fungal culture – Lablogatory

Microbiology Case Study: Male Patient on Steroids Presenting with Myasthenia Gravis Exacerbation and Respiratory Symptoms

Case History

A 41-year-old male was admitted with myasthenia gravis exacerbation and respiratory difficulty. He was diagnosed with myasthenia gravis in 2004, underwent a thymectomy in 2010, and was taking prednisone (20 mg daily). He presented to another hospital one month ago with fever, chest pain, and shortness of breath. He was treated with antibiotics and antifungals for pneumonia, along with 5 days of immunoglobulin for a possible myasthenia gravis crisis, but showed no improvement. After admission, a CT scan revealed diffuse bilateral mixed airspace and ground-glass opacities without pleural effusions, most concerning for multifocal pneumonia. A bronchoalveolar lavage (BAL) with Gomori methenamine silver (GMS) staining showed hyphal and yeast elements. Both BAL and urine Histoplasma antigen tests were positive. He was started on posaconazole (300 mg daily), which led to an improvement in symptoms, and he was subsequently discharged. After 6 weeks of culture, macroscopic and microscopic features of the colony confirms Histoplasma capsulatum (Figure 1).

Figure 1. Macroscopic analysis reveals dark brown color on reverse (top). Lactophenol cotton blue preparation of mold reveals tuberculated, macroconidia (bottom).

Discussion

Histoplasma capsulatum is a dimorphic fungus responsible for histoplasmosis, a respiratory infection. As with other dimorphic molds, this fungus exists as a mold in the environment at 25°C and converts to a yeast form once it infects a host at 37°C. It is primarily endemic in areas with rich soil, particularly in the Ohio and Mississippi River valleys in the United States, but has also been reported in Maryland, Pennsylvania, Delaware, West Virginia, Virginia, and North Carolina. Outside the United States, histoplasmosis incidence is best understood and highest in parts of Mexico and South and Central America and is largely driven by the AIDS pandemic^1,2. The fungus thrives in bird and bat guano due to the high nitrogen and organic content; common exposures include bird roosts, chicken houses, caves, and old buildings that are commonly visited by bats. Humans typically become infected through the inhalation of aerosolized spores. Demolition or building construction around these soiled environments can lead to outbreaks.

While most infections are asymptomatic, histoplasmosis can lead to severe illness in immunocompromised individuals. Histoplasma spores are deposited in the lungs, where they convert to the yeast form. These yeast cells are phagocytosed by alveolar macrophages, where they survive and multiply inside the cells. The fungus can then disseminate via the lymphatic and bloodstream systems to other organs ³. The clinical presentation of histoplasmosis varies depending on the host’s immune status and the level of fungal exposure. In most cases, the infection is asymptomatic or manifests as mild flu-like symptoms, including fever, fatigue, and cough. However, in cases of acute pulmonary histoplasmosis, patients may experience pneumonia-like symptoms such as chest pain, cough, and fever. Chronic pulmonary histoplasmosis can mimic tuberculosis, presenting with symptoms such as weight loss, night sweats, and a chronic cough. Pleural thickening and apical cavitary lesions are common. Extrapulmonary manifestations include granulomatous mediastinitis, fibrosing (sclerosing) mediastinitis, and broncholithiasis¹. In severe disseminated cases, particularly in immunocompromised individuals, the infection can spread to multiple organs. Adrenal involvement may manifest as adrenal masses, adrenal insufficiency, or electrolyte imbalances. Gastrointestinal involvement is relatively common but rarely produces clinical symptoms⁴. Skin involvement occurs in up to 15% of cases in studies, more frequently in patients with AIDS. Central nervous system (CNS) involvement occurs in 5-20% of cases⁵.

Diagnosing histoplasmosis involves clinical suspicion, radiological studies, and laboratory tests. The most common radiographic findings are diffuse reticulonodular pulmonary infiltrates. Cavitations are seen in chronic cavitary pulmonary histoplasmosis, and mediastinal or hilar lymphadenopathy is often present. In immunocompetent individuals, infections may lead to development of granulomas that may or may not be necrotizing.

For laboratory testing, antigen detection in urine or serum using enzyme immunoassays has a sensitivity of 90%⁶ in disseminated disease and 75%⁷ in acute pulmonary disease. Antibodies may take 2-6 weeks to appear in circulation and are useful in chronic cases to confirm suspicions of infections but less effective for detecting acute infection and in immunosuppressed patients with a poor immune response.

Culturing Histoplasma from clinical specimens is definitive, but it can take 4-8 weeks due to the slow growth of the fungus. The colonies appear white at young growth, have a cottony, cobweblike-aerial mycelium and can mature into brown or grey color on reverse (Figure 1, top) ⁸. Microscopic examination of mold colony using lactophenol cotton blue preparations reveals the characteristic large, rounded, tuberculate macroconidia (circular structures with roughened/spiked edges) originating from short, hyaline conidiophores (Figure 1, bottom). Histoplasma capsulatum appears as small (2-5 μm), oval, intracellular yeast cells typically found within macrophages. The yeast may exhibit a clear space which may appear like a capsule, but is actually a retraction artifact due to the processing of the specimen. Staining with GMS or periodic acid-Schiff (PAS) highlights the yeasts⁹. Molecular methods such as PCR and sequencing have been shown to detect cases of Histoplasma as well.

Treatment varies depending on the severity of disease. Mild cases, particularly those involving acute pulmonary histoplasmosis, often resolve without specific treatment, although antifungal therapy (e.g., itraconazole) is recommended in more severe cases. For chronic or disseminated histoplasmosis, initial treatment with amphotericin B is often followed by long-term itraconazole therapy. The prognosis is favorable for immunocompetent individuals with mild disease, but disseminated histoplasmosis can be fatal in immunocompromised patients if not treated promptly.

