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.


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.


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

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

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

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

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

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

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

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

By the Book

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

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

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

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

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

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

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

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

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

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

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

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

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

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.


  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

-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.


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).


  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.

Lab Safety Whiplash

The world seemed like a brighter place just a few short weeks ago. The pandemic seemed to be nearing an end, and life was returning to normal. In laboratories, the COVID-19 testing volumes decreased, wearing surgical masks all day long at work was no longer the norm, and the workday had that old feeling of familiarity again. Then, suddenly, it all came roaring back. The COVID-19 Delta Variant, loading its victims with over 1000 times more viral particles than the original could, came to visit. Now masking and social distancing are back with a vengeance, and everyone holds their collective breath as we wait to see what other cancellations and restrictions will come our way. It is almost worse this time because we know what the future will bring, and it isn’t pretty.

So how do we deal with it in the laboratory? How do we manage our lab safety program as our staff deals with this physical and mental whiplash? Many labs already saw the fatigue workers exhibited in the past 18 months. People stopped distancing from each other, they became less diligent about hand hygiene in the department, and PPE use became a bigger compliance issue than it had been when the pandemic began.

Fortunately, this is not a new challenge for lab safety professionals. Even without a pandemic, maintaining an awareness for the importance of lab safety has been a consistent need. Those who have been in the field for years and have never had a chemical exposure or a needle stick become complacent about the hazards where they work. Formaldehyde is treated like it was water, and contaminated blood tubes are handled with no gloves. This “disease” spreads also, when new employees observe these poor safety behaviors and emulate them. A poor safety culture does not have to become a pandemic, however, there is a cure, even in times such as these.

First, determine where your lab safety culture lies on the spectrum- is it very broken, or does it just need a little boost? Make an assessment of the overall culture using surveys or by talking to lab staff and leadership directly. Review your findings with the staff so that they are clear about why you are tackling the issues. That act alone raises awareness in the department. If possible, obtain a commitment from staff to improve the overall safety culture. Find safety champions who will work with you on the on-going project. Be sure safety is being discussed daily and is placed in front of the staff. Use huddles, e-mails and safety boards to promote a positive culture.

Unsafe behaviors in the laboratory can easily have consequences that may affect others in the department. Spills and exposures are just some incidents that may occur. Messy lab areas can create trips or falls, and improper storage of chemicals or hazardous wastes can be dangerous as well. Perhaps laboratory staff don’t think enough about the dangerous consequences because there isn’t enough training about them. Perhaps they don’t think about the potential consequences to others because they haven’t been told about the possible physical, environmental, or financial consequences. Maintaining awareness of these issues is always key.

The COVID-19 pandemic and its apparent rebound has made for some very long months for employees in healthcare, and the struggles do not appear to be ending anytime soon. As safety leaders, it is important for us to do what we can to help staff build resilience against the whiplash and to reinvigorate them to continue with good safety practices. We must remind them that despite all of the changes in safety guidelines in the recent past that the basics – PPE use, using engineering controls and work practice controls- are there to help us get safely through the day so that we can still go home healthy and to be able to enjoy our lives so that we can see the end of these unusual times.

Dan Scungio, MT(ASCP), SLS, CQA (ASQ) has over 25 years experience as a certified medical technologist. Today he is the Laboratory Safety Officer for Sentara Healthcare, a system of seven hospitals and over 20 laboratories and draw sites in the Tidewater area of Virginia. He is also known as Dan the Lab Safety Man, a lab safety consultant, educator, and trainer.

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.


  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.

A COVID Reflection

Usually, I talk about some of the more administrative happenings in the laboratory world (accreditation, competency, etc.). Today, however, as there is seemingly a glimmer of light at the end of the nation’s pandemic tunnel, I thought I would reflect on what we have collectively experienced.

 Like much of the nation, it has been a difficult journey for laboratorians. It has been particularly trying for those who were asked, who were required, to rise up and meet the unprecedented challenges of the times while suffering from the same burdens of fear, uncertainty, and physical ailments as those they were serving.

Dying Alone

One year ago, my uncle died from COVID-19. He died alone and afraid in the nursing home where he never wanted to be. We visited him after being given special permission from the president of the company operating the nursing home. After being told about how unusual it was to be allowed to see him, we dressed in full PPEs and went into his room. We found him curled in a fetal position, dead and cold to the touch. It was so unfair.

