2 degrees Celsius, blood pressure is 96/65 mmHg, pulse is 102 beats/minute, and respiratory rate is 20 breaths per minute. Physical exam is negative for nuchal rigidity and Kernig sign. Funduscopic exam is negative for papilledema. CBC shows leukocyte count of 16,000/mm3. The patient’s blood culture is positive.

Laboratory Identification

Image 1. Short gram positive bacilli identified on Gram stain of blood culture (100x oil immersion).
Image 2. Aerobic growth of round and translucent colonies with a narrow zone of beta hemolysis subcultured from positive blood culture bottle to sheep blood agar plate.

The blood culture was positive for short, gram positive bacilli. Sheep blood agar plate grew round and translucent colonies which have a narrow zone of beta hemolysis as shown on our plate. The organism was catalase positive and motile at 25 degrees Celsius. It showed end over end tumbling motility in a wet prep and an umbrella pattern in semi-solid motility medium. It was identified by MALDI-ToF as Listeria monocytogenes.


Listeria monocytogenes is a gram positive bacillus that is isolated from the environment and a variety of animals. It is associated with foodborne outbreaks from dairy and meat products. The most common foods associated with listeriosis outbreaks include unpasteurized raw milk, cold deli meat, hot dogs, raw sprouts, smoked seafood, and soft cheese.1

Listeria commonly infects pregnant women, immunocompromised individuals, and elderly 65 years or older.1 Among pregnant women, Listeria can lead to miscarriages, stillbirths, and newborn meningitis resulting in death.1 In 1985, an outbreak of Listeria due to soft cheese resulted in 142 individuals sick, 10 newborn deaths, 18 adult deaths, and 20 miscarriages.1 Among the immunocompromised and elderly, Listeria can cause septicemia and meningitis. In 2011, a cantaloupe outbreak due to Listeria resulted in 147 people sick in 28 states and 33 deaths.1 The infected population was mostly over the age of 65 years.1 In addition, Listeria can cause acute febrile gastroenteritis in healthy individuals.2 Patients typically present with fever, watery diarrhea, nausea, headache, and pain in joints and muscles.2 Symptoms start 24 hours after the ingestion of bacteria and resolve by themselves in 2 days.2

Treatment of Listeria depends on the severity of symptoms. Although pregnant women with Listeria infection typically present with a self-limited flu-like illness, they are treated with IV ampicillin to prevent infection of the fetus.1 For patients other than pregnant women, the treatment of Listeria infection depends on the severity of symptoms.


  1. Information for Health Professionals and Laboratories. (2017, June 29). Retrieved on March 1st, 2018 from https://www.cdc.gov/listeria/technical.html
  2. Say Tat Ooi, Bennett Lorber; Gastroenteritis Due to Listeria monocytogenesClinical Infectious Diseases, Volume 40, Issue 9, 1 May 2005, Pages 1327 1332, https://doi.org/10.1086/429324


-Ting Chen, MD is a 1st 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 45 Year Old Woman with Breast Abscess

Case History

Our patient is a 45-year-old female who presents to the Emergency department with breast pain. She was diagnosed with granulomatous mastitis 3 months prior. She was treated with 3 weeks of steroids, but they were stopped when the mass was unchanged and the patient was experiencing increasing breast tenderness. Since then she and has undergone several procedures to drain her right breast abscess, the most recent being five days prior. The woman has been treated with sequential courses of sulfamethoxazole–trimethoprim and metronidazole without improvement. On this visit, the abscess was again drained and sent to the microbiology laboratory for culture. The Gram stain showed no bacteria and 3+ polymorphonuclear cells. After 48 hours incubation there was scant growth on the blood agar plate and no growth on the chocolate, MacConkey or CNA plates. The colonies growing on the blood plate were tiny, white, and lipophilic (Image 1).

Image 1. Small, white, lipophilic colonies growing on blood agar plate at 48 h incubation.  There was no growth of this organism on chocolate or MacConkey agars this time point.


The organism was identified as Corynebacterium kroppenstedtii.

