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

Case History

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

Microbiology

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

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

Discussion

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

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

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

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

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

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

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

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

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

References

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

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

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

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

Case History

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

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

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

Discussion

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

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

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

References

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

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

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

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

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

Case History

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

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

Discussion

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

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

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

References

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

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

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

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

Case History

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

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

Microbiology

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

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

Discussion

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

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

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

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

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

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

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

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

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

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

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

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

Discussion

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

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

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

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

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

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

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

References

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

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

Microbiology Case Study: An Adult Male with HIV Presents with Shortness of Breath

Case History

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

Pneumocystis jirovecii

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

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

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

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

Sources:

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

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

Microbiology Case Study: 70 Year Old Man with a History of Papillary Urothelial Carcinoma

Case Description

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

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

Discussion

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

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

References

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

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

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

-Clare McCormick-Baw, MD, PhD is an Assistant Professor of Clinical Microbiology at UT Southwestern in Dallas, Texas. She has a passion for teaching about laboratory medicine in general and the best uses of the microbiology lab in particular.

Microbiology Case Study: An 80 Year Old Man with a Violaceous Leg Lesion

An 80 year old male with a PMH of myelodysplasia with progression to AML and neutropenic fever presented with severe leg pain and violaceous leg lesion spanning a large portion of the left medial thigh and knee. There was no reported trauma. A radiograph demonstrated significant gas/air tracking in the tissues of the left medial thigh. He was given vanc/zosyn/clinda and subsequently transferred to UVMMC for further evaluation. He underwent debridement in the OR and fluid was sent for culture. Primary Gram stain showed no neutrophils and few gram positive bacilli. The blood agar plate and leg lesion are shown below.

Image 1. Growth on blood ager plate (BAP).
Image 2. Patient’s leg lesion.

This organism was identified on MALDI-MS as Clostridium septicum, consistent with the spreading colonies with irregular margins on blood agar. Box-car GPB morphology was present on gram stain, and although subterminal spores can be an additional clue to the identify C. septicum, they were not seen in this particular case.

Important diagnostic features of Clostridium septicum in the microbiology lab include:

  • GPB with rare subterminal spores (Image 3)
  • Gray/white colonies with irregular margins, translucent, β-hemolytic
  • Can show swarming growth pattern in < 24 hours on Schaedler plate (Image 4)
    • This swarming phenomenon is due to the ability to differentiate into giant hyperflagellated swarm cells, which can participate in migrations across surfaces
    • Major changes in virulence characteristics during swarming are reported
  • Lecithinase negative
  • Lipase negative
  • Gelatinase positive
  • Maltose positive
  • Indole negative
  • Urease negative
Image 3. Subterminal spores seen on Gram stain.
Image 4a. Swarming.
Image 4b. Swarming.

C. septicum is classically associated with GI malignancies and perforations when isolated in blood cultures. While less common than C. perfringens, Clostridium septicum can also cause gas gangrene (as in this case) along with C. novyi, C. histolyticum, C. sporogenes, and C. bifermentans. Importantly, in cases of nontraumatic gas gangrene, C. perfringens is the most common cause and classically presents as a primary perineum or extremity infection following bacterial translocation from the gastrointestinal tract. Patients with immune suppression, specifically neutropenia, are at increased risk of spontaneous gas gangrene due to C. septicum and have a high mortality rate.

To remember the colony morphology of the different Clostridial spp, you can think:

  • While C. Septicum Swarms, C. Perfringens Perforates (aka makes a double zone in the agar).

Edited to add: C. septicum is the most common cause of nontraumatic gas gangrene.

References

  1. McPherson, R, and M Pincus. (2011). Henry’s Clinical Diagnosis and Management By Laboratory Methods (22nd Edition, pp. 1155-1184). Philadelphia, PA: Elsevier Saunders.
  2. Murray PR et al: Medical Microbiology. 8th ed. Philadelphia: Elsevier, 2016
  3. Macha, Kosmas & Giede-Jeppe, Antje & Lücking, Hannes & Coras, Roland & Huttner, Hagen & Held, Jürgen. (2016). Ischaemic stroke and Clostridium septicum sepsis and meningitis in a patient with occult colon carcinoma – a case report and review of the literature. BMC Neurology. 16. 10.1186/s12883-016-0755-4.
  4. Stevens DL, Aldape MJ, Bryant AE. Life-threatening clostridial infections. Anaerobe. 2012;18(2):254–259.
  5. Macfarlane S, Hopkins MJ, Macfarlane GT. Toxin synthesis and mucin breakdown are related to swarming phenomenon in Clostridium septicum. Infect Immun. 2001;69(2):1120-1126. doi:10.1128/IAI.69.2.1120-1126.2001

-Nicole Mendelson, MD is a 3rd year Anatomic and Clinical Pathology Resident at the University of Vermont Medical Center.