I think about all of the laboratorians who had to endure similar or worst experiences: those who lost close family members and even those who themselves suffered through the disease.

Unseen Warriors

Laboratorians have always been the silent warriors in the life-long battle to defeat pain and disease. More often, nurses and doctors received public gifts of admiration and praise for their service to patients. With quiet satisfaction, laboratory technologists, technicians, and support personnel are dedicated 24 hours a day, seven days a week, to providing the information on which 70% of medical decisions are based. Information that no other group of professionals can provide.

I think about all of the effort and skill required in the mad rush to set up tents and collection sites needed across the nation. And then, too, there were the laboratories needing to scale up testing or create entirely new testing areas with new instruments and new tests kits. The chaos was magnified by constantly changing guidelines, reagent shortages, and a lack of trained personnel.

Amid all the confusion, misinformation, and anger, laboratorians were themselves experiencing disease, death, and social isolation. Yet still, they delivered the results the nation needed to understand the pandemic’s depth and breadth.

Needless Death

Now the Delta variant has taken hold just when the nation thought the disease, if not bested, had at least been brought under some semblance of control. Unfortunately, the refusal of many to get vaccinated contributes to the virus’s persistence. More will suffer, and more will die.

How many needless deaths will the nation have to experience? Will there ever be a point when everyone who can be vaccinated will be? Or, two years later, will we be mourning preventable COVID-19 deaths. Will we still have to watch our loved ones perish with a tube down their throat, or worst, alone in a room far away surrounded by cold walls and quiet indifference?


Regardless of where this pandemic leads or how the nation reacts, laboratorians will continue to remain steadfast in their dedication to their profession and their patients. We have often considered ourselves the stepchild of the healthcare industry because, despite the criticality of what we do, we go unnoticed and unremarked on as long as we deliver the results our patients need. We are okay with that.

We are also tired and worn.


Thanks, fellow laboratorian, for reading this minor soliloquy of frustration and sadness. I will probably be back next quarter discussing inspections, competency, or some other administrative aspect of laboratory operations. I hope, also, to discuss how the nation has reached or is close to reaching the theoretical goal of herd immunity because of high vaccination levels. However, if I were honest, I know the likelihood of this happening is disappointingly low.

If you can get vaccinated, please do.

-Darryl Elzie, PsyD, MHA, MT(ASCP), CQA(ASQ), has been an ASCP Medical Technologist for over 30 years and has been performing CAP inspections for 15+ years. Dr. Elzie provides laboratory quality oversight for four hospitals, one ambulatory care center, and supports laboratory quality initiatives throughout the Sentara Healthcare system.

Pitfalls of Artificial Intelligence for COVID-19 Variant Classification

While you have surely heard about all of the SARS-CoV-2 variants and how concerning they are, I would bet that you may not know how they are classified. Sure, from my last post, the technical aspects of whole genome sequencing and targeted approaches have been described, but bioinformatic (big data) analyses are essential to assign lineages. Furthermore, the advances of machine learning have been integrated into this system for SARS-CoV-2 lineage assignment.

How VOC lineages are given

First, phylogenetic trees (circular example below) are formed to demonstrate relatedness of strains based on how many mutations they share. The more similar they are, the closer they are together. These trees are not new nor do they rely on artificial intelligence, but they can give visual clues as to whether a lineage is new.  For instance, when the first variant of concern B.1.1.7 (now called Alpha) was discovered, it branched away from other limbs of the phylogenetic tree.

Within these new viral variants, there are a set of mutations that are present in most of the viral variants. For instance, there are 17 protein coding changes in Alpha variant. However, these exact 17 mutations may not be in every Alpha variant. Individually, mutations may be present in 98% of isolates or lower; the spike gene deletion of amino acids 242-244 of the Beta variant (B.1.351, South Africa origin) is only present in 88% of specimens sequenced. This could be due to issues in sequencing, data processing, or just the prevalance/biology of the virus.

As there are many mutations that fit into certain variants, it would be difficult for a human to process all of this information in a probabilistic manner to assign lineages. Thus, machine learning tools (most common SARS-CoV-2 program is Pangolin) have been added onto the end of bioinformatic analyses to assign the lineage to a sample.