Colonies on the blood agar plate were identified as Corynebacterium kroppenstedtii using MALDI-TOF mass spectrometry for identification. C. kroppenstedtii is catalase positive, non-motile and a facultative anaerobe. It grows better on 5% sheep blood agar than chocolate agar, as is the case for many Corynebacterium spp. Corynebacterium come in two varieties, lipophilic such as Corynebacterium jeikeium, and luxuriantly growing, such as Corynebacterium straitum. C. kroppenstedtii is part of the former lipophilic group, forming small colonies after extended incubation.  Lipids such as Tween-80 can added to the media to improve growth of lipophilic Corynebacterium such as C. kroppenstedtii, but clinically this is not routinely performed. When viewed on a gram stain, the bacteria are rod-shaped gram positive diptheroids with typical coryneform morphology. Both MALDI-TOF and 16S rRNA sequencing can accurately identify C. kroppenstedtii to the species level.

C. kroppenstedtii is a relatively newly recognized species within the Corynebacterium genus. It was first described in a case series of young Polynesian women with histological evidence of lobar mastitis, from which C. kroppenstedtii was identified from >40% of the patients’ abscesses. Since that time, isolation of C. kroppenstedtii has been clinically associated with breast abscesses and granulomatis mastitis. C. kroppenstedtii is a highly lipophilic bacterium. Its cell wall lacks many mycolic acids, which may explain its propensity to grow in lipid-rich sites such as mammary glands. C. kroppenstedtii typically affects women of reproductive age and can be difficult to diagnose due to the slow growing nature of the lipophilic organism and the relatively few organisms present in abscess specimens.

Prior to identification by MALDI-TOF MS and 16s rRNA sequencing this patient’s culture would have been reported as rare or 1+ “dipthroid,” “coryneform,” or “Corynebacterium spp.” Without knowing the clinical significance of this organism, the culture results could easily be dismissed as contaminating skin flora.

It is very difficult to treat C. kroppenstedtii in abscesses, with the most effective treatment requiring both surgical drainage of the abscess and long term antibiotic use. It is fairly difficult to get antibiotics to the site of infection, so antibiotics that test as susceptible in the laboratory may not eradicate the pathogen. Our patient’s isolate of C. kroppenstedtii was susceptible to ciprofloxacin, clindamycin, doxycycline, and intermediate to penicillin. She remains on ciprofloxacin therapy, but has ongoing right breast tenderness. She had another surgical drainage of her breast abscess a week after this case, and the culture also grew 1+ C. kroppenstedtii with 3+ PMN seen on Gram stain, so her infection has not yet been resolved.

  1. Tauch, Andreas, et al. “A Microbiological and Clinical Review on Corynebacterium Kroppenstedtii.” International Journal of Infectious Diseases, vol. 48, 2016, pp. 33–39., doi:10.1016/j.ijid.2016.04.023. ScienceDirect.
  2. Johnson, Matthew G., et al. “The brief case: recurrent granulomatous mastitis due to Corynebacterium kroppenstedtii.” Journal of clinical microbiology 54.8 (2016): 1938-1941.
  3. Paviour, Sue, et al. “Corynebacterium species isolated from patients with mastitis.” Clinical Infectious Diseases 35.11 (2002): 1434-1440.



-Carolyn Wiest, MT(ASCP) graduated from Michigan State University with a BS in molecular genetics and is a medical technologist at NorthShore University HealthSystem.  Her interests are in microbiology and molecular diagnostics. 

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.

Microbiology Case Study: A 28 Year Old Female with Perirectal Abscess

Case History

A 28 year old female with a history of Ulcerative Colitis on humira and azathioprine presented with proctitis and a recent perirectal abscess. The patient reported a two week history of progressively worsening pain and swelling in the perianal region. In addition, she reported recent purulence excreted with bowel movements.  On physical exam, the patient was afebrile and negative for rash, oral lesions, joint pain, or abdominal pain. A perirectal abscess was identified and drained. Abscess culture was positive. Patient reported recently engaging in high-risk sexual behavior with multiple male sexual partners often without protection.