References

1. Wheat LJ, Azar MM, Bahr NC, Spec A, Relich RF, Hage C. Histoplasmosis. Infect Dis Clin North Am. 2016 Mar;30(1):207-27. doi: 10.1016/j.idc.2015.10.009. PMID: 26897068.
2. Bahr NC, Antinori S, Wheat LJ, Sarosi GA. Histoplasmosis infections worldwide: thinking outside of the Ohio River valley. Curr Trop Med Rep. 2015 Jun 1;2(2):70-80. doi: 10.1007/s40475-015-0044-0. PMID: 26279969; PMCID: PMC4535725.
3. Horwath MC, Fecher RA, Deepe GS Jr. Histoplasma capsulatum, lung infection and immunity. Future Microbiol. 2015;10(6):967-75. doi: 10.2217/fmb.15.25. PMID: 26059620; PMCID: PMC4478585.
4. Sarosi GA, Voth DW, Dahl BA, Doto IL, Tosh FE. Disseminated histoplasmosis: results of long-term follow-up. A center for disease control cooperative mycoses study. Ann Intern Med. 1971 Oct;75(4):511-6. doi: 10.7326/0003-4819-75-4-511. PMID: 5094067.
5. Wheat LJ, Batteiger BE, Sathapatayavongs B. Histoplasma capsulatum infections of the central nervous system. A clinical review. Medicine (Baltimore). 1990 Jul;69(4):244-60. doi: 10.1097/00005792-199007000-00006. PMID: 2197524.
6. Wheat LJ, Kauffman CA. Histoplasmosis. Infect Dis Clin North Am. 2003 Mar;17(1):1-19, vii. doi: 10.1016/s0891-5520(02)00039-9. PMID: 12751258.
7. Wheat LJ. Laboratory diagnosis of histoplasmosis: update 2000. Semin Respir Infect. 2001 Jun;16(2):131-40. doi: 10.1053/srin.2001.24243. PMID: 11521245.
8. Azar MM, Hage CA. Laboratory Diagnostics for Histoplasmosis. J Clin Microbiol. 2017 Jun;55(6):1612-1620. doi: 10.1128/JCM.02430-16. Epub 2017 Mar 8. PMID: 28275076; PMCID: PMC5442517.
9. Hage, C. A., Ribes, J. A., Wengenack, N. L., Baddour, L. M., Assi, M., McKinsey, D. S., … & Wheat, L. J. (2010). A multicenter evaluation of tests for diagnosis of histoplasmosis. Clinical Infectious Diseases, 50(4), 508-512.

-Xingbang Zheng, M.D., was born and raised in Hefei, China. He attended the Peking University Health Science Center, where he received his doctorate degree. He then worked as an OB/GYN physician in China, primarily focusing on female infertility and reproductive surgery. His clinical research concentrated on subtle distal fallopian tube abnormalities and their relationship with endometriosis and infertility. In his free time, Xingbang enjoys swimming, visiting museums, and spending time with his family. Xingbang is currently pursuing AP/CP training.

-Rebecca Yee, PhD, D(ABMM), M(ASCP)^CM is the Chief of Microbiology, Director of Clinical Microbiology and Molecular Microbiology Laboratory at the George Washington University Hospital. Her interests include bacteriology, antimicrobial resistance, and development of infectious disease diagnostics.

Microbiology Case Study: A 30 Year Old with Fever Post Stem Cell Transplant

Case History

A thirty year old female with refractory acute leukemia was admitted to undergo allogeneic stem cell transplantation. Her initial admission had multiple infectious complications and chemotherapy-induced pancytopenia with profound absolute neutropenia. The patient was placed on prophylaxis/therapy including bacterial, viral, and fungal coverage. On hospital day 14, the patient was febrile to 38°C; vancomycin/piperacillin-tazobactam were added as empiric therapy due to concern for sepsis with fluctuation in mental status. CT Brain without contrast revealed a large intracranial hematoma with mass effect. Her mental status continued to decline and intubation was required for airway protection. An emergent decompressive hemicraniectomy was performed where necrotic brain tissue with hemorrhage/clot were found.

Due to ongoing fevers, empiric antimicrobial therapy was further broadened with meropenem, doxycycline, trimethoprim/sulfamethoxazole, and liposomal amphotericin B (L-AMB). Repeat cultures and imaging studies were ordered to evaluate for infection as a fever source. CT Angiography Chest (Image 1A-C) was performed and revealed an extensive non-enhancing area of ground glass opacity with peribronchovascular consolidation (“Reversed Halo” sign) in the right lung concerning for angioinvasive fungal infection. A tracheal aspirate was sent for bacterial and fungal culture. Subsequent bronchoscopy revealed extensive necrosis involving all visualized airways of the right tracheobronchial tree to the first subsegmental level (Image 1D). By contrast, the left lung appeared relatively normal and uninvolved. Bronchial washings of the right lung were also submitted for culture.

Image 1. Computed Tomography (CT) Angiography Chest images: Coronal (A), Cross-section (B) and Sagittal (C) sections reveal a large central ground glass opacity surrounded by a dense consolidation in the shape of a ring “Reversed Halo” sign. Bronchoscopy (D) revealed extensive necrosis and friable mucosa in the visible airways of the right lung.

Laboratory Identification

Respiratory specimens were sent to the microbiology laboratory for bacterial and fungal cultures. Hyphal elements were observed on the Gram stain of the submitted tracheal aspirate (Image 2A). Robust fungal growth was noted within 48 hours on Brain Heart Infusion and Inhibitor Mold agars from both the tracheal aspirate and bronchial wash specimens (Image 2B). A lactophenol cotton blue prep revealed broad hyphae with few septations, consistent with a member of the Mucorales (Image 3). Sporangiophores were noted to be long, dark and branched with round sporangia. Few rhizoids were observed and located between the sporangiophores. A definitive identification of Rhizomucor sp. was obtained through the use of matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS).

Image 2. A: Gram stain (400X) of direct sample from tracheal aspirate with broad, irregular, long, pauciseptate hyphae consistent with infection from a fungal species belonging to the order Mucorales. B: Fluffy “cotton candy” appearing light brown-grey colonies grew rapidly (<4 days) on brain heart infusion agar from both tracheal aspirate (pictured) and bronchial washing specimens.