How machine learning works

The subject of machine learning has been discussed in a previous post about Protein folding prediction. Briefly, it is helpful to remember that machine learning is a process to create algorithms that give an outcome based on training data. The more diverse, large, and well curated the data, the better the accuracy of the program. One pitfall is they are based on previous data, which works well for many situations: using AI to find a lung cancer on chest x-ray would work well, because lung cancers have consistent characteristics.

However, with COVID-19, new variants keep arising and current variants are evolving (think Delta and Delta “plus”). Furthermore, if the classifier Pangolin is trained on high quality data, then trying to interpret lower quality data (missing genome regions, few sequencing reads) may confuse Pangolin and lead to inaccurate results. What follows is an example of how this occurred at our institution.

Case study

We have been sequencing COVID-19 positive specimens at UT Southwestern for the last several months. Many of the cases have been the Alpha variant (B.1.1.7, origin U.K.). However, it was around this time that Delta (B.1.617.2, origin India) cases started to arise. In one week, we found two specimens that were classified as B.1.95. This was an unusual variant I had not heard of before. There are several “wild type” strains that are B.1.1/ B.1.2 and other derivations, but I had not seen anything like this before.

Clinical history

Two specimens sequenced belonging to Hispanic, adolescent brothers whose mother had recently been hospitalized with COVID-19. There was information on mother’s travel history.

Therefore, I performed manual review of the specific variants. Many of the diverse mutations occur in the spike protein, so this was analyzed first. Immediately, I noticed two classic mutations of the Delta variant: a 2 amino acid deletion in the spike gene (S:Del157_158) and a receptor binding site mutation (S:L452R) also seen in the variant from California (B.1.429). Other mutations could be evaluated, but the combination of these two mutations is unique to Delta variant.

One suspected cause was that the Pangolin lineage classifier had an issue. Specifically, it had not been updated since February 2021- when Delta did not exist. Thus, there was no data for the program to classify the variant properly. Upgrading to the latest version of Pangolin provided the correct lineage classification.

A Few weeks later…

Once again, I was checking the lineages reported by the classifier and there were several B.1.617.2 and B.1.617.1. Both of these are variants from India (before the helpful WHO Delta designation), but they are distinct sub-variants. It was odd to see B.1.617.1, because this was found to be less infectious compared to the dominant B.1.617.2 variant (later named Delta) and B.1.617.1 was not spreading across the globe.


Therefore, I once again went to the sequence data for the spike protein to compare some mutations. Although these are sub-variants from the same original variant, they have several mutually exclusive mutations in the spike protein. The figure below compares the prevalence of specific mutations in the spike protein of B.1.617.1 and B.1.617.2 (dark purple = common in a variant, white = rare).

Upon manual review, all of the spike gene mutations were specific to B.1.617.2. So why was there an issue in classification? Again, there were few sequences for either of these sub-variants at that time, so the classifier wasn’t as well trained. Updating the Pangolin version brought the benefit of new data and more accurate classifications.

Take away messages

  1. Updating Lineage classification software (Pangolin) on a regular basis is needed for accurate results.
  2. Manual review is essential for any abnormal findings- a typical process for pathologists, but also plays an important role in COVID-19 variant monitoring.
  3. Know what you’re looking for and know which mutations differentiate the variants.
  4. Delta is now the dominant strain in the U.S. (graphic below).


  1. Outbreak.info
  2. https://pangolin.cog-uk.io/

Jeff SoRelle, MD is Assistant Instructor of Pathology at the University of Texas Southwestern Medical Center in Dallas, TX working in the Next Generation Sequencing lab. His clinical research interests include understanding how lab medicine impacts transgender healthcare and improving genetic variant interpretation. Follow him on Twitter @Jeff_SoRelle.

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).


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


  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.

Microbiology Case Study: Salads, Stool, and Special Staining Studies

Case History

A woman in her 40s presented to her primary care physician in summer 2020 with mild abdominal pain, diarrhea, nausea, and headache. She experienced loose bowel movements 3 – 4 times per day for the past 18 days. She denied bloody stools, travel, consumption of raw or undercooked meats or unpasteurized dairy, contact with animals, or recent antibiotic use. SARS-CoV-2 PCR was negative. A stool sample was collected and sent for an enteric panel PCR (Salmonella, Shigella, Campylobacter, and Shiga toxin), a bacterial stool culture (Aeromonas, Plesiomonas, and Vibrio), and an ova and parasite (O&P) exam with a request to perform a modified acid-fast stain. While the enteric panel and bacterial stool culture were negative, the following organism was observed on the modified acid-fast stain (Image 1). This organism measured approximately 9 µm, was variably modified acid-fast, and had a wrinkled-cellophane appearance. The organism was identified as a Cyclospora cayetanensis oocyst. The patient later shared that she had consumed a bagged salad mix that was implicated in the ongoing Cyclospora outbreak.

