Microbiology Case Study: A 3 Year Old Girl with Abdominal Pain and Fever

Case History

A 3 year old girl initially presented with abdominal pain and fevers. Ultrasound identified a left kidney mass, leading to a left radical nephrectomy and excision of retroperitoneal mass. Pathology showed a Wilms’ tumor, diffuse anaplasia type. Staging uncovered multiple pulmonary metastases and involvement of a supraclavicular lymph node. She received chemotherapy and radiation without regression of disease.  She then presented to the ED and was admitted for neutropenic fever. She was started on broad-spectrum antibiotics. She continued to spike fevers so an antifungal, micafungin, was added.  While admitted, she developed scattered erythematous papules. Infectious disease was consulted and a skin biopsy from the left forearm was obtained.

Laboratory Results

  • Bacterial cultures, blood: negative
  • Fungal cultures, blood: negative
  • Blastomyces urine antigen: negative
  • Skin biopsy: slight epidermal hyperplasia with follicular dilatation, mild vascular ectasia, and focal erythrocyte extravasation. Negative for organisms.
  • Bacterial culture, tissue: no growth.
  • Gram stain: rare budding yeast forms seen
  • Fungal culture, tissue: no growth to date
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The gram smear made from skin biopsy tissue for bacterial culture displayed rare broad-based budding yeast forms, consistent with Blastomyces dermatiditis.

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The gram smear made from skin biopsy tissue for bacterial culture displayed rare broad-based budding yeast forms, consistent with Blastomyces dermatiditis.

 

Discussion

Blastomyces dermatitidis is a dimorphic fungus found in moist soil and decomposing matter. It is endemic within the Mississippi and Ohio River valleys as well as the Great Lakes region and Southern United States.  There are reports of infection in Africa and India. The fungus is transmitted by inhalation of as few as 10-100 conidia. Once in the lungs, the spores convert to yeast and multiply. Infection usually results in a flu-like illness with pulmonary involvement 3-15 weeks post-exposure. Hematogenous spread can further result in involvement of the skin, bone, genitourinary tract, and central nervous system.

The gold standard for diagnosis is culture or cytopathology/histology. However, the organism is a slow grower, which can take 2-4 weeks, and may fail to grow in one-third of cases. On culture at room temperatures (25-30°C), the mold form appears wrinkled and waxy and is cream to tan in color. Microscopically, they form septate hyphae with short or long conidiophores bearing small round to pear-shaped conidia (2-10 microns). This arrangement of the mold is described as a “lollipop” appearance. At 35-37°C, the fungus is a yeast (8-10 microns) with classic broad-based budding and double contoured walls.

Antigen testing is available on urine, serum, bronchoalveolar lavage fluid, and CSF. Antigen testing is more rapid, utilizing enzyme immunoassay, but has a lower sensitivity. Antigen testing is most sensitive in patients with isolated pulmonary disease. Serial urine antigen testing can be used to indicate disease regression or relapse.

A real-time PCR assay is available for confirmation of B. dermatitidis. The probe targets the promoter region of the BAD1 gene, which encodes an adhesin molecule and virulence factor. This method can be performed in five hours, but is only available at reference laboratories.

Mild to moderate pulmonary and extrapulmonary blastomycosis can be treated with oral itraconazole. Severe cases, CNS involvement, or infection of immunosuppressed patients, pregnant women, or children require amphotericin B followed by step-down therapy with itraconazole for 6-12 months.

Upon report of the mold on gram smear, micafungin was discontinued and amphotericin B treatment initiated. Her fever and rash resolved. The patient was transitioned to oral itraconazole prior to discharge and will remain on therapy for 12 months.

REFERENCES

  1. https://www.cdc.gov/fungal/diseases/blastomycosis/index.html
  2. Frost HM, Novicki TJ. Blastomyces Antigen Detection for Diagnosis and Management of Blastomycosis. Journal of Clinical Microbiology. 2015;53(11):3660-3662. doi:10.1128/jcm.02352-15.
  3. Sidamonidze K, Peck MK, Perez M, et al. Real-Time PCR Assay for Identification of Blastomyces dermatitidis in Culture and in Tissue. Journal of Clinical Microbiology. 2012;50(5):1783-1786. doi:10.1128/jcm.00310-12.
  4. Chapman SCAW, Dismukes WE, Proia LA, et al. Clinical Practice Guidelines for the Management of Blastomycosis: 2008 Update by the Infectious Diseases Society of America. Clinical Infectious Diseases. 2008;46(12):1801-1812. doi:10.1086/588300.