Lab Identification

Image 1. Kidney-bean shaped gram negative cocci identified on Gram staining of perirectal abscess culture (100x oil immersion).  
Image 2. Aerobic growth of flat, gray-white, and moist colonies from perirectal abscess on chocolate agar plate.  

Abscess culture grew kidney-bean shaped gram negative diplococci. Colonies on chocolate agar plate appeared medium sized, flat, gray-brown, and moist. The organism was oxidase positive and identified by MALDI to be Neisseria gonorrhoeae.


Neisseria gonorrhoeae is a kidney-bean shaped gram negative diplococci for which humans are the only host. The organism causes gonorrhea, a common sexually transmitted disease, among young people between the ages of 15-24 years. Gonorrhea is spread by sexual contact or through the birth canal. The most common site of infection is the urogenital tract.2 Males commonly present with dysuria with penile discharge.2 Females commonly present asymptomatically or with symptoms such as mild vaginal mucopurulent discharge and severe pelvic pain2. In addition, gonorrhea can cause infections of the anus, conjunctiva, pharynx, ovary and uterus.2 In the neonate, the culprit organism can lead to ophthalmia neonatorum.2 Lastly, gonorrhea causes disseminated disease such as arthritis, endocarditis, meningitis, and skin lesions on extremities.2 CDC currently recommends treating gonorrhea with dual therapy, a single dose of 250 mg intramuscular ceftriaxone and 1g of oral azithromycin.1

Antibiotic resistance in gonorrhea is an increasing public health concern. The World Health Organization has a program that monitors the global antimicrobial resistance of gonorrhea.3 The data from 77 countries between 2009 and 2014 showed that 66% of reporting countries had encountered gonorrhea strains with either resistance or reduced susceptibility to ceftriaxone.3 81% of reporting countries had encountered gonorrhea strains resistant to azithromycin.3 Given these data, it is important to improve gonorrhea prevention and continue to monitor gonorrhea antibiotic resistance at both the national and global levels.


  1. Gonorrhea treatment and care. (2017, Oct 31st). Retrieved on March 1st, 2018 from https://www.cdc.gov/std/gonorrhea/treatment.htm
  2. Miller KE. Diagnosis and Treatment of Neisseria gonorrhoeae Am Fam Physician. 2006 May 15:73 (10): 1779-1784.
  3. Wi T, et al. Antimicrobial resistance in Neisseria gonororheae: Global surveillance and a call for international collaborative action. PLoS Med 14(7): e1002344.https://doi.org/10.1371/journal.pmed.1002344


-Ting Chen, MD is a 1st 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.

Hematopathology Case Study: A 16 Year Old Male with Fatigue, Fevers, and Weight Loss

Case History

16 year old male with a history of chronic pilonidal cyst presented with fatigue, fevers and weight loss. He was febrile and noted to have cervical and inguinal adenopathy. Labs were significant for a white count of 77,000 with 85% peripheral blasts, anemia and thrombocytopenia.

Bone marrow aspirate
Bone marrow core biopsy
Flow cytometry myeloid markers
Flow cytometry cytoplasmic markers
Flow cytometry T-cell markers


The bone marrow aspirate shows cellular spicules with sheets of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

The bone marrow core biopsy shows a greater than 95% cellular marrow, hypercellular for age with approximately 90% of the cellularity composed of an interstitial population of intermediate-to-large sized mononuclear cells with irregular nuclei, distinct nucleoli, dispersed chromatin, and scant to generous amphophilic cytoplasm, with occasional vacuoles, consistent with blasts.

Flow cytometry shows leukemic cells that express immaturity markers (TdT, CD34, CD117, HLA-DR), T cell lineage markers (CD2, CD7 cCD3), and multiple myeloid markers (CD13, CD117, and variable CD15 and CD11b as well as MPO in a small subset).

Bone marrow core biopsy staining (not shown) had similar findings with blasts showing dim-to-strong positivity for myeloperoxidase, lysozyme, CD34 and CD117, as well as strong positivity for TdT. CD7 was weakly positivity, as well as CD3. CD4 and CD5 were negative.

