Microbiology Case Study–Back and Flank Pain in a Transplant Patient

Clinical Summary:

59 year old male with a history of acute myeloid leukemia, status post allogeneic bone marrow transplant complicated by graft-versus-host disease and relapse presented to the ED complaining of flank and back pain. His work-up identified multiple pulmonary lesions, thought to be infarcts and a left upper extremity deep vein thrombosis. He was started on cefepime for neutropenic fever and lovenox for the emboli. His symptoms worsened and imaging revealed progressive lesions in the lung that were concerning for invasive aspergillosis. A biopsy of one of the lesions was non-diagnostic but an aspergillus serum antigen test was positive. He was started on voriconazole. He remained hospitalized and began showing improvement, but then again developed a febrile neutropenia and became hypotensive with a decreased hemoglobin level. A CT of the abdomen showed typhlitis in the cecum and possible liver phlegmons, and a CT of the chest showed bilateral pulmonary effusions. He went into respiratory failure and passed away. An autopsy was performed and they sent lung tissue for fungal cultures.

Microbiology:

Plates:

Potato flake agar shows a brown, rapid growing mold that was raising the lid of the plate. No growth on the Mycosel plate.
Potato flake agar shows a brown, rapid growing mold that was raising the lid of the plate. No growth on the Mycosel plate.
Scotch-tape prep shows ribbon-like hyphae with few septations.
Scotch-tape prep shows ribbon-like hyphae with few septations.

Temperature Studies:

The mold grew at both 37° and 42°C.

Discussion:

The above findings lead to the classification of a Zygomycete which are hyaline, pauciseptate molds that include Rhizopus, Mucor, Absidia, Rhizomucor, Synecephalastrum, Cunninghamella, and others.

The colonies are fluffy, white to gray or brown. They are rapid-growers and diffusely cover the agar within 24-96 hours. The hyphae appear to be coarse and fill the entire culture dish with loose, grayish hyphae dotted with brown or black sporangia. It is not possible to differentiate the organisms based on colony morphology. Temperature studies can differentiate between some species: Rhizopus grows best between 40-50°C; Rhizomucor grows best around 38-58°C; Mucor grows best at less than 37°C; Absidia grows between 45-50°. Since our specimen grew at both 37° and 42°C, that would lead us to have Rhizomucor high on our differential.

Zygomycetes produce large, ribbon-like hyphae that are irregular in diameter and contain occasional septae. Classification of specific organisms relies on identifying the characteristic saclike fruiting structures called sporangia. The sporangia produce sporangiospores, which are within the sporangia and are spherical and yellow or brown. Each sporangium is formed at the tip of a sporangiophore which is a supporting structure. The sporangiophores are connected by hyphae with occasional septations called stolons. These are contact points where rootlike structures called rhizoids attach to the hyphae. The presence and location of the rhizoids helps to identify the organism. Rhizopus has unbranched sporangiophores with rhizoids at their base where the stolon arises. Mucor has singularly produced or branched sporangiophores that have round sporangium filled with sporangiospores at their tips. It does not have rhizoids or stolons. Absidia has rhizoids that are between sporangiophores, and the sporangia are pyriform and have a funnel-shaped area called apophysis at the junction of the sporangium and the sporangiophore. Usually a septum is formed in the sporangiophore just below the sporangium. Our microscopic exam did not show any rhizoids at first, but a second exam after a longer growth period showed potential rhizoids at the base of the sporangiophores, which would lead us to have Rhizopus on our differential. Our case did not clearly define itself at the species level, so it was signed out as a Zygomycete and there were no treatment implications.

Zygomycetes are not a common cause of infection, but are an important cause of morbidity and mortality in patients who are immunocompromised. They have a worldwide distribution and are commonly found on decaying vegetable matter, soil, or old bread. Infection occurs by inhalation of spores, and once established, it is rapidly progressive, particularly in patients with diabetes mellitus who have infections that involve the sinuses. The organisms have a propensity for vascular invasion and rapidly produce thrombosis and necrosis of tissue. A common presentation is invasion within the nasal mucosa, palate, sinuses, orbit, face, and brain showing massive necrosis with vascular invasion and infarction. Perineural invasion can also occur which can spread retro-orbitally into the brain. They can also infect the lungs and GI tract as well as have disseminated infection. They can cause skin infections in patients who have severe burns and infections of subcutaneous tissue of patients who have undergone surgery.