Image 3. Lactophenol cotton blue stain obtained by tape preparation direct from fungal colony (A; 200X, B-D; 400X). Numerous branched dark brown non-apophysate sporangiophores with spherical columella were seen along with numerous broad, irregular pauciseptate hyphae with right angle branching (A, B). Rudimentary rhizoids developing off stolons (C, D) were rarely identified.

Discussion

Members of the order Mucorales can be identified in the laboratory by their rapid, robust growth and cotton candy-like appearance on conventional fungal media excluding those containing cycloheximide.² Microscopically, these molds exhibit pauciseptate, broad (9-15 μm wide) hyphae with sporangia. Some species elaborate root-like structures called rhizoids (e.g Rhizopus, Rhizomucor) while others lack them (e.g. Mucor).² Rhizomucor sp. can be differentiated microscopically from other related members of the Mucorales by its branched, dark brown sporangiophores with absent apophysis, round columella and the presence of few, short, rudimentary rhizoids (Image 3A-D). In practice, the rhizoids can be challenging to identify microscopically.² Some species can also grow at elevated temperatures (~38-58°C) which can be utilized as a tool for use in identification.² Newer methods including the use of MALDI-TOF MS and DNA probes allow for rapid, accurate identification of these fungi, but are not routinely available in all laboratories.

Infections caused by Mucorales (Mucormycosis) usually involve immunocompromised patients with defects in cell-mediated immunity. Rapid and often fatal, these infections can prove extraordinarily difficult to manage. They are known to be angioinvasive and can widely disseminate. Debridement of involved tissues and higher-level antifungal agents (e.g. posaconazole, amphotericin B) are mainstays of therapy. The most recognized group of patients where these infections are identified are uncontrolled diabetics, frequently in diabetic ketoacidosis, often with nasal/orbital sinus involvement. Patients with leukemia/lymphoma who are undergoing stem cell transplantation are another group often affected. In addition to the nasal/orbital sinuses, the gastrointestinal tract, skin and lungs also serve as important sites for these infections, especially if mucositis occurs.¹

Rhizomucor sp. are an occasional cause of mucormycosis, but have a predilection for leukemic patients such as in this case.² Given the significant bronchoscopy findings and the intraoperative presence of necrotic brain tissue, there was substantial clinical concern for invasive pulmonary mucormycosis with possible central nervous system involvement. Isavuconazole was discontinued, and posaconazole and micafungin were added to her antifungal therapy (L-AMB). Granulocyte infusions were used in an attempt to increase her cell-mediated immune response to the mold. Cardiothoracic surgery evaluated the patient but the lesion was deemed unresectable. Due to the presence of epistaxis, the otolaryngology service evaluated the patient for invasive fungal sinusitis; however, nasal endoscopy did not reveal any nasal/sinus involvement. The patient never regained significant neurological function and continued to medically decline during the hospitalization. She was placed on comfort care where she died shortly afterwards.

References

Love GL, Ribes JA. 2018. Color Atlas of Mycology, An Illustrated Field Guide Based on Proficiency Testing. College of American Pathologists (CAP), p. 244-274
Walsh TJ, Hayden RT, Larone DH. 2018. Larone’s Medically Important Fungi, A Guide to Identification. ASM Press, p. 185-190

-John Markantonis, DO is the current Medical Microbiology fellow at UT Southwestern and will be completing his Clinical Pathology residency in 2022. He is also interested in Transfusion Medicine and parasitic diseases.

–Kim Stewart BS, MT(ASCP)SM holds a bachelor’s degree from Texas Tech University and is medical technologist in the microbiology section at UT Southwestern Medical Center with 35 years’ experience.

-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.

Microbiology Case Study: A 40 Year Old Woman with Fever, Chills, and Leg Pain

Clinical History

A 40 year old African American female with a history of sickle cell disease presented to an outpatient clinic with fever, chills, and leg and back pain consistent with a sickle cell crisis. Her past medical history was also significant for asthma and seizures. She rated her pain as 10 out of 10, her vitals showed a temperature of 101.0°F, and she was also tachycardic and hypotensive. Her white blood cell count was 23.0 TH/cm², hemoglobin 8.4 g/dL, hematocrit 26.0%, and platelets 619,000 TH/cm². In clinic, she received pain medications and a fluid bolus, two sets of blood cultures were collected, and she was transferred to the emergency department for further work up.

Laboratory Identification

Image 1. Gram stain from a positive blood culture bottle showing small, gram positive budding yeast (1000x oil immersion).

Image 2. A mucoid, salmon-colored yeast grew on Sabouraud dextrose and chocolate agars.

Blood culture bottles were positive after approximately two days on the automated instrument. The Gram stain showed small, gram positive budding yeast (Image 1). The BioFire FilmArray for blood culture identification was negative for Candida albicans, C. glabrata, C. krusei, C. parapsilosis, and C. tropicalis. At this time, she was started on micafungin for antifungal therapy. A mucoid, salmon colored yeast grew on both Sabouraud dextrose and chocolate agars (Image 2) and was identified by Vitek 2 as Rhodotorula spp.

Discussion

Rhodotorula spp. are basidiomycetous yeasts that make up the normal microbiota on moist skin and can be found in bathtubs and on shower curtains. Rhodotorula spp. are usually considered contaminants, but can rarely cause fungemia in patients with central lines, endocarditis, peritonitis, and meningitis, especially in those that are immunocompromised. R. mucilaginosa, R. glutinis, and R. minuta are the species commonly associated with human disease.

In the laboratory, Rhodotorula spp. grow as a mucoid, salmon colored yeast within 1-3 days of incubation. On Gram stain or lactophenol cotton blue prep, the yeast is small and round to oval with multilateral budding. Pseudohyphae are not usually present. Rhodotorula spp. produce urease and fail to ferment carbohydrates. R. mucilaginosa is negative for nitrate assimilation. Identification can also be confirmed by commercial kits, automated systems, and MALDI-TOF mass spectrometry. Rhodotorula spp. are intrinsically resistant to echinocandins and fluconazole.