Genetics diagnostics
NGS panel

With the expression of MPO by flow cytometric analysis and immunohistochemistry, a final diagnosis of acute leukemia with myeloid and T lymphoid phenotypic features, most consistent with T/Myeloid Mixed Phenotype Acute Leukemia (MPAL) was rendered. 


Most acute leukemias are definitively assigned to either myeloid, T or B lymphoid lineages. However, approximately 2-5% of patients diagnosed with acute leukemia display an ambiguous lineage after immunophenotyping. A portion of these cases are classified under the category of mixed phenotype acute leukemia (MPAL) by the current WHO nomenclature.1

In a study of 117 MPAL patients by Yan et al, 55% of the cases had combined B/Myeloid, while 33% had T/Myeloid, and 12% had B/T/Myeloid. CD34 was strongly positive in 82% of cases, which reinforces the idea that the cell of origin is a multi-potent stem cell capable of differentiating into both myeloid and lymphoid progenitors. Cytogenetic analysis revealed no chromosomal abnormality in 36% of the patients with MPAL, while 64% had complex karyotypes (>3 aberrations). Translocation (9;22) was the most common abnormality, found in 15% of patients. Monosomy 7, a common finding in myelodysplastic syndromes as well, was found in 7.6% of patients. Mutational analysis revealed IKZF1 deletions in 13% of patients, ASXL1 in 6.5% of patients and a variety of other mutations including ETV6, NOTCH1 and TET2.2

In 2016, Eckstein and colleagues demonstrated epigenetic regulatory genes such as DNMT3A, IDH2, TET3 and EZH2 are the most commonly mutated in MPAL. RAS mutations including NRAS and KRAS and tumor suppressors, such as TP53 and WT1, were frequently identified as well.3

Interestingly enough, the genetic features of MPAL often overlap with early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). ETP-ALL is a high-risk subgroup, representing 10% of adult T-lineage acute lymphoblastic leukemia. It is defined by a characteristic immunophenotype (CD1a/CD8 negative with weak CD5) and distinct gene expression associated with early arrest in T-cell development. This subgroup, called the LYL1 group, expresses the early hematopoietic marker CD34 as well as myeloid antigens (CD13 or CD33), but lacks expression of both CD4 and CD8. These leukemias are associated with a poor prognosis, with a 10- year overall survival of 19% compared to 84% for all other T-ALLs.4

Zhang et al in 2012 performed whole genome sequencing on ETP-ALL cases and found a high frequency of mutations in factors mediating cytokine receptor, tyrosine kinase and RAS signaling. It also showed inactivating mutations in genes encoding transcription factors (GATA3, ETV6, RUNX1, IKZF1) as well as genes involved in histone modification, such as EZH2.5

Overall, the genetic features of both ETP-ALL and MPAL display an identical genomic pattern that involves multiple pathways, including tyrosine kinase signaling, cytokine receptor response, RAS pathway activation, and loss of function in tumor suppressors. These findings give credence to the hypothesis that the early T-cell precursor actually displays more of a pluripotent stem cell profile that is similar to myeloid neoplasms, thus confounding findings found during molecular profiling. With this paradigm in mind, molecular diagnostics cannot differentiate between ETP-ALL and in this case, MPAL.



  1. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed., International Agency for Research on Cancer, 2017.
  2. Yan et al. Clinical, immunophenotypic, cytogenetic, and molecular genetic features in 117 patients with mixed-phenotype acute leukemia defined by WHO-2008 classification. 2012 November;97(11):1708-12.
  3. Eckstein OS et al. Mixed Phenotype Acute Leukemia (MPAL) Exhibits Frequent Mutations in DNMT3A and Activated Signaling Genes. Exp Hematol. 2016 August; 44(8):740-744.
  4. Ferrando AA et al.  Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia. Cancer Cell. 2002. 1:75–87.
  5. Zhang J et al. The genetic basis of early T-cell precursor acute lymphoblastic leukemia. Nature. 2012 Jan 11;481(7380):157-63.