Image 1. Cyclospora cayetanensis.

Cyclospora cayetanensis

Cyclospora cayetanensis, a coccidian protozoan, is transmitted through ingestion of food or water contaminated with infectious oocysts. While infected humans shed oocysts in their stool, these oocysts are unsporulated and non-infectious at the time of excretion. In order to sporulate and become infectious, these excreted oocysts must incubate in the environment for 7 – 15 days post-excretion. Due to the required incubation post-excretion, direct fecal-oral transmission cannot occur.

Endemic areas include Central and South America, Middle East, South East Asia, and the Indian subcontinent. In non-endemic areas, travelers make up a large proportion of cases. Local outbreaks in non-endemic areas are often due to contaminated food sources. Most commonly the source of these outbreaks arise from consumption of raw fruits and vegetables that are difficult to thoroughly clean. These include leafy green vegetables (salad mixes, lettuce), herbs (basil, cilantro), and raspberries. Moreover, Cyclospora is resistant to many disinfectants used in the food industry. As exposure to this parasite is through contaminated food and water, infected patients are also at risk for other food and waterborne parasites including Cryptosporidium.

Once infectious oocysts are ingested, symptoms are typically observed after a one week incubation. Clinical presentation of Cyclospora infection includes diarrhea, nausea, fatigue, low grade fever, and weight loss. Although Cyclospora causes infections in both immunocompromised and immunocompetent individuals, symptoms may be severe and prolonged in immunocompromised patients, particularly in HIV and AIDS patients. Children and elderly individuals are also at higher risk for severe disease. Trimethoprim-sulfamethoxazole is the standard treatment. If untreated, symptoms can last for 10 – 12 weeks and may exhibit a relapsing pattern.

Stool samples should be submitted to the clinical microbiology laboratory for microscopic and/or molecular studies. To increase recovery of the organism during intermittent or low burden shedding, multiple stool specimens should be submitted over 2 -3 days. When viewed under a UV fluorescent microscope, Cyclospora oocysts autofluoresce and appear blue or green. While safranin-based stains or UV fluorescent microscopic examination can be used, modified acid-fast staining is commonly performed for the microscopic identification of Cyclospora. Cyclospora oocysts are modified acid-fast variable and measure 8 – 10 µm in diameter, unlike Cryptosporidium oocysts which are modified acid-fast positive and measure 4 – 6 µm in diameter. It is important to alert the clinical microbiology lab if suspecting Cyclospora as stains used in routine O&P exam, including trichrome stains, are not effective in highlighting Cyclospora. Although lab developed tests and FDA cleared multiplex gastrointestinal pathogen panels including Cyclospora are available, molecular assays are not yet routinely used for identification of Cyclospora due limited widespread availability.


  1. Almeria S, Cinar HN, Dubey JP. Cyclospora cayetanensis and Cyclosporiasis: An Update. Microorganisms. 2019;7(9):317. Published 2019 Sep 4. doi:10.3390/microorganisms7090317
  2. Hadjilouka A, Tsaltas D. Cyclospora Cayetanensis-Major Outbreaks from Ready to Eat Fresh Fruits and Vegetables. Foods. 2020;9(11):1703. Published 2020 Nov 20. doi:10.3390/foods9111703
  3. Ortega YR, Sanchez R. Update on Cyclospora cayetanensis, a food-borne and waterborne parasite. Clin Microbiol Rev. 2010;23(1):218-234. doi:10.1128/CMR.00026-09
  4. Garcia LS. Diagnostic Medical Parasitology. 6th Edition. 2016.
  5. Casillas SM, Bennett C, Straily A. Notes from the Field: Multiple Cyclosporiasis Outbreaks — United States, 2018. MMWR Morb Mortal Wkly Rep 2018;67:1101–1102. DOI: http://dx.doi.org/10.15585/mmwr.mm6739a6

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.