In the case of our patient, she was switched to intravenous amphotericin B after the identification of Rhodotorula spp. was made. Reference laboratory testing identified the isolate as R. mucilaginosa with high minimum inhibitory concentrations (MIC) to fluconazole and echinocandins. Amphotericin had an MIC of 0.5 µg/ml. She successfully completed a 14 day course with close monitoring of creatinine, electrolytes, and platelet count. Repeat blood cultures were negative and no other focuses of infection were found on CT scans, transthoracic echocardiogram, and ophthalmology exam.

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

Microbiology Case Study: A 24 Year Old Male with No Past Medical History Returning from Guatemala with Fevers, Myalgia, and Cough

Case History

A 24 year old male with no past medical history presented with fevers, myalgia, and cough following return from a 1-week trip to Guatemala where he spent significant time within caves. The patient described his cough as persistent, non-productive, and associated with mild shortness of breath at rest that significantly worsens with activity. In the emergency department, the patient was afebrile with a WBC of 10.2, Transaminitis, and chest X-ray showed diffuse reticular pattern. He underwent a bronchoscopy and BAL washout.

Laboratory Findings

Histoplasmosis Urine Antigen test came back positive.

Image 1. Fungal culture with white/tan, fluffy mold (growth at day 7).

Image 2. Scotch tape prep with tuberculate macroconidium. This mold was morphologically identified as Histoplasma capsulatum and sent to Mayo Laboratories for further confirmatory testing.

Discussion

Histoplasma capsulatum is an intracellular, thermally dimorphic fungus (grows as a yeast at body temperature/37°C in humans or culture media and as mold at 25°C in the environment/culture media). Histoplasma is found in soil, particularly in areas containing bird and bat droppings, such as caves. Within the United States Histoplasma in found in central and eastern states with a predominance in the Ohio and Mississippi River Valleys. This fungus is also found in parts of Central and South America, Africa, Asia, and Australia.

Infection with Histoplasma capsulatum causes significant morbidity and mortality worldwide. Upon inhalation of conidia, H. capsulatum transforms into the pathogenic yeast phase. This form replicates within macrophages that carry the yeast from the lungs to other organs. Histoplasmosis has three main forms:

Acute primary histoplasmosis which presents as a pneumonia with fever, cough, myalgia.
Chronic cavitary histoplasmosis which is characterized by pulmonary lesions that often resemble cavitary tuberculosis.
Progressive disseminated histoplamosis that spreads to infect many organs in immunocomprimised patients.

In the laboratory, culture of blood, tissue and respiratory specimens may be completed. In addition, a test for H. capsulatum antigen is sensitive and specific when simultaneous serum and urine specimens are tested. It is important to note that cross-reactivity with other fungi (Coccidioides immitis, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei) has been identified.

Growth on fungal culture shows white/tan, fluffy mold that turns to brown to buff with age. The organism may also produce wrinkled, moist, or heaped yeast-like colonies that are soft and cream when grown at 37°C on certain media. Scotch tape preparation of the mold form shows tuberculate macroconidia, a diagnostic structure of Histoplasma capsulatum. The mycelia are septate and produce microconidia and macroconidia. Yeast forms of Histoplasma capsulatum are small (2 to 4 μm) and reproduce by budding. These budding forms may be seen on histology specimens. A commercially available DNA probe can be performed on culture material to confirm identification.

Patients with mild-moderate histoplasmosis can often have resolution of their symptoms without treatment. Those with more moderate-severe disease require antifungal agents including amphotericin B or itraconazole.

-Nicole Mendelson, MD is a 1^st 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 30 Year Old Male with Fevers and Cough

Clinical History

A 30 year old African American male presented to the emergency department (ED) with fevers and cough. His past medical history was significant for type 1 diabetes & diabetic nephropathy requiring a kidney/pancreas transplant three years prior. He is compliant with his immunosuppressant regimen. He described the cough as non-productive and denied shortness of breath or chest pain. He denied sick contacts, recent travel, and has no pets. After hospital admission, he became septic, developed severe hypotension (70/30s), and was transferred to the intensive care unit (ICU). Chest x-ray showed multifocal consolidations in bilateral lung fields and a small pleural effusion consistent with pneumonia. He was empirically started on vancomycin, piperacillin-tazobactam, azithromycin, and micafungin. Infectious diseases was consulted and recommended a board variety of tests and cultures given the patient immunosuppressed status.

Laboratory Identification

The following results were obtained:

Sputum culture: normal respiratory flora, negative for fungi and acid fast bacilli

Streptococcus pneumoniae & Legionella pneumophila antigens: negative

Histoplasma & Blastomyces urinary antigens: negative

Fungitell: negative

Respiratory viral PCR panel: positive for adenovirus, coronavirus, and rhinovirus

Image 1. Cryptococcal lateral flow assay showing positive (left) and negative (right) results. The patient had a positive result from the serum with a titer of 1:20.

Given this positive serum result for Cryptococcus neoformans despite multiple negative sputum cultures, a bronchoalveolar lavage & lumbar puncture were performed and bacterial & fungal cultures were performed.

Image 2. Discrete, mucoid, cream colored colonies of Cryptococcus neoformans growing on Sabouraud dextrose agar after the third week of incubation at 25°C from the bronchoalveolar lavage specimen.

Discussion

Cryptococcus neoformans is an encapsulated yeast that is most commonly acquired through inhalation and can infect & disseminate to multiple organ systems including the lungs, central nervous system, skin and bones, especially in immunocompromised patients such as those with HIV or organ transplant patients. The thick polysaccharide capsule gives colonies of C. neoformans a mucoid appearance, serves as a major virulence factor, and also plays an important part in various laboratory identification methods.

In the lab, C. neoformans will grow a variety of selective and non-selective agars including blood, chocolate, Sabouraud dextrose, and cornmeal agars as discrete, cream colored colonies (Image 2). On microscopic examination, the C. neoformans yeast are gram positive with narrow based budding and a thick capsule. The yeast vary in size from 2-20 µm and are evenly spaced from one another due to the capsule. C. neoformans is positive for both urease and phenoloxidase. Historically, India ink stain was performed on CSF specimens to highlight the capsule using direct microscopy. Grocott’s methenamine silver (GMS), mucicarmine, and Fontana-Masson histochemical stains are all positive for C. neoformans.