Marcus, Chelsea_099-Edit

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Microbiology Case Study: A 27 Year Old Male with Unintended Weight Loss

Case History

A 27 year old African American male presented to the emergency department with confusion and abdominal pain. His past medical history was significant for a 100 pound unintended weight loss and oral candidiasis which prompted a recent diagnosis of HIV. He was prescribed anti-retroviral therapy and antibiotic prophylaxis with which he reported compliance. Currently, he had no fever or chills. An abdominal CT scan showed an enlarged liver & spleen, generalized lymphadenopathy and a small amount of fluid. Significant lab work included anemia with a platelet count of 18,000 TH/cm2, absolute CD4 100 cells/cm2 (reference range: 506-3142 cells/ cm2) and a HIV viral load of 4,871 vc/mL. Given the concern for an infectious process, the infectious disease service was consulted and the patient underwent a thorough infectious work up including lumbar puncture, was started on board spectrum antibiotics and antifungals and was placed in airborne isolation until Mycobacterium tuberculosis could be ruled out.

Laboratory Identification

Image 1. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).
Image 2. Direct smear from a stool specimen showed beaded acid fast bacilli (Kinyoun stain, 1000x oil immersion).

Initial diagnostic testing for bacterial, fungal and viral pathogens was negative. Three concentrated sputum AFB smears as well as a TB PCR were negative. The quantiferon gold TB test was also negative. The physician additionally ordered AFB blood and stool cultures. The direct smear from the stool specimen showed rare, beaded acid fast bacilli in a background of bacteria and yeast normally present in the stool via Kinyoun stain (Images 1 & 2). The specimen was sent to the department of health for additional work up. There was growth after 21 days incubation and Mycobacterium avium complex was identified by high performance liquid chromatography (HPLC).


Mycobacterium avium complex (MAC) is a slow growing nontuberculous mycobacteria (NTM) frequently involved in human disease. Historically, it was classified as Runyon group III which are non-chromogens and do not produce pigment regardless of culture conditions. The group encompasses two taxa, M. avium and M. intracellulare. The species M. avium can further be classified into four subspecies: subsp. avium, subsp. silvaticum, subsp. paratuberculosis and subsp. hominissuis. Of interest, M. avium subsp. paratuberculosis can often be seen in association with Crohn’s disease.

In general, MAC organisms have low pathogenicity, but in the setting of those with lung disease (including cystic fibrosis), heavy smokers, immunocompromised patients and those with HIV, it is a well-known cause of disease. Infections with MAC can range from localized mycobacterial lymphadenitis and isolated pulmonary disease to bacteremia with dissemination to almost any organ. The organisms are located in circulating monocytes and further spread most commonly to the lungs, gastrointestinal tract and lymph nodes. In the case of HIV positive patients, MAC is the most common environmental NTM causing disease, especially in those with CD4 counts less than 100 cells/mm3 who are more likely to have disseminated disease.

In order to diagnosis MAC infections, specimens from blood, sputum, lymph nodes and other tissues are preferred. In addition, stool may also be an acceptable alternative in HIV patients if other specimens are negative or unable to be obtained. However, the sensitivity of a direct stool smear is only 32 to 34% making it not a very effective approach to identifying those at risk for disseminated infections. Once the culture has growth, various methods can be used to identify MAC, including phenotypic methods, DNA probe testing, HPLC, pyrosequencing and other forms of PCR & sequencing.

In the case of our patient, he was started on M. tuberculosis therapy: rifabutin, isoniazid, pyrazinamide & ethambutol (RIPE) until TB was ruled out. At that time, he was removed from isolation and switched to a drug regimen that included azithromycin, rifabutin and ethambutol. He showed clinical improvement and his cell counts, renal function and liver enzymes trended to normal ranges.



-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories.  Her interests include infectious disease histology, process and quality improvement and resident education.

Chemistry Case Study: Hypercalcemia in Sarcoidosis

Our patient is a 47-year-old female with a history of type II diabetes mellitus, hypertension, pancreatic insufficiency, systemic sarcoidosis with lung and liver involvement. She was admitted into the ED for severe hypercalcemia, hypokalemia and hypomagnesemia. Her total calcium concentration was at 15.1 mg/dL (ref range: 8.3-10.2 mg/dL, critical: >13.0) at admission and albumin was low. Further testing revealed 25-hydroxy vitamin D (25(OH)D) of 23.2 ng/mL, which is considered insufficient, and decreased PTH of < 15 pg/mL (ref range: 15 – 65). From these results, primary hyperparathyroidism was ruled out. PTH related peptide (PTHrp) was tested given her sarcoidosis history.