 

 

-Prajesh Adhikari, MD is a 2nd 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: A 72 Year Old Man with Extreme Fatigue

Patient History

A 72 year old Caucasian man with diabetes presented to his primary care physician in late September complaining of recent extreme fatigue while baling hay on his farm in southern New England. A thorough interview revealed recent anorexia with a 15 pound weight loss, dyspnea on exertion, joint pain and easy bruising. Blood work demonstrated pancytopenia with 17% blasts on peripheral smear. Further work-up established a diagnosis of acute myelomonocytic leukemia (AML) and the patient was started on chemotherapy.  During his admission, he spiked a fever (101°F) and based on a chest x-ray showing a left basilar consolidation was consistent with pneumonia and he treated with vancomycin and Zosyn. Symptoms persisted despite the addition of acyclovir and anidulafungin. Given an infectious etiology was continually suspected based on a chest CT showing right upper and left lower lobe opacities, infectious disease (ID) was consulted.

The detailed ID work-up noted an exposure history including interaction with chickens and cows, though the veterinarian reported that the livestock were avian flu negative and vaccinated against brucellosis, respectively. Further, it was revealed that the farmer’s hay had recently been infested with voles and that mold had been found growing in his home and barn. ID recommended a bronchoscopy and a CT-guided lung biopsy to characterize the patient’s pathology more completely. The patient remained febrile (102.9°F) and his condition deteriorated to the point of requiring MICU admission. A new chest CT showed rapid progression of airspace opacities.

Gross findings from the bronchoscopy raised concern for an invasive fungal infection but all specimens obtained for cytology and culture were negative for a fungal process.  The patient continued to decline with multisystem organ failure, the development of new hypoechoic liver lesions on ultrasound and a brainstem mass without evidence of herniation on head CT. His fever peaked at 106.2°F at which time he was transitioned to comfort measures only and passed away shortly after.

Pathology Identification

Post-mortem, a limited autopsy was performed and gross, histologic and special stains finding are shown in Figure 1.

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Figure 1. Gross and histologic lung images from autopsy. Target shaped lesions on the lung surface can be seen in this gross photograph of the upper and middle lobes of the right lung in situ (A). A sample of lung parenchyma was sectioned and H&E staining revealed broad, irregularly branched, pauci-septate fungal elements admixed with a brisk inflammatory infiltrate within damaged alveoli (B). Depiction of angioinvasion as the fungal organisms penetrate the walls of pulmonary vessels; GMS and PAS fungal stains, respectively (C and D).

Grossly, both lobes were abnormal. Specifically, as seen in Figure 1A, the right upper lobe exhibited multiple target shaped lesions on its surface in addition to a thin fibrinous coat involving the visceral and parietal pleurae. Moreover, the right upper lobe was adhered to the chest wall. Histologic examination of the lesions demonstrated abundant inflammatory cells and board fungal hyphae that were irregular and “ribbon like” with occasional septations within the disrupted lung parenchyma and invading into blood vessels as seen in Figure 1B-D.  Tissue from the lung lesions were cultured at autopsy and grew Lichtheimia spp.

A battery of additional tests collected premortem for Streptococcus pneumoniae, Cryptococcus neoformans, Mycoplasma, Histoplasma, Blastomyces and Toxoplasma gondii were all negative.  The AFB culture from the BAL specimen grew Mycobacterium avium after 8 weeks of incubation.

Discussion

Lichtheimia is a fungal genre, which shares the order Mucorales with variety of other clinically relevant organisms including Rhizopus, Rhizomucor and Mucor. It was formerly referred to as Absidia and many resources still use this term. As a saprophyte, Mucorales species live freely in the environment and can often be found indoors as well as outdoors. In the healthcare setting, infections are most frequently encountered in diabetic patients. In fact, along with its other Mucorales relatives, Lichtheimia spp. demonstrate a particular tropism for high glucose environments. Immunosuppressed individuals, especially those with hematologic malignancy, are also commonly infected with the fungi of this order – an observation, which speaks both to the ubiquity of these organisms in the human environment as well as the crucial role played by the immune system in their control. Today, the infection caused by Lichtheimia and its relatives is referred to as mucormycosis, though the related term zygomycosis remains deeply ingrained in the medical lexicon. The most striking presentations of mucormycosis are those involving the rhino-orbital-cerebral tissues. However, Mucorales organisms can also colonize many other organ systems. Relevant to this case, pulmonary mucormycosis is a particularly severe form of the infection with a mortality rate nearing 90%. Further, in a patient with pulmonary mucormycosis, the likelihood of concomitant disseminated mucormycosis is also very high (nearing 50%). Though in the present case the post-mortem examination was limited to only particular lung lobes, clinicoradiographic findings preceding the patient’s death strongly suggested that the infection also involved the liver and brain.

Diagnosis of pulmonary mucormycosis can be tricky and often hinges upon histopathologic findings and culture results. As in the present case, Mucorales organisms are differentiated from other common fungi with similar presentations based on their broad hyphae with limited septations and irregular branching. By contrast, Aspergillus spp. exhibits more narrow hyphae with septations and regular, acute-angle branching patterns. Additionally, Mucorales organisms, like Lichtheimia, are often angioinvasive and histologic examination may demonstrate fungal elements entering vascular lumina and even inducing thrombotic infarctions (both noted in the current case). In the laboratory, if a Mucorales is in the differential diagnosis, the tissue specimen should be minced instead of ground in order to preserve viability of the organisms. Mucorales grow rapidly as non-descript, whitish-gray molds within 4 days and are described as “lid lifters” due to their predilection to completely fill the plate. Due to its highly infectious nature, plates should be wrapped in parafilm and only examined in certified biosafety cabinets. On a lactophenol cotton blue prep, the sporangiophores of Lichtheimia and Rhizomucor spp. arise internodally between the rhizoids. This is in contrast to Rhizopus spp. in which the sporangiosphores arise directly over the rhizoids and Mucor spp. where rhizoids are not produced.

While there is little doubt that the patient in the above case was particularly susceptible to environmental Lichtheimia spores as a consequence of his immunosuppressed condition and status as a diabetic, the contribution played by his occupational exposures is less clear. Though provocative, it would be challenging to establish a link between the patient’s terminal infection and his agricultural encounters with decaying vegetable matter and the associated molds.

lavik

-JP Lavik, MD/PhD, is a 3rd year Anatomic and Clinical Pathology Resident at Yale New Haven Hospital.

Stempak

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

Microbiology Case Study: A 50 Year Old Male with High Fevers and Chronic Cough

Case History

A 50 year old male of Indian descent presents to the pulmonary clinic with complaints of high fevers and chronic cough. The cough has persisted for the past month and recently became productive with green sputum.  His fevers are cyclic in nature and reach 104°F.  He denies hemoptysis, unintentional weight loss or chest pain.  He has tried over the counter decongestants and cough suppressants as well as a course of levofloxacin, with minimal improvement. His past medical history is significant for rheumatoid arthritis, which is currently treated with methotrexate and prednisone. He works as a long distance truck driver and is a non-smoker.  A recent chest x-ray demonstrated a left hilar opacity with a nodular appearance. A computed tomography scan of the chest shows focal consolidation of the left lower lobe along with mediastinal and hilar adenopathy. Also, there are innumerable non-calcified nodules seen throughout bilateral lung fields. A bronchoscopy with bronchoalveolar lavage (BAL), transbronchial biopsy, and fine needle aspiration (FNA) of the enlarged lymph nodes were performed. BAL fluid was transported to the microbiology lab for bacterial, fungal and mycobacterial cultures.

Laboratory Identification

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Figure 1. Histologic evaluation of the lung biopsy showed diffuse necrotizing granulomas which contained large yeast-like forms (red arrow) (H&E, 100x).

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Figure 2. The large yeast-like forms measured between 10-25 µm in size and demonstrated a thick walled capsule (Grocott’s methenamine silver (GMS), 600x).

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Figure 3. White mycelium with a downy texture and faint brown reverse grew on Mycosel agar after 28 days of incubation at 25°C.

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Figure 4. Numerous coarse, septate hyphae producing thick walled, “barrel shaped” arthroconidia (lactophenol cotton blue stain, 1000x oil immerson).

Histology of the lung biopsy specimen showed necrotizing granulomas with occasional large, yeast-like spherules which measured between 10-25 µm in diameter (Figures 1 & 2). The spherules had a thickened capsule and endospores were not visualized. The fungal BAL cultures grew a white mycelium with a downy texture and light brown reverse after incubation for 28 days at 25°C on Mycosel agar (Figure 3). Microscopic morphology of a lactophenol cotton blue prep illustrated alternating thick walled arthroconidia suggestive of Coccidioides immitis/posadasii (Figure 4). The dimorphic mold was confirmed by DNA probe testing. Due to the findings on histology and the unusually slow growth of this particular isolate, Coccidioides IgM and IgG antibodies were performed by ELISA in the interim. They were found to be 5.4 and 4.4, respectively, suggestive of a current or recent infection. Laboratory studies for Aspergillus galactomannan, Fungitell, Cryptococcus antigen, and Histoplasma & Blastomyces urinary antigens were all negative. A Quantiferon Gold for Mycobacterium tuberculosis and all other cultures were also negative.

Discussion

Coccidioides immitis is often considered a thermally dimorphic mold geographically distributed to the arid climate of the southwestern United States and Mexico. It is morphologically identical to the C. posadasii, a species which is more widespread and endemic in South America. The two species can only be differentiated by molecular methods, although it is not routinely necessary as there is no difference in symptoms and treatment between the two.