Cryptococcal antigen tests directed to the capsular polysaccharide can also be used to diagnosis C. neoformans infections from both serum and CSF specimens. Common methods include immunochromatographic lateral flow assays or particle agglutination. Advantages to these methods include increased sensitivity and the ability to provide semi-quantitative titer results which can be used to monitor the patient’s response to therapy. Rarely, false negative results can occur due to extremely high concentrations of the cryptococcal antigen. In order to combat the prozone effect, the sample should be diluted prior to repeating the test if there is a high suspicion of cryptococcal infection. False positive results may also occur when macroglobulins are present in the sample due to disease states such as rheumatoid arthritis or lupus. Use of pronase can prevent the interference of macroglobulins on serum test results. False positive test results have also be documented due to interferences from various collection devices such as anaerobic vials.

In the case of our patient, as C. neoformans is intrinsically resistant to echinocandins, he was switched from micafungin to fluconazole. He responded well and after completing the therapeutic course, he continued on a prophylactic dose of fluconazole. His cerebrospinal fluid culture showed no growth and the cryptococcal lateral flow assay was negative on the CSF specimen.

-Charles Middleton, MD, is a first year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.

Microbiology Case Study: A 14 Year Old Immunocompromised Male with Pneumonia

Case History

A 14 year old male with a history of selective IgG deficiency, asthma, and GE reflux s/p Nissen Fundoplication, presented to the pediatric pulmonary clinic with 2 weeks of cold like symptoms that progressed to a wet sounding cough productive of sputum and chest pain. 5 days prior he had seen his primary care physician who diagnosed him with pneumonia and started him on amoxicillin and 5 days of prednisone (60mg daily). He is using his albuterol nebulizer every 3 hours, and feels as though his asthma may be contributing to his symptoms but is not the main cause as he has not had a wet cough and chest heaviness and pain with previous asthma attacks. He has had low grade fevers for the past several days but denies chills, sweats, or hemoptysis. Of note his immune deficiency is treated with IVIG and long term Bactrim which was recently stopped. Pediatric pulmonology elected not to restart the Bactrim but changed his antibiotic to Augmentin. The patient continued to have chest pain and coughing, so the decision was made to proceed with bronchoscopy

Laboratory Identification

zygo-1 — Image 1. Scotch tape prep showing broad hyphae with rare septation and round sporangia.

zygo-2 — Image 2. Colonies with growth at 30° and 37° C showing “fluffy” growth with darkening in the center of the colony.

Cultures from the BAL revealed a rapidly growing fungi with broad hyphae (5 to 15 micron diameter) that are irregularly branched, and have rare septations. These features, paired with the morphology of the sporangia are diagnostic for a Zycomycete; further identification was attempted however, rhizoids characteristic of Rhizopus, and branching characteristic of Mucor were not identified in this culture.

Discussion

Zygomycetes (Mucormycetes) are widely distributed in the environment in soil and vegetation and infection is through inhalation of spores. Typically, the spores are transported by the muco-cilliary escalader to the pharynx, are swallowed, and then are broken down by the GI tract. In immunocompromised patients, the spores can settle in the nasal turbinates and alveoli causing disease (1). The most common sites of infection are rhino-orbital-cerebral and pulmonary and typically occur in immunocompromised hosts and diabetics (2). The Zygomycetes are also known to invade blood vessels making tissue infarction and necrosis one of the hallmarks of the disease (3). These fungi grow rapidly and are often referred to as “lid lifters” when cultured. Because of this rapid growth, infection with Zygomycetes typically progress quickly and cause periorbital edema, proptosis, and blindness. Facial numbness can occur if there is infarction of sensory branches of the fifth cranial nerve. Infection can spread from the ethmoid sinus to the frontal lobe and result in obtundation. If infection spreads from the sphenoid sinuses to the adjacent cavernous sinus, it can result in cranial nerve palsies, thrombosis of the sinus, and involvement of the carotid artery (4). When the spores are inhaled into the lung, pneumonia with infarction and necrosis results. The infection can spread to contiguous structures, such as the mediastinum and heart, or disseminate hematogenously to other organs such as the GI tract, kidney, and brain (5). Infections are treated with a combination of antifungal drugs such as Amphotericin B, and aggressive surgical debridement. In some situations removal of the palate, nasal cartilage, and orbit are necessary for cure. In patients with pneumonia, often lobectomy is needed for cure (5).

The genera most commonly found in human infections are Rhizopus, Mucor, and Rhizomucor. Rhizopus organisms have an enzyme called ketone reductase, which allows them to thrive in high glucose, acidic conditions, such as in individuals with diabetic ketoacidosis. Rhizopus will have root like rhizoids and are typically located near the sporangiophores. The post-mature sporangiophores can undergo “umbrella like” collapse which is characteristic of Rhizopus. Mucor is more likely to have branching and is often identified when all other species are ruled out; they do not produce a rhizoid.
References

Ferguson BJ. Mucormycosis of the nose and paranasal sinuses. Otolaryngology Clinics of North America. 2000;33(2):349.
Kauffman CA, Malani AN. Zygomycosis: an emerging fungal infection with new options for management. Current Infectious Disease Reports. 2007;9(6):435.
Greenberg RN, Scott LJ, Vaughn HH, Ribes JA. Zygomycosis (mucormycosis): emerging clinical importance and new treatments. Current Opinions in Infectious Diseases. 2004;17(6):517.
Yohai RA, Bullock JD, Aziz AA, Markert RJ. Survival factors in rhino-orbital-cerebral mucormycosis. Survey of Ophthalmology. 1994;39(1):3.
Tedder M, Spratt JA, Anstadt MP, Hegde SS, Tedder SD, Lowe JE. Pulmonary mucormycosis: results of medical and surgical therapy. Annals of Thoracic Surgery. 1994;57(4):1044.