PTHrp is produced by some cancers, especially kidney, breast and lung cancers, and as well as lymphoma and leukemia. It has the same N-terminal and binds to the same receptor as PTH, therefore sharing some functions of PTH. In patients with hypercalcemia associated with malignancy, PTHrP may be evaluated. There are also case reported sarcoidosis-related hypercalcemia due to production of PTHrp. In the case, PTHrp was normal at 0.4 pmol/L (ref range: < 2.0).

Further tests showed that 1, 25-dihydroxyvitamin D (DHVD) was elevated at a concentration of 93.3 pg/mL (ref: 18.0 – 78.0). In the presence of decreased 25(OH)D, this result suggested that the 1-alpha-hydroxylase could be the cause of hypercalcemia. DHVD is the active form of vitamin D. It promotes intestinal calcium absorption and, in concert with PTH, skeletal calcium deposition. 25(OH)D converts to DHVD via 1-alpha-hydroxylase, which is almost exclusively expressed in the kidney, but can also be found in some extrarenal tissues, including inflammatory cells of the monocyte/macrophage lineage commonly seen in sarcoidosis and other granulomatous diseases. DHVD produced in extrarenal tissues is PTH-independent, and moreover, elevated calcium induced by extrarenal DHVD can inhibit PTH production via calcium-sensing receptor (CaSR) on parathyroid cells.

Sarcoidosis is a multisystem inflammatory disease of unknown etiology manifests as granulomas found predominantly in the lungs and lymph nodes. Hypercalcemia is seen in about 10-13% of patients. Overproduction of 1-alpha-hydroxylase and production of PTHrp can both contribute to the hypercalcemia in some patients with sarcoidosis. In this case, PTHrp was normal and elevated 1-alpha-hydroxylase was found to be the cause of hypercalcemia.

In addition to treatment of the underlying disorder, treatment of hypercalcemia in sarcoidosis is aimed at reducing intestinal calcium absorption and DHVD synthesis. Besides dietary interventions, glucocorticoids and bisphosphonates have also been used successfully to treat hypercalcemia in sarcoidosis:

  • Glucocorticoids: inhibit DHVD synthesis by the activated mononuclear cells (major contribution), inhibit intestinal calcium absorption and osteoclast activity
  • Bisphosphonates: inhibit the resorption of bone by osteoclasts



  1. https://emedicine.medscape.com/article/301914-overview
  2. https://www.uptodate.com/contents/hypercalcemia-in-granulomatous-diseases
  3. https://www.uptodate.com/contents/diagnostic-approach-to-hypercalcemia
  4. https://www.ncbi.nlm.nih.gov/books/NBK109831/



-Rongrong Huang, PhD is a first year clinical chemistry fellow at Houston Methodist Hospital. Her interests include general clinical chemistry, genetic biochemistry and applications of mass spectrometry in clinical laboratories.


-Xin Yi, PhD, DABCC, FACB, is a board-certified clinical chemist, currently serving as the Co-director of Clinical Chemistry at Houston Methodist Hospital in Houston, TX and an Assistant Professor of Clinical Pathology and Laboratory Medicine at Weill Cornell Medical College.

Microbiology Case Study: An Elderly Woman with a Cough Develops Skin Nodules

Case History

Our patient is an 83 year old female with previous history of arterial hypertension, atrial flutter and chronic obstructive pulmonary disease who presented with dry cough (~2 weeks), fever (102ºF), and cutaneous ulcerated plaques with elevated borders on forearm, foot, leg, and neck. Chest radiographs and chest CT scan showed numerous bilateral nodular consolidations compatible with pneumonia. Additionally, mild leukocytosis (14,200 cells /mm3) and hypohemoglobinemia (10.9 mg/dl) were documented. A skin biopsy was taken from the forearm lesion. Periodic acid–Schiff (PAS) and Grocott-Gomori’s (or Gömöri) methenamine silver (GMS) stains identified rare budding yeast (PAS, Image 1). Acid-fast bacilli (AFB) and Gram stains were negative for mycobacteria and bacteria, respectively.