Inhalation of infectious arthroconidia occurs as a result of environmental exposure to dust, sand and soil that has been disturbed. While many immunocompetent individuals who are exposed to C. immitis will show mild flu-like symptoms which resolve with no treatment, a portion of patients will go on to have pulmonary disease. A severe disseminated infection can occur in individuals with underlying immune system disorders, including rheumatologic diseases, HIV and transplant recipients on immunosuppression. C. immitis can have direct invasion of adjacent structures and can cause eruptive chronic granulomatous cutaneous disease. Women who are diagnosed with Coccidiomycosis during pregnancy are also at high risk for disseminated disease due to the presence of estrogen-like receptors in the fungus.

In the environment and when cultured in the laboratory at 25°C, C. immitis grows as a hyphal mold with alternating barrel-shaped arthroconidia (3-6 µm) separated by disjunctor cells. The arthroconidia are highly infectious and cultures in the laboratory must be worked up in a biological safety cabinet to minimize the risk of accidental exposure. The yeast-like phase occurs in tissue at temperatures above 35°C and is characterized as the transformation of arthroconidia in to large spherules (10-80 µm) which become filled with endospores. The cell wall of the spherule ruptures and the endospores are released into the tissue to become additional spherules.

Most patients with primary Coccidiomycosis do not require specific therapy as the disease will resolve on its own. For those patients who are immunocompromised, or whom exhibit severe disease, treatment is amphotericin B followed by fluconazole or itraconazole as maintenance therapy. In the case of our patient, he was placed on oral fluconazole twice daily for at least 3 months.

 

ka

-Kristen Adams, MD, is a fourth year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center. 

Stempak

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

Microbiology Case Study: A 57 Year Old Woman with Recurrent Fever

Case History

A 57 year old female presents to the hospital with complaints of a recurrent fever for the past few days. Her past medical history is significant for acute myeloid leukemia (AML). On physical examination, she has multiple, warm, erythematous, non-painful papules and nodules involving her extremities. One of the lesions, located on the dorsal aspect of her finger, had ulcerated. She is found to be neutropenic with a white blood cell count of 0.36 TH/cm2 (reference range 4.0-10 TH/cm2). A chest CT scan is performed and reveals multiple, small hyperdense pulmonary nodules.  As part of the work up for febrile neutropenia, blood cultures are collected. Dermatology was also consulted and a skin biopsy at the advancing edge of the lesion was performed.

Laboratory Identification

fus1

Figure 1. Review of the deep portion of the punch biopsy demonstrates panniculus with a deep, dilated vessel containing fibrin and possible fungal organisms (H&E, 40x).

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Figure 2. Deep dilated blood vessel with fibrin and fungal forms consistent with a hyaline septate mold were identified (H&E, 400x). Mycotic organisms were confirmed by a Grocott’s methenamine silver (GMS) stain.

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Figure 3. Gram stain from a positive blood culture vial highlighting narrow hyphal elements with acute angle branching consistent with a hyaline septate mold (400x).

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Figure 4. Lactophenol cotton blue preparation highlighting septate hyphae and numerous macroconidia that can be described as canoe or banana shaped (400x).

 

All of the specimens were collected on the same day and the results from the skin biopsy which showed fungal elements consistent with a hyaline septate mold were reported first (Figure 1). After 2 days of incubation, multiple blood culture bottles were positive for a mold similar to what was seen on tissue biopsy, indicating a disseminated fungal infection in this severely immunocompromised patient (Figure 2). Fungal cultures were set up from the blood and after 5 days of incubation at 30°C, lavender cottony colonies with lighter periphery grew on Sabouraud’s dextrose agar (SDA). The lactophenol cotton blue preparation showed many macroconidia with three to five septa (Figure 4). All of the findings are consistent with Fusarium spp.

Discussion

Fusarium spp. are filamentous fungi which are classified as a rapidly growing, hyaline septate mold. This opportunistic mold can be found all over the United States in the soil and on plants. In immunocompetent individuals, Fusarium spp. can cause localized infections, most commonly as the result of traumatic inoculation. Frequently, the eye is the site of infection, leading to keratitis. This can be due to trauma, contamination of contact lenses or solution, or corticosteroid drops. Fusarium has been reported as the infectious pathogen and has many clinical manifestations such as pneumonia, sinusitis, and wound infections. However, in immunocompromised patients, Fusarium spp. pose a greater threat for invasive and disseminated infections. Of the fungal organisms routinely implicated in fungemia (in addition to Candida, Cryptococcus and Histoplasma), Fusarium spp. have a high frequency of positive blood cultures.