-Casey Rankins, DO, is a 1^st 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 Associate Professor at the University of Vermont.

Microbiology Case Study: A 49 Year Old with HIV and CNS Lymphoma

Case History

A 49 year old African American female was transferred from an outside hospital due to orbital cellulitis. Her past medical history was significant for HIV, CNS lymphoma, for which she was taking methotrexate & rituximab, and type II diabetes. Her vitals were: blood pressure 181/145, heart rate 145, temperature 98.6°F and respiratory rate 20. On physical examination, her right eye was bulging, with conjunctiva & eyelid swelling, and her iris was non-reactive. Scant serous drainage was noted. Admission labs showed a normal white blood cell count (9.8 TH/cm²), glucose of 211 mg/dL (normal: 74-106 mg/dL), hemoglobin A1C of 7.7% (normal: 4.2-6.0%) and platelets were low at 41,000 TH/cm². An infection was suspected and the patient was started on vancomycin and piperacillin-tazobactam. She had a head CT scan which showed right periorbital cellulitis and diffuse sinus disease but no abscess formation. Nasal endoscopy was performed and extensive adhesions & black colored, necrotic tissue of the right nasal cavity was noted in addition to whitish debris, consistent with fungal overgrowth extending into the nasopharynx. Biopsies were taken for frozen section and bacterial & fungal culture and Infectious Disease was consulted for management of a probable rhinocerebral fungal infection.

Laboratory Identification

rhiz1 — Image 1. Biopsy of the right nasal wall showed tissue invasion and necrosis with broad, ribbon like hyphae that were pauciseptate and branched at right angles (H&E, 40x).

rhiz2 — Image 2. Fluffy, white fungal growth on Sabouraud Dextrose and Sabouraud Dextrose with Chloramphenicol agars at 72 hours of incubation at 25°C. There was no growth on the Mycobiotic agar slant.

rhiz3 — Image 3. Tape prep showed a round sporangium containing small sporgangiospores located directly below the rhizoids of the mold which is consistent with the diagnosis of *Rhizopus* spp. (lactophenol cotton blue, 40x).

Discussion

Rhizopus spp. belong to the order Mucorales, are ubiquitous in the environment and are the most common etiologic agents of mucormycosis. Rhizopus spp. typically cause invasive infections in the nasal sinus, brain, eye and lung, particularly in patients that have uncontrolled diabetes, HIV or are immunosuppressed. Mucorales are angioinvasive, exhibit perineural invasion and there is usually thrombosis, infraction and necrosis of surrounding tissue. As the illness can progress quite rapidly, prompt diagnosis and treatment is necessary.

If a Mucorales is suspected, tissue specimens obtained during a surgical procedure should be sent for frozen section, direct examination with calcofluor white/KOH and fungal culture. On histologic exam or microscopic exam in the microbiology laboratory, the hyphae of Rhizopus spp. are wide & ribbion-like with few to no septations (pauci- or aseptate) and wide angle branching (90°) (Image 1). Further classification requires culture.

If a Mucorales is suspected, the tissue submitted for fungal culture should be minced into small pieces and directly applied to the appropriate fungal media. Grinding of tissue will kill the hyphae and result in no growth from culture. Mucorales will not grow on media containing cycloheximide. Rhizopus spp. grow rapidly within 1-4 days and start as white, fluffy colonies that become grey or brown in color as they mature (Image 2). The Mucorales are described as “lid lifters” due to their rapid growth and “cotton candy” like colonies that fill the plate. On lactophenol cotton blue prep, Rhizopus spp. have unbranching sporangiophores that terminate in a round sporangium and arise directly under well-developed rhizoids (Image 3). The sporangium ruptures when mature and releases many oval sporangiospores.

Treatment of patients with mucormycosis is usually a dual approach with wide surgical excision and amphotericin B, which has been shown to be an effective anti-fungal drug in the majority of Mucorales. In contrast, voriconazole has poor activity against these isolates. If susceptibility testing is needed, CLSI provides reference broth microdilution guidelines. In the case of our patient, due to the grave prognosis of her condition, in addition to her other comorbidities, the family elected for comfort care measures only and board spectrum anti-fungals were not started.

Stempak

Microbiology Case Study: A 60 Year Old Female with Right Ear Pain

Case History

60 year old female presents to the emergency department with increased pain in her right ear and decreased hearing. She denies ear discharge. She endorses vertigo for 7 months that is precipitated by sudden changes in head position. On physical exam, the right ear canal is obscured by a foreign body. Ear swab is positive for growth on fungal culture.

Lab Identification

Image 1. Salt and pepper fungal colonies isolated from ear swab.

aspniger — Image 1. Salt and pepper fungal colonies isolated from ear swab.

aspniger2 — Image 2. Septate hyphae with unbranched condidiophore connected to a swollen vesicle covered in phialides that produce chains of conidia.

The identification of Aspergillus niger is made based on macroscopic colony morphology and microscopic structures. On the potato flake agar, Aspergillus niger grows salt and pepper colonies. For microscopic examination, a slide is made by touching the colonies with a piece of clear tape, putting a drop of lactophenol analine blue on a glass slide, and placing the tape on the slide. Microscopically, Aspergillus niger appears as septate hyphae with long smooth unbranched conidiophores. Compared with other Aspergillus species, the phialides of niger cover the entire vesicle and form a “radiate” head, which splits into several loose columns.

Discussion

Aspergillus is a common mold that lives both indoors and outdoors. The Aspergillus genus is composed of 180 species, among which 34 are associated with human disease.¹ A. fumigatus is the most common cause of aspergillosis syndromes. A. terreus is a species of particular concern due to its resistance to amphotericin. An invasive disease due to A. terreus has a poor prognosis.¹

Healthy individuals inhale hundreds of conidia of Aspergillus per day without illness. However, people with a weakened immune system or lung disease are at higher risk of developing infections from inhaling the condidia. Presentations of aspergillosis range from allergy to fungal balls, to dissemination.¹ Examples of aspergillosis include asthma, allergic bronchopulmonary aspergillosis, and allergic sinusitis.¹

Invasive otitis externa due to Aspergillus is a rare, potentially life-threatening invasive fungal infection affecting immunocompromised patients.² It spreads from the external auditory canal to adjacent anatomical structures such as soft tissues, cartilage, and bone.² The condition can lead to osteomyelitis of the base of the skull with progressive cranial nerve palsies, irreversible hearing, and neurological impairment.² The infection can be treated with antifungals.