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

Microbiology Case Study: A 59 Year Old Male with Acute Prostatitis

Clinical history

A 59 year old male with a past medical history of benign prostatic hypertrophy who presented to his primary care physician with complaints of dysuria, urgency, nocturia, and a weak stream. He was referred to a urologist who diagnosed him with acute prostatitis. The patient was given Flomax, a medication that relaxes the muscles in the prostate and bladder, and sulfamethoxazole-trimethoprim, an antibiotic for his possible bladder infection. The patient continued to have urinary symptoms despite antibiotics and multiple urinary cultures that were negative for growth of bacteria. Eventually the patient’s symptoms worsened and he presented to the emergency department with symptoms of fever (102.8ºF), bladder spasms, drenching night sweats, and painful urinary retention. His lab results showed a mild leukocytosis (15,500 cells /mm3) and increased leukocyte esterase, leukocytes, and erythrocytes in his urine. He was admitted and a pelvic computed tomography scan showed multiple prostatic abscesses. The infectious disease team ordered a urine fungal culture. The fungal stain showed broad-budding yeast forms (Image 1).

Image 1. Calcofluor white fungal stain highlighting brad-based budding yeast (100x oil immersion).

Laboratory identification

A cytospin Gram stain was made of the urine specimen which showed rare large yeast forms, numerous red blood cells and neutrophils (Image 2). These yeast forms are consistent with Blastomyces dermatitidis which are 10-15 µm in diameter and have thick contoured cell walls. The fungal culture grew a dirty white leathery mold on inhibitory mold agar after ten days. The lactophenol cotton blue adhesive tape preparation highlighted short to long conidiophores with large pear-shaped conidia at the tips of delicate conidiophores and septated hyphae. A positive urine antigen test supports the identification of the organism as Blastomyces dermatitidis.

Image 2. Large yeast with thick refractile walls seen on Gram stain. Flattened areas suggest recent separation.

Discussion

Blastomyces dermatitidis is a dimorphic fungus commonly found in the eastern half of the United States and Canada, specifically the Mississippi and Ohio River valleys, and Great Lakes Region. It typically grows as a mold in damp soil and decomposing vegetation; outdoor activities that disrupt the soil can increase the risk of infection. Infection occurs when the reproductive spores known as conidia are inhaled into the alveoli of the lungs. There the conidia transform into yeast and multiply causing the disease blastomycosis. Approximately 50% of those infected with Blastomyces are asymptomatic and clear the infection without trouble. For the other 50%, symptoms depend on the course of infection with a range of flu-like illness over a few days to chronic or severe illness that can last for months. Symptoms include fever, chills, headache, joint and muscle pains, shortness of breath, cough, night sweats, weight loss, and pleuritic chest pain. Twenty to forty percent of symptomatic patients will have disseminated disease. The common extra-pulmonary sites include skin, bone, urogenital, and the central nervous system, however, it has been reported in all organ systems.

In our case, the patient presented with prostatic blastomycosis which was quickly identified by calcofluor white fungal stain and urine antigen test. The patient was placed on itraconazole and a transurethral prostatic resection was performed. Histopathologic analysis showed necrotizing granulomas with fungal elements that are consistent with Blastomyces dermatitidis (Image 3). After a few days of treatment, the patient was discharged and is being followed on an outpatient basis.

Image 3. PAS stained tissue showing three budding yeast forms in a granuloma with extensive neutrophilic reaction.

– Joshua Wodskow, DO is a 1st year clinical and anatomic pathology resident at University of Chicago (NorthShore). Academically, Joshua has a particular interest in hematopathology and informatics. In his spare time, Joshua enjoys board games with his family and listening to podcasts.

-Erin McElvania, PhD, D(ABMM), is the Director of Clinical Microbiology NorthShore University Health System in Evanston, Illinois. Follow Dr. McElvania on twitter @E-McElvania. 

Microbiology Case Study: A Fishy Situation

Case History

The microbiology laboratory received a tissue specimen from a male patient in his 60s. The tissue was biopsied from the patient’s hand during surgery at an outside hospital, but no further clinical information was available. No organisms were observed on the Gram stain, fungal smear, or fluorochrome stain and no white blood cells were observed on the Gram stain. No organisms were recovered from the aerobic or anaerobic bacterial culture or fungal culture.