Blood cultures positive for a septate mold and the presence of characteristic skin lesions are highly indicative of a disseminated Fusarium infection, especially in a severely neutropenic patient. To support the diagnosis of fusariosis, it is reassuring to have two different specimens each growing the same pathogen. In the case of our patient, both blood and skin tissue cultures grew Fusarium spp. with the same colony and microscopic morphology. The patient was unable to produce sufficient sputum for respiratory cultures, but the lung abnormalities were attributed to the fungal process as well.

In the laboratory, Fusarium spp. grow relatively rapidly on Sabouraud’s dextrose agar and can usually be identified within 3 to 5 days. Colonies are typically cottony in appearance and develop a pink to lavender color as they mature. The reverse of the plate is usually light. Fusarium spp. produce both macroconidia and microconidia. The characteristic macroconidia have been described as canoe, banana or sickle shaped and are separated by 3 to 5 transverse septa. The microconidia arise from short conidiophores and are more oval in shape, containing zero to one septa and can be single or arranged in clusters.

In cases of invasive fusariosis, anti-fungal agents such as voriconazole or high-dose amphotericin B are therapies of choice. In the case of neutropenic patients, growth factors (G-CSF or GM-CSF) or granulocyte transfusions are potential treatment options as well. Surgical debridement of necrotic tissue has shown benefit in patients with large abscesses. Additionally, if the cause of the fungemia is thought to the result of an infected line, the catheter should be removed.

Disseminated involvement of Fusarium spp. has a high rate of mortality associated with the infection.  Often times the prognosis is related to the extent of infection and the degree immunosuppression.  It is important to suspect Fusarium infections in the clinical setting of a severely neutropenic patient with skin lesions and having a low threshold for beginning anti-fungal therapy. An accurate and prompt diagnosis will lead to appropriate treatment and improved outcomes.

 

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-Katie Tumminello, MD, is a fourth year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center. 

Stempak

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

 

Microbiology Case Study: A 68 Year Old with Refractory Pneumonia

Case History

After failing to improve on outpatient treatment for community acquired pneumonia, a 68 year-old New England man was admitted to the hospital for refractory pneumonia. Despite the initiation of triple antibiotic therapy, he continued to spike fevers and his respiratory status progressively declined. Notably, the patient reported recent travel to Arizona.

Laboratory Identification

This sputum sample was received from an outside hospital with no accompanying clinical history. After a couple days, the sample grew wooly, white-beige colonies on both potato flake and mycobiotic agars. A cellophane tape test performed on day 6 revealed narrow septate hyphae with alternating arthrocondia and empty cells. No conidiophores were present.

 

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Sputum fungal culture on Potato Flake Agar
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Sputum fungal culture on Mycobiotic agar
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Cellophane tape test

 

While the overall morphology was most consistent with Malbranchea species, rare slightly swollen arthrocondia were identified prompting increased handling precautions and further investigation into the patient’s clinical course at the referring hospital. After several more days of growth, a repeat cellophane tape test (Figure 3) demonstrated thick-walled, barrel-shaped arthrocondia alternating with empty cells as is characteristic of Coccidioides species.

The organism was then confirmed as Coccidioides immitis/posadasii by PCR.

Discussion

Coccidioides immitis and Coccidioides posadasii are pathogenic soil fungi with limited geographic distribution. Coccidioides immitis is found primarily in California’s San Joaquin Valley, Arizona, and Mexico while Coccidioides posadasii is slightly more widespread throughout arid regions of the Americas. Although genetically distinct, the two species are clinically and morphologically identical.

Coccidioides species cause Coccidioidomycosis, also known as Valley fever, which typically presents as a primary pulmonary infection about 1-4 weeks after exposure. While the majority of infected individuals will be entirely asymptomatic, 40% of cases result in a mild, self-limiting influenza-like illness with fever, sore throat, cough, headache, pleuritic chest pain, and occasionally a maculopapular rash on the trunk and limbs.  The fatigue and arthralgia associated with disease may persist for months after resolution of the pulmonary infection and is therefore sometimes referred to as “desert rheumatism”.

As with many infectious diseases, immunocompromised patients are at greater risk for developing more severe forms of the disease including extra-pulmonary manifestations.  Other risk factors for disseminated disease include high inoculum exposure, chronic illness, and primary infection during pregnancy.  While most infections will resolve without treatment, an extended course of azole therapy is recommended in these more complicated cases.

Since Coccidioidomycosis has relatively nonspecific symptoms, obtaining a history of exposure is often the key to the initiation of appropriate laboratory identification. Serologic testing for Coccidioides is available and is often the method of choice in the ambulatory setting. While highly specific, these tests are not very sensitive due to delayed formation and rapid clearance of the targeted antibodies. Therefore, a negative test result should not be used to exclude a Coccidioides infection especially early in the course of the disease.