Image 1. Budding yeast with a thick double cell wall were identified microscopically surrounded by prominent supurative granulomatous reaction (PAS, 100x).


A diagnosis of disseminated blastomycosis was made based broad based budding yeast seen on PAS stain (Image 1).

Blastomycosis infection most commonly affects persons living in the Mississippi and Ohio River valleys, Great Lakes Region of the United States, and southern Canadian provinces. It is a fungal infection that can cause asymptomatic infection, isolated pulmonary disease, or serious and potentially fatal disseminated disease. B. dermatitidis can infect every organ of the body giving great variety of clinical manifestations, which is the reason why it is known as “the great pretender.” More than half of infected patients are asymptomatic. Symptomatic patients generally present with pulmonary symptoms, and the development of disseminated disease after hematogenous spread is common (~25 to 40% of symptomatic cases). The most common extra-pulmonary locations are: skin, bone, genitourinary tract, and central nervous system (CNS). Unlike histoplasmosis, most cases of blastomycosis are seen in immunocompetent patients, although immunocompromised patients may be at higher risk to develop severe forms of the disease.

Blastomyces is a thermally dimorphic fungus that grows as a yeast in the body and as filamentous fungi with septate hyphae in the environment. Recent phylogenic analysis has divided the Blastomyces genus into 2 species, B dermatitidis and B gilchristi . Culture of B. dermatitidis from the environment is extremely difficult, and much of what we know is conjecture from a few documented outbreaks, of which several occurred in wooded areas near waterways. These investigations found that exposure to dust clouds associated with construction or crop harvesting were the only identified risk factors for infection. Blastomycosis infection occurs through aerosolization of conidia from the environment causing respiratory infection or less commonly through direct inoculation into cutaneous abrasions. Once in the host, the conidia transform into yeast. The specific proteins expressed during the yeast phase allow the evasion of phagocytic and CD4+ cells.

Laboratory diagnosis

The most expedient method to diagnose blastomycosis remains examination of stained fluid or tissue specimens. Yeast are 8-15 µm in size with broad based buds of 4-5 µm and have a characteristic refractory double cell wall. Fluid can be stained with 10% potassium hydroxide plus calcofluor white, whereas formalin fixed paraffin embedded tissue samples can be stained with GMS or PAS. B. dermatitidis yeast can be difficult to visualize with Gram or hematoxylin and eosin (H&E) stains, but if found, the characteristic broad-based budding pattern of yeast can lead to presumptive diagnosis before culture and non-culture based diagnostic test results are available.

Culture of B. dermatitidis provides a definitive diagnosis of pulmonary and extra-pulmonary disease. B. dermatitidis grows well on routine fungal media such as Sabouraud dextrose agar, potato dextrose agar, and brain–heart infusion media. The yeast phase is inhibited by media containing cyclohexamide. Culture typically demonstrates growth in as little as 4-7 days. Colonies will initially appear yeast-like, but then develop white cottony aerial mycelium and turn tan with age. Mature growth is achieved around day 14 and the reverse of the colony is a tan color. At 25-30°C, B. dermatitidis forms septate hyphae with round or pear-shaped conidia attached to the hyphae by short or long conidiophores. This gives the characteristic appearance of “lollipops.” Scedosporium spp. and Chrysosporium spp. are common confounders because they make similar structures. Definitive identification of Blastomyces sp. can be made by conversion of the mold phase to the yeast phase by incubation at 37°C. An alternative to conversion is using a DNA probe assay.



  1. Medically Important Fungi, 5th edition
  2. Principles and Practices of Infectious Disease, 7th edition

Julio picture

-Julio Diaz-Perez, MD is a 1st year anatomic and clinical pathology resident at University of Chicago (NorthShore).

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois.