2020 NPA-02

This case was originally published in 2020. The information provided in this case was accurate and correct at the time of initial program release. Any changes in terminology since the time of initial publication may not be reflected in this case.

A 64-year-old man with a past medical history of idiopathic cardiomyopathy status post-heart transplant presented with altered mental status and severe cough. Imaging studies revealed multiple bilateral pulmonary cavitary lesions and multiple brain lesions. He quickly deteriorated and expired. An autopsy was performed.

Tissue Site
Brain, multifocal

The whole slide images provided are H&E and Grocott’s methenamine silver stained images of the brain from an autopsy.

This case is an example of CNS fungal infection caused by Aspergillus in an immunocompromised patient. The typical CNS lesions are multifocal, hemorrhagic, and necrotic, as seen in the gross autopsy photograph (Image A). On low magnification routine H&E-stained sections, brain parenchyma with extensive inflammation, necrosis, and hemorrhage is visible (Image B). On higher magnification, abundant branching fungal hyphae are seen in a background of necrotic inflammatory debris (Image C and Image D). Grocott’s methenamine silver (GMS) stain highlights multiple clusters of branching, septate hyphae at both low and high magnification (Image E and Image F). The fungal hyphae can be seen characteristically traversing the blood vessels (Image G).

As the use of immunosuppressive drugs increases, so too does the incidence of CNS fungal infections. Aspergillus species are ubiquitous, worldwide, and the most common cause of fungal infection in the immunosuppressed. Those who have undergone organ or bone marrow transplant are at the greatest risk. Other at-risk groups include those with diabetes, chronic obstructive pulmonary disease, alcoholism, intravenous drug use, malnutrition, or burns. Paradoxically, CNS Aspergillus infection in AIDS patients is exceedingly rare. Most cases of CNS Aspergillus infection are due to A. fumigatus or A. flavus; other less common species include A. niger and A. oryzae. Other CNS fungal infections include yeasts (Cryptococcus neoformans, Candida spp., Trichosporon spp.), other monomorphic molds (Fusarium), mucormycetes (Mucor spp., Rhizopus spp.), dimorphic fungi (Blastomyces dermatitidis, Coccidioides spp., Histoplasma capsulatum), and dematiaceous fungi (Cladophilaophora bantiana, Exophila dermatitidis). Precise fungal typing requires culture or polymerase chain reaction (PCR). Because Aspergillus is morphologically similar to other molds, a diagnosis of “invasive septate hyphae consistent with aspergillosis” is appropriate when based on histomorphology alone.

Clinical presentations vary greatly depending on extent and location but may include headache, neurologic deficit, seizure, or increased intracranial pressure. The patient may develop meningitis, encephalitis, hydrocephalus, cerebral abscesses, stroke, or cavernous sinus syndrome. Mycotic disease of the CNS is typically caused by hematogenous spread from a primary focus elsewhere in the body. Lesions are frequently multifocal, ranging from a few millimeters to several centimeters, and do not discriminate between cortex, white matter, basal ganglia, or meninges. Fewer infections are the result of direct spread from sinuses, ears, orbit, or via direct inoculation from trauma or surgery. Airborne spores derived from the soil, water, or rotting vegetable matter typically gain entrance through the lungs, nose, or sinuses. Aspergillus produces elastase, which allows it to invade through the blood vessel walls and spread hematogenously, thus gaining access to the brain, heart, and other organs. Of patients with disseminated disease, 60% to70% have CNS lesions. Once within the CNS, Aspergillus can produce mycotic aneurysm and multifocal hemorrhage.

Rapid identification of mycotic CNS infection is imperative to patient survival, allowing targeted therapy to be initiated. Sterile culture of the organism, ideally in combination with evidence of tissue invasion on histopathology, provides the most definitive evidence of invasive aspergillosis. Aspergillus is a rapid grower, providing culture results within one to three days. Confirming the diagnosis of CNS aspergillosis, however, can be difficult, as invasive procedures are often precluded by neutropenia and thrombocytopenia. Cerebral spinal fluid (CSF) fluid analysis can be attempted but frequently shows only mild pleocytosis and rarely contains organisms. The serum galactomannan antigen assay is relatively specific for invasive Aspergillus; however, the sensitivity is low. Beta-D-glucan assay is positive in the setting of various invasive fungal infections, including candidiasis, but does not provide species-specific information. Aspergillus-specific PCR assay and other emerging molecular assays can be performed on CSF of patients with suspected CNS disease. Aspergillus-specific PCR has been shown to have a sensitivity approaching 100% and a specificity of greater than 90%. For this reason, PCR testing of CSF samples is recommended if the clinical condition precludes invasive procedures. If the patient presents with clinical and radiologic findings suggestive of primary pulmonary infection with disseminated disease, sputum culture or bronchoscopy with bronchoalveolar lavage and lung biopsy should be pursued.

In the brain, Aspergillus tends to form either abscesses or granulomas. The type of associated inflammation depends on the chronicity of infection and immune status of the patient. Inflammation may be totally absent if the patient is severely immunocompromised. Neutrophils predominate in early phase of disease, and macrophages appear later; frank pus may be seen in the lesion center with abundant neutrophils at the edges. Abscesses are more common with hematogenous spread and may be single or multifocal. Grossly, abscesses are pale, soft, hyperemic areas with poorly-defined borders, necrosis, and hemorrhage (Image F). These may resemble acute hemorrhagic infarct and are most common in the distributions of the anterior or middle cerebral arteries. Granulomas, which are more likely to be associated with direct contiguous