In tissues and body fluids, Coccidioides is identified as round, thick-walled spherules (10-80µm) containing multiple endospores (2-5µm). When cultured at both 25°C and 37°C, Coccidioides forms wooly, white-grey colonies which may turn brown as they mature. The coarse hyphae are hyaline and septate with alternating thick-walled, barrel-shaped arthroconidia and empty cells. Although colonies usually form within three to five days, the distinctive arthroconidia may take up to 2 weeks to fully mature. Due to this delayed maturation, Coccidioides is often initially misidentified as its non-pathogenic look-a-like Malbranchea.  Confirmatory testing by PCR may be performed on both bodily fluids and paraffin embedded tissue.

Coccidioides was once considered a “select agent” with the potential to pose a severe threat to human health but advancements in diagnostic techniques and antifungal medications resulted in the loss of that status in 2012. However, since inhalation of even just 10 of its highly infectious arthroconidia may result in disease, Coccidioides is still a major source of laboratory-acquired infections. Technologists should use increased biosafety precautions whenever handling specimens suspicious for Coccidioides.

 

-Elaine Amoresano, MD, is a 2nd 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: A 61 Year Old Male with Productive Cough and Altered Mental Status

Case History

A 61 year old African American male presents to the emergency department with complaints of a productive cough, dyspnea and altered mental status. His past medical history is significant for HIV and currently he is non-compliant with his anti-retroviral medications. On arrival, he is found to be hypoglycemic (glucose 49 mg/dL) and tachycardic (heart rate between 160-180 beats/min). He lives in a group home and they report decreased oral intake for several days but he denies fever, chills, chest pain or abdominal pain. He is a tobacco smoker and admits to previous illicit drug use. On physical exam, he is lethargic and respiratory auscultation reveals coarse lung sounds, bilaterally. A chest x-ray shows bilateral interstitial and airspace opacities suggestive of an infectious process. His CD4 count is markedly decreased at 3 cells/cmm. Peripheral blood and bronchoalveolar lavage (BAL) fluid are sent to the hematology and cytology laboratories for microscopic examination. Blood and BAL specimens were also transported to the microbiology lab for bacterial, fungal and mycobacterial cultures.

Laboratory Identification

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Figure 1. Peripheral blood smear highlighting small, intracellular yeast forms with narrow based budding (Giemsa stain, 1000x oil immersion).
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Figure 2. Fluid from a bronchoalveolar lavage showing macrophages filled with numerous small yeast forms that have an “acorn-like” appearance (Giesma stain, 1000x oil immersion).
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Figure 3. White colonies with a fine, cottony texture growing on Mycosel agar after 21 days of incubation at 25°C.
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Figure 4. Numerous tuberculate, thick-walled macroconidia with septate hyphae in the background (Lactophenol cotton blue stain, 400x).

Both the peripheral blood smear and BAL showed intracellular, small, ovoid yeast cells with narrow based budding (Figures 1 & 2). The yeast forms measured between 2-4 µm in diameter. The characteristic “acorn-like” appearance of the yeast cells surrounded by a thin halo is the result of staining fixation. The blood and the BAL cultures grew white colonies with a fine, cottony texture after incubation for 21 days at 25°C (Figure 3). The mold form grew on Sabouraud dextrose, SAB with chloramphenicol and Mycosel agars. Microscopic morphology of a lactophenol cotton blue prep illustrated septate hyphae bearing round to pear-shaped microconidia as well as tuberculate, thick-walled macroconidia, which measured between 8-15 µm in size (Figure 4).  The dimorphic mold was confirmed to be Histoplasma capsulatum by DNA probe testing. The patient also had a positive Histoplasma urinary antigen and fungitell was found to be >500 pg/ml.

Discussion

Histoplasma capsulatum is a thermally dimorphic fungus and the most common endemic mycosis in North America. In the United States, the disease is most prevalent in areas surrounding the Mississippi and Ohio River valleys. Inhalation of conidia occurs as a result of environmental exposure to soil contaminated with bird dropping or exploring caves and other dwelling inhabited by bats. Pulmonary infections are the most frequent manifestation of disease; however, disseminated infection can occur in individuals with underlying cell-mediated immunological defects, including those with HIV, transplant recipients, and individuals receiving tumor necrosis factor alpha inhibitors for rheumatologic conditions. Other extra-pulmonary sites from which H. capsulatum has been isolated include the skin, liver, spleen, central nervous system and bone marrow.

In the environment and when cultured in the laboratory at 25°C, H. capsulatum is a filamentous mold and exhibits both pear shaped microconidia (2-5 µm) and thick walled macroconidia that display characteristic tubercles or projections on their surface (8-15 µm). The yeast phase occurs in tissue and at temperatures above 35°C. The yeast phase is characterized as small oval budding cells, 2-4 µm in diameter and are  often found in clusters within macrophages. Historically, mold to yeast culture conversion was used to confirm the diagnosis, but with the advent of more rapid DNA probe technologies, this has been discontinued. Other rapid tests routinely utilized include a urine test to detect the Histoplasma antigen.