References

Barnes PD, Marr KA. Aspergillosis: spectrum of disease, diagnosis, and treatment. Infect Dis Clin North Am. 2006 Sep;20(3):545-61, vi.
Parize, P. et al. Antifungal Therapy of Aspergillus Invasive Otitis Externa: Efficacy of Voriconazole and Review. Antimicrobial agents and chemotherapy. 2018 April; 62(4). http://aac.asm.org/content/53/3/1048.long

-Ting Chen, MD is a 1^st 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 Associate Professor at the University of Vermont.

A Candida Comeback?

Hello again everyone! And special thanks to the readers who read, commented, shared, and reached out to me from my last post “A Serious Aside,” talking about physician burnout and health worker suicide. Numerous people had so much to say in support of this topic—and it’s well deserved—sharing their personal stories and relating their own connects, so I truly appreciate it.

This time, how about something different? In the past few months, I’ve been working through my clinical rotations at a major community hospital in New York City, in the Bronx. A CDC-sponsored screensaver image keeps appearing at terminals throughout floors, services, and clinics; and it directly addresses healthcare professionals to monitor hygiene practices to eliminate Candida infections. I’ll have to admit—innocuous stuff—I’ve been seeing health-message PSA-like screensavers at work for years, about a myriad of topics. Who hasn’t seen those? “Keep beds out of the hallways,” “Protect you and your patients from MRSA,” “Make sure lab requisitions are filled out properly…” the list is endless. But having seen my aforementioned screensaver about Candida one too many times, I had to find out what this was about. You might have thought that, since I spent time working in an HIV clinic, this was a simple PSA for those patients otherwise immunocompromised. Right? Nope.

This particular PSA from the CDC warns about Candida auris, a true blue (or pinkish gold, rather) member of everyone’s favorite budding, germ-tube positive, yeast family. C. auris has been in literature for roughly the past decade. Having etiologic origins in southeast Asia and spreading west through the Middle-East, all throughout Africa, and even the UK, this bug has caught the eyes of epidemiologists around the world. Two years ago, the CDC¹ and Public health England² issued warnings about this pathogen, its multi-drug resistance, and its virulence in healthcare-associated infections. Last fall, the NY State Department of Health published their official update for guiding clinicians and laboratory staff.³ In this report, they discussed infection control, prevention, and detection limitations.

canaur1 — Image 1. CDC screensaver on hospital computers. Due to increased incidence of reported cases, epidemiologic data suggest prevention measures would benefit patients. *C. auris* is associated with healthcare-related infections and can live in the environment for an extended period of time.

canaur2 — Image 2. NY State Department of Health, Report on *C. auris.* Informing clinicans and laboratory staff about epidemiology, prevention, detection in the laboratory, and associated implications of limitations and multidrug resistance.

So what’s so scary about C. auris? The two most challenging features of this emerging pathogen are its multi-drug resistance and its relatively difficult identification.

This yeast has been shown to show resistance to many antifungal/antimicrobial agents including fluconazole, voriconazole, amphotericin-B, echinocandins, and even flucytosine. Even more concerning is that nearly half of the C. auris strains collected in research done in Asia, Africa, and South America demonstrated multi-drug resistance patterns to two or more combination therapies. These are most of our first-line standard of care therapies for invasive candidiasis in patients!

Image 3. UpToDate recommendations summary for candidemia and invasive candida infections.

There are various other recommendations regarding therapies to C. auris specifically, as its potential for resistance are known, but infection control along with empiric therapy seem to be the current standard.

The major risk factors for C. auris infections include the relative status of individual patients: intensive care, acute renal failure, immunocompromised status, localized or systemic infections, and colonization. Simply being hospitalized is an associated risk. On my current service of patients I’m part of a nephrology/medicine team. There are several chronic infection, ESRD, immunocompromised, or otherwise applicable patients to these risk stratifications. No wonder we’ve got those screensavers!

Concerns for identifying C. auris take us back to the lab. Detecting this bug is not as simple as a couple microscopic morphologies and a yeast API strip—sorry to my old mycology professors. C. auris based on chemical tests like these can produce confounding results. Even VITEK identification (unless you’re running Vitek 2 with Biomerieux software) or culture growth can yield non-specifics like C. haemulonii or Saccharomyces cerevisiae. C. auris has a very high salt and temperature tolerance, and with no particular morphologic identifiable features, it remains a challenging identification. It can be grown on dulcitol agar or CHROMagar, but you do not get clear results. What’s the way to get the ID then? Ultimately MALDI-TOF, PCR, and molecular testing is the answer. There are already available C. auris sequences you can obtain for in-house validation if you’re using MALDI already. And when it comes to susceptibility, fear not: as far as I’ve been able to read E-Tests still work.

Image 4. Definitely not a definitive CHROMagar result.

I was very impressed with MALDI when I was working in Chicago, and a community hospital I was with just finished validating when I left for medical school. I am glad to see it again with this emergent pathogen, and it definitely demonstrates the next wave of instrumentation. Extremely rapid and very accurate.

The variable drug susceptibility, virulence, and ability to thrive in the environment actively threaten those with long inpatient stays. This microorganism is treated with standard precautions and infection control measures. Currently NY leads the nation by far in purported cases of C. auris. So … please wash your hands. A lot. I know I am.

Thanks! See you next time!