After 20 days of acid-fast bacilli (AFB) culture, yellow colonies grew on the Lowenstein Jensen (LJ) media and the 7H10 media (Image 1) and acid-fast bacilli were noted on the Kinyoun stain prepared from the colonies (Image 2).

Image 1. Yellow colonies on the 7H10 media.
Image 2. Kinyoun stain of the colonies demonstrating acid-fast bacilli. This is characteristic of Mycobacteria spp. Species-level identification is not possible based on morphology alone.

The ordering physician was contacted regarding the positive Kinyoun stain. Upon hearing that acid-fast bacilli grew in culture, the physician shared that he has a high suspicion for Mycobacterium marinum as this patient developed a wound infection after he cut his hand with a fish bone while cooking. MALDI-TOF identified this organism as Mycobacterium marinum, confirming the astute physician’s hypothesis. 

Mycobacterium marinum

Mycobacterium marinum is a slow growing non-tuberculous mycobacterium (NTM) that causes skin and soft tissue infections. M. marinum can be found in both fresh and saltwater environments and is commonly associated with contact with fish, fish tanks, marine shells, and non-chlorinated swimming pools, earning the names “swimming pool granuloma” and “fish tank granuloma.” Infections are normally localized to the skin and occur following trauma, with the limbs and extremities most often infected. Nodules or ulcers around the site of inoculation are most common—these are typically single nodules or ulcers but may spread along the lymphatic vessels to regional lymph nodes, similar to sporotrichosis. Rare manifestations of M. marinum include osteomyelitis and disseminated disease, with non-cutaneous manifestations occurring predominately in immunocompromised populations. M. marinum is a slowly progressing disease and the patient may not present to their provider until months after the initial trauma, often complicating the link to water or fish exposures. M. marinum, while it can be transmitted from fish to human, is not transmitted person-to-person and thus does not pose an infection control risk.

AFB cultures are typically incubated at 37 degrees C. However, M. marinum grows optimally at 30C with scant or no growth at 37degrees C. Thus, when skin and soft tissue samples are received for AFB culture, specimens should be held at both 37 degrees C and 30 degrees C to support growth of most NTM and to facilitate the growth of M. marinum, respectively. Recovery of M. marinum in culture from primary specimen takes several weeks to grow, but subculture may take less than 7 days.

M. marinum can be identified by phenotypic characterization, molecular identification, or mass spectrometry. M. marinum is a photochromogen, meaning that colonies possess a yellow pigment when grown in light and no pigment when grown in the dark. This is characteristic of group I of Runyon’s classification. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), which has been used for years in most clinical microbiology laboratories for the identification of bacteria and yeast, can be used to identify Mycobacteria to the complex or species level. While the processing of Mycobacteria for MALDI-TOF is more laborious than for bacterial identification, the use of MALDI-TOF for identification of Mycobacteria is increasing in use. Whole genome sequencing can be performed on pure isolates for molecular identification, although this is more expensive and less common than MALDI-TOF or phenotypic identification.

A thorough history identifying risk factors for M. marinum should be conducted by the physician. Given the length of time for growth, clinical suspicion due to water or fish exposures can guide treatment before the organism grows in culture.  In addition to providing a diagnosis and guiding clinical care, a history of M. marinum infection is also critical to note for latent tuberculosis screening as M. marinum can cross react with tuberculin skin testing and interferon gamma release assays.

References

  1. Aubry A, Mougari F, Reibel F, Cambau E. Mycobacterium marinum. Microbiol Spectr. 2017 Apr;5(2). doi: 10.1128/microbiolspec.TNMI7-0038-2016. PMID: 28387180.
  2. Wu TS, Chiu CH, Yang CH, Leu HS, Huang CT, Chen YC, Wu TL, Chang PY, Su LH, Kuo AJ, Chia JH, Lu CC, Lai HC. Fish tank granuloma caused by Mycobacterium marinum. PLoS One. 2012;7(7):e41296. doi: 10.1371/journal.pone.0041296. Epub 2012 Jul 20. PMID: 22911774; PMCID: PMC3401166.
  3. Franco-Paredes C, Marcos LA, Henao-Martínez AF, Rodríguez-Morales AJ, Villamil-Gómez WE, Gotuzzo E, Bonifaz A. Cutaneous Mycobacterial Infections. Clin Microbiol Rev. 2018 Nov 14;32(1):e00069-18. doi: 10.1128/CMR.00069-18. PMID: 30429139; PMCID: PMC6302357.

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.