H. capsulatum var. duboisii, which is endemic in central and western Africa, is also implicated in causing disease in humans. It can be distinguished from H. capsulatum var. capsulatum due to its larger diameter in tissue where the yeast form of H. capsulatum var. duboisii measures between 8-15 µm in diameter as opposed to 2-4 µm for var. capsulatum. Caution is recommended, however, due to the yeast forms of the two variants being the same size when grown in culture.

Amphotericin B is the antifungal agent used to treat disseminated histoplasmosis infections.  In cases of less severe disease, itraconazole is effective and commonly utilized. In the case of our patient, he received 14 days of amphotericin B infusion as an inpatient and was then transitioned to oral itraconazole upon discharge.

 

JK

-Joy King, MD, is a third year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center. 

Stempak

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

An Emerging Trend: Multi-Drug Resistant Fungus

Over on her blog, Maryn McKenna discusses the latest emerging microscopic threat: drug-resistant fungal infections. It mentions the organism Candida auris and states: “The Centers for Disease Control and Prevention (CDC) is so concerned that it recently sent an alert to U.S. hospitals, even though only one possible case of the resistant fungus has been identified in the United States so far.” 

 

Microbiology Case Study: A 44 Year Old Woman with Abdominal Tenderness

Case History

A 44 year old woman presented to an outside hospital with a chief complaint of abdominal tenderness and increased abdominal girth over the past few days. Her past medical history was significant for type II diabetes mellitus and associated complications including retinopathy and chronic kidney disease. As a result of her renal failure, she was currently undergoing peritoneal dialysis. Due to concern for infectious peritonitis, a paracentesis was performed and the resulting fluid obtained was sent to the Microbiology laboratory for Gram stain and bacterial culture. Because of difficulty in identifying the isolate, it was sent to our mycology section for further work up.

Laboratory Identification

prowick

Figure 1. Many sporangia of various sizes containing endospores arranged in a “soccerball” like pattern or morula configuration (Lactophenol cotton blue prep, 400x).

 

The isolate was received on potato dextrose agar (PDA) and appeared as discrete cream colored colonies, resembling a yeast. Upon transfer to cornmeal agar and incubation at 30°C, the isolate grew rapidly over the course of two days. The organism did not grow on media containing cycloheximide. A lactophenol cotton blue prep was performed (Figure 1) and showed many sporangia, ranging in size from 4-15 µm. In the larger forms, individual endospores were able to visualized and they were arranged in a “soccerball” like pattern. No budding or hyphae were present. Given these characteristics, the organism was identified as Prototheca wickerhammii.

Discussion

Prototheca wickerhammii is classified as an achlorophyllous algae and is known to cause human infections involving the skin and subcutaneous tissues, bursa of the elbow joint (olecranon bursitis) and rarely, systemic infections. P. wickerhammii is ubiquitous in nature and infection is usually the result from traumatic inoculation. Both immunocompetent and immunocompromised hosts can be affected, although more severe or systemic disease occurs in those who have defects in cell mediated immunity.

Prototheca spp. grow rapidly on PDA after incubation at 30°C for 2-3 days. Initially, it may be confused with a yeast based on plate morphology as they are cream colored and have a yeast-like consistency. When a lactophenol cotton blue prep is viewed under the microscope, sporangia of various sizes are identified (3-30 µm) that contain endospores arranged in a “soccerball” or symmetric daisy like pattern. Size of the sporangia, assimilation tests (such as the API 20C) and automated yeast identification systems (such as the Vitek yeast ID by bioMérieux) are helpful in identifying Prototheca to a species level. Both P. wickerhammii (4-11 µm in diameter) and P. zopfii (9-28 µm in diameter) can cause disease in humans, but P. wickerhammii is more common. While P. wickerhammii has symmetric internal divisions as seen in the above image, P. zopfii has random internal divisions.

The treatment of P. wickerhammii usually includes a combination of surgical management and anti-fungals depending on the site of involvement. For superficial skin infections, localized excision and the use of topical or systemic anti-fungals (azoles and amphotericin B) has been shown to have success. The treatment for olecranon bursitis focuses on bursectomy. In the case of systemic infections, amphotericin B has been the most successful treatment modality. Susceptibility testing is not routinely recommended to guide treatment of initial infections, as studies are few and results don’t always correlate with outcome.

 

Stempak

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

Microbiology Case Study: A 30 Year Old Male with Painful Right Eye

Case History:

A previously healthy 30 year old man presents with a painful right eye. Three weeks before he had been cutting concrete with an electric saw when a piece of hard concrete hit him in the eye. The eye became painful the following day. He was treated with empiric antibiotics but the eye pain failed to improve. He was eventually seen by ophthalmology, where ophthalmologic exam demonstrated findings suspicious for fungal keratitis. Corneal scraping was performed and sent for bacterial and fungal culture.