Follow up:

Lung tissue, area of necrosis; H&E stain, 10x
Lung tissue, area of necrosis; H&E stain, 10x
Lung tissue, vasculature; H&E stain, 40x
Lung tissue, vasculature; H&E stain, 40x
Lung tissue, vasculature; silver stain, 10x
Lung tissue, vasculature; silver stain, 10x
Lung tissue vasculature; silver stain, 40x
Lung tissue vasculature; silver stain, 40x
Lung tissue, areas of necrosis; silver stain, 10x
Lung tissue, areas of necrosis; silver stain, 10x

The histology on H&E stain shows areas of necrosis with faint septate hyphae as well as broad, ribbon-like hyphae within the vasculature. The silver stain nicely highlights the broad hyphae which we can identify as a zygomycete. The silver stain also accentuated the massive amounts of thinner hyphae with parallel walls and 45 degree branching which is consistent with aspergillus. This patient was found to have both an aspergillus infection which caused the positive serum antigen test, but then also developed a zygomycete infection which led to his death. We did not identify aspergillus on our fungal culture which may be explained by several possibilities. Our patient had been treated with voriconazole for a potential aspergillus infection which may make it more difficult for the aspergillus to grow on fungal culture. Zygomycetes are rapid growers which could have inhibited the growth of another organism or could have inhibited our ability to identify a second organism growing on the plate.

 

Kirsten Threlkeld, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Microbiology Case Study

Patient History:

81 year old man with a history of systemic vasculitis (present for the past 10 years ANCA negative, ANA negative, Rheumatoid factor <20) on immunosuppression (plaquenil with prednisone 40mg for flares about every 6 months), type 2 diabetes, and hypertension presented to an outside hospital with weakness and dyspnea. He was found to have a widespread purpura, ulcerative lesions, acute kidney injury (creatinine 4.7), and 3 days of hematochezia. He was started on 7 days of levoquin and zosyn for a presumed pneumonia and with no improvement was transferred to our institution. On admission, a CT scan of the chest demonstrated bilateral multifocal pneumonia and multiple cavitary nodules within the lungs. A thoracentesis was performed and was transudative (wbc 1883, N 63%, protein 2.6).

Laboratory findings:

  • WBC 7000/cmm
  • Hemoglobin 9 g/dL
  • Platelet count 104 K/cmm
  • Bacterial culture blood, no growth
  • Cryptococcal antigen negative
  • Pleural fluid bacterial culture and smear negative
  • Pleural fluid AFB culture and smear – no acid fast bacilli, modified acid fast bacilli seen from bottle
  • Pleural fluid fungal culture and smear – no fungi seen, rare modified acid fast bacilli growing
  • Histoplasma urinary antigen positive
  • Histoplasma antibodies negative
  • Blastomyces urinary antigen negative
Gram stain of growth from the AFB bottle showing beaded, branch Gram positive bacilli.
Gram stain of growth from the AFB bottle showing beaded, branch Gram positive bacilli.
Modified acid fast stain of growth from the AFB bottle showing modified acid fast bacilli.
Modified acid fast stain of growth from the AFB bottle showing modified acid fast bacilli.
Isolated growth on BCYE media.
Isolated growth on BCYE media.

Discussion:

Based on Gram stain and modified acid fast stain, modified acid fast bacilli suggestive of Nocardia species was reported. Nocardia are strict aerobic, gram positive, filamentous rods that stain partially acid fast. This is due to the mycolic acids in the cell wall which are shorter than those of mycobacteria. Nocardia species produce many virulence factors including Cord factor (prevents intracellular killing), catalase and superoxide dismutase (which inactivate reactive oxygen species that would otherwise prove toxic to the bacteria).

Nocardia grow well on buffered charcoal yeast extract agar and at 30oC. They produce aerial hyphae and can have a chalky colony appearance. Species level identification is best done with molecular methods. This isolate was identified as Nocardia farcinica at a reference laboratory.

Nocardia species are ubiquitous in the soil. They can cause infections in immunocompromised hosts usually after inhalation or direct inoculation. Infections include bronchopulmonary disease and cutaneous infections. With bronchopulmonary disease, cavitation and spread to the pleura is common, which fits with our patient. Dissemination is also seen with common sites being brain and subcutaneous tissue.

Our patient had a positive Histoplasma urinary antigen, but negative Histoplasma antibodies. The working diagnosis was disseminated Histoplasmosis and he was being treated with amphotericin B. He expired and no postmortem exam was performed. Fungal cultures from the pleural fluid were not growing fungus at the time of this post. Fungal cultures were not obtained from sputum and a BAL was not performed.