References

Centers for Disease Control and Prevention. Clinical Alert to U.S. Healthcare Facilities – Global Emergence of Invasive Infections Caused by the Multidrug-Resistant Yeast Candida auris. https://www.cdc.gov/fungal/diseases/candidiasis/candida-auris-alert.html
Public Health England. Candida auris identified in England. https://www.gov.uk/government/publications/candida-auris-emergence-in-england/candida-auris-identified-in-england
NY State Department of Health https://www.health.ny.gov/diseases/communicable/c_auris/docs/c_auris_update_for_lab_staff.pdf

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–Constantine E. Kanakis MSc, MLS (ASCP)^CM graduated from Loyola University Chicago with a BS in Molecular Biology and Bioethics and then Rush University with an MS in Medical Laboratory Science. He is currently a medical student at the American University of the Caribbean and actively involved with local public health.

Microbiology Case Study: A 59 Year Old Female with Fevers, Weakness, and Altered Mental Status

Case History

A 59 year old African American female presented to the emergency department with fevers, weakness, fatigue and altered mental status. Her past medical history was significant for hypertension, diabetes mellitus (type 2) with end stage renal disease and a recent cerebrovascular accident the month prior. Her surgical history included a mitral valve repair surgery three years ago and a renal transplant two years ago. Current medications included prednisone, mycophenolate and tacrolimus immunosuppressive agents. Physical examination was unremarkable except for a temperature of 101°F and she was oriented to person, place and time. Pertinent labs included a WBC count of 13.2 TH/cm², microcytic anemia, and a creatinine of 1.51 mg/dL. Due to previous cardiac surgery, a transesophageal echocardiograph (TEE) was performed and showed a large vegetation (1.6 x 1.5 cm) on the mitral valve. A diagnosis of endocarditis was made and the patient was started on broad-spectrum antibiotics & taken to surgery for a mitral valve replacement. Multiple blood cultures were negative to this point. Portions of the mitral valve were submitted to surgical pathology and the microbiology laboratory for bacterial, fungal and AFB cultures.

Laboratory identification

Surgical pathology received an aggregate of tan-yellow, fibrous tissue fragments (3.1 x 1.5 x 1.1 cm). Histologic assessment showed a confluent mass containing abundant narrow, septate hyphae consistent with a fungal infection (Image 1). No definitive pigment was identified. Grocott’s methenamine silver (GMS) stain also highlighted the narrow hyphae with numerous septations (Image 2). In the microbiology laboratory, a darkly pigmented mold grew after 5 days of incubation on Sabouraud dextrose agar (Image 3). Lactophenol cotton blue prep showed pigmented, curved conidia with 2-3 transverse septations consistent with Curvularia spp (Image 4). All blood cultures were finalized as no growth after 5 days. Fungitell was found to be greater than 500 pg/ml and Aspergillus galactomannan was negative (<0.5).

Image 1. Sections of mitral valve tissue showed a confluent mass of abundant hyphal elements (H&E, 4x).

Image 2. Special stains of the fungal mass highlighted narrow hyphae with numerous septations and acute angle branching (GMS, 4x).

Image 3. A darkly pigmented mold grew of Sabouraud dextrose agar after 5 days of incubation at 25°C.

Image 4. Many pigmented, curved conidia with multiple transverse septations were seen (lactophenol cotton blue prep, low power).

Discussion

Curvularia spp. belong to a heterogeneous group of dematiaceous or black molds. The presence of pigment in this category of molds is due to melanin in the hyphae. Dematiaceous molds are ubiquitous in nature and can occasionally cause human infections. These molds have a characteristic dark appearance on fungal media that is often dark gray, brown or black in color. In addition, when the reverse of the plate or slant is examined, the under surface is also pigmented. Based on their growth rate, the dematiaceous fungi are divided into the fast growers, such as Curvularia, Bipolaris and Alternaria spp., which are mature in 5-7 days. The second group is slow growers that take between 7-25 days to fully mature. Examples of slow growers include Phialophora, Exophila/Wangiella, Cladosporium, Fonsecaea and Rhinocladiella spp.

Most commonly, dematiaceous molds infections usually present as phaeophyphomycosis, chromoblastomycosis or mycetomas. These three entities are cutaneous or subcutaneous mycoses that are obtained by traumatic implantation but vary from one another based on clinical features and histologic features of the mold in tissues. They are most frequently cause infection in male agricultural workers in rural areas of tropical or subtropical climates. These infections are indolent in nature but can lead to significant morbidity over time, as they are difficult to treat effectively.

In addition to the above superficial infections, Curvularia spp. has also be known to cause keratitis, sinusitis and wound infections. In immunosuppressed individuals, disseminated infections with spread to the lungs and brain have been documented. Endocarditis due to Curvularia spp. is quite rare with very few cases previously reported in the literature. On those documented, Curvularia spp. infections tend to have a predilection for prosthetic heart valves or occur after cardiac surgery. Diagnosis of infective endocarditis is difficult as symptoms are indolent and blood cultures do not have a high yield. Therefore, culture of the vegetation may be the only way to make a diagnosis.

In the microbiology laboratory, Curvularia spp. will grow on routine fungal media as a darkly pigmented mold in a relatively short time. On lactophenol cotton blue prep, Curvularia spp. produce large conidia that usually contain 4 cells that are divided by transverse septations. The conidia take on a curved appearance due to swelling of the subterminal cell, which is often the largest and most deeply pigmented. If identification is necessary beyond the genus level, panfungal PCR assays followed by sequencing of ribosomal genes may be useful in providing a species level diagnosis from fresh or paraffin embedded tissue.

For localized infections, surgical treatment alone may be adequate in some cases. In infections that are extensive or if there is dissemination, treatment with newer triazoles, such as posaconazole or voriconazole, have shown a broad spectrum of activity against dematiceous molds. Amphotericin B is also another effective option. While susceptibility testing can be performed on clinically significant Curvularia spp. infections, interpretative breakpoints have not been defined and clinical correlation is lacking.

In the case of our patient, she remained on a ventilator following surgery and with the identification of mold on histology, she was started on micafungin. She was switched to amphotericin B after the mold was classified as Curvularia spp. Her condition did not improve despite therapy and she died 3 weeks after surgery.

-Azniv Azar, MD, is a fourth year anatomical 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.