Laboratory Identification:

Bacterial culture showed no growth. Fungal culture demonstrated rapid growth of multiple white, cottony molds on potato flake agar. Over time these white colonies turned dark grey/brown. The reverse surface of the agar was white at first but also turned dark grey/brown over time.

Microscopically (scotch tape preparation), there were thin hyphae with single conidia arising directly off the tips of tapered conidiophores of variable length. Occasional conidia were also observed arising directly off of the sides of hyphae. The conidia were small and oval in shape with a truncated base, and somewhat darker as compared to the surrounding hyphae. Sexual forms were not observed.

scedo1
Potato flake agar showing a white, cottony mold with a central dark gray/brown coloration.

 

scedo2
Scotch tape preparation demonstrating thin hyphae with single oval-shaped conidia with truncated base. The conidia are small and have a “sperm-like” or “tadpole-like” morphology.

Discussion:

The clinical history and laboratory findings are characteristic of Pseudallescheria boydii/Scedosporium apiospermum.

Pseudallescheria boydii/Scedosporium apiospermum is an environmental mold which can be isolated from rural soils, polluted waters, manure and compost. Infection occurs secondary to local trauma. In immunocompetent individuals, infection is limited to the site of trauma, with some of the more common presentations being fungal keratitis, endophthalmitis, eumycotic mycetoma, sinusitis and pneumonia in the setting of near drowning. In immunocompromised individuals, infection can disseminate and involve any organ.

Pseudallescheria boydii/Scedosporium apiospermum is the most common cause of eumycotic mycetoma. Mycetoma is a chronic granulomatous infection of the subcutaneous tissue, usually involving the distal lower extremities, which can be caused by either a fungus (eumycotic mycetoma) or an actinomyces species bacteria (actinic mycetoma). On clinical exam there are multiple draining sinus tracts. The causative microorganisms aggregate into macroscopically visible groups (“granules”) which can be white, yellow or brown in color. Mycetoma may progress over time to involve underlying soft tissue, muscle, fascia and bone. Other less common causes of eumycotic mycetoma include Madurella spp., Acremonium spp., Fusarium spp., and Curvularia spp.

This organism has two names because historically different names were assigned to the sexual state (Pseudallescheria boydii) and to the asexual state (Scedosporium apiospermum). Morphologically, Pseudallescheria and Scedosporium are identical, the only difference being the presence of the sexual form, cleistothecia, in Pseudallescheria. Cleistothecia, when present, can be recognized as very large, dark-brown asci containing numerous ascospores. Technically, in the absence of cleistothecia the correct diagnosis would be Scedosporium boydii, however in clinical practice both names are usually listed regardless of whether cleistothecia are seen.

Pseudallescheria boydii/Scedosporium apiospermum is resistant to amphotericin B, but should be susceptible to azole therapy. The patient is being treated with topical voriconazole and oral fluconazole and currently (two weeks of antifungal therapy) has near complete resolution of symptomatology.

-Javier De Luca-Johnson, MD is a 3rd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-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: 7 Year Old Male with Rash on his Scalp

A 7 year old Congolese male presented with pruritic, erythematous, non-flaky rash on top of his scalp for the past 3 weeks. The rash in non-painful, but continues to spread. His mother has been applying hydrocortisone cream nightly, with no improvements.

Colony morphology on fungal media.
Colony morphology on fungal media.
Organism morphology on lactophenol analine blue scotch tape prep.
Organism morphology on lactophenol analine blue scotch tape prep.

 

Laboratory Identification

A hair sample was obtained for fungal culture. Colonies were yellow and waxy with feet-like projections. Microscopic morphology on lactophenol analine blue scotch tape prep revealed broad hyphae with tortuous branches. The hyphae lacked obvious micro and macro conidia, raising the suspicion for Trichophyton violaceum.

 

Discussion

Trichophyton violaceum is an anthropophilic fungus seen predominantly in North Africa, East Asia and parts of the Middle East. It forms slow growing with glabrous colonies. Microscopically, broad tortuous hyphae are seen. Microconidia and Macroconidia are notably absent. T. violaceum causes Tinea Capitis, which can be acquired through scalp contact with the dermatophyte, either with direct contact with an infected individual or an object. It can also affect skin, nails and beards. It manifests clinically as pruritic scaly patches with alopecia, often producing black dots. Affected hairs demonstrate an endothrix infection.

 

-Mustafa Mohammed, MD is a 2nd year anatomic and clinical pathology resident at the University of Vermont Medical Center.

Wojewoda-small

-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.