-Dan Olsen, MD is a 4th year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

There’s a Fungus Among Us

A 53 year old man with history of stroke, alcoholism, heart failure, hypertension, and atrophic right kidney presented to the ED with acute urinary retention and complained of dysuria and frequency. He was afebrile, denied nausea/vomiting or headaches. His labs at admission are listed below:

  • WBC: 21 k
  • Na: 122
  • Cr: 3 (baseline 1.2)

Urinalysis showed innumerable white blood cells, leukocyte esterase 3+ and negative nitrite.

A catheter was placed and drained 1 L of yellow cloudy urine. The patient refused admission and he was prescribed ciprofloxacin 500 mg BID empirically and was sent home with a foley catheter in place with plans to follow up with Urology. He returned to the ED the following day because his foley catheter was not draining urine and he noted leaking around his catheter. CT scan was obtained and showed ill-defined areas of increased and decreased attenuation within the urinary bladder lumen and left hydroureteronephrosis.

fungusball1

Urine cultures obtained during his initial presentation grew >100,000 yeast and he was treated with fluconazole. The patient was taken to the operating room 11 days after first presentation to diagnose and treat the mass in the bladder. A tan-brown mass was removed and send to surgical pathology. Representative section (H&E stain) of the specimen is shown below:

fungusball2

Which of the following statements regarding Candiduria is true?

  1. Most patients with candiduria are asymptomatic and the yeasts merely represent colonization
  2. The presence of pseudohyphae in the urine or the number of colonies growing in culture help to distinguish colonization from infection
  3. The most commonly involved organ in disseminated candidiasis is the heart
  4. There is a higher propensity for fungal ball formation in adults than children

The correct answer is 1. Most patients with candiduria are asymptomatic and the yeast merely represent colonization. Infected patients may have symptoms (dysuria, frequency, suprapubic discomfort) while others might not. Pyuria is so common in patients with a chronic indwelling bladder catheter that it cannot be used to indicate infection.

Neither the presence of pseudohyphae in the urine nor the number of colonies growing in culture (unlike bacterial cultures) help to distinguish colonization from infection. Ascending infections are rare but usually subacute or chronic, unilateral and can cause perinephric abscesses.

Fungus balls in adults are uncommon with less than 10 adult cases reported in the literature. Risk factors include uncontrolled diabetes, prolonged use of antibiotics or steroids and immune compromise. Classic laboratory findings include marked leukocytosis, pyuria, hematuria and a concomitant bacterial urinary tract infection. Most cases are caused by Candida species although Aspergillus has been implicated in a few cases.

The kidneys are the most commonly involved organ in disseminated candidiasis and there is a higher propensity of fungus ball formation in neonates.

-Agnes Balla, MD is a 1st year anatomic and clinical pathology resident at the University of Vermont Medical Center.

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

Why Do Two When One Will Do?

Today I attended a great session on transfusion case studies by Carolyn D. Burns, MD, FASCP, and Phillip J. DeChristopher, MD, PhD, FASCP. The speakers were dynamic, personable, and made learning fun. They presented cases on hematology/oncology, transplant recipients, and HLA antibodies, among others. I won’t go over each case—honestly, there was so much great information I’m afraid I won’t do it justice—but I’d like to share tidbits I found interesting.

-A fact that I had forgotten from my blood banking class oh-so-long-ago: the platelets your body makes live for eight to ten days, an autologous platelet transfusion last four days, and a non-autologous transfusion would last three. If a patient has an immune response to a platelet reaction, those platelet might only live a day.

-Fellows and residents in transfusion medicine don’t actually know how to transfuse a unit of blood product. They aren’t aware of what happens in a blood bank or a transfusion center. Laboratory professionals need to be cognizant of this and be open with information. Use teaching moments when they present themselves.

-Eliminate unnecessary transfusions through dialogue with doctor and pathology. Hence the title of this post: “why do two when one will do?” It’s a mantra for the blood banker to live by.

-Don’t be afraid to question orders. Medical technologists might be the first line of defense, so to speak, and are essential when bringing questionable orders to the attention of pathologists. Don’t be afraid to speak up when your instincts are telling you something is off. Hone your critical thinking skills.

-Blood transfusion is like marriage. It should not be entered upon lightly, wantonly or more often than is absolutely necessary.

-This couldn’t be stressed enough: keep the lines of communication open. Ask the doctor and/or nurse questions about the patient; have a open relationship with your medical director; don’t be afraid to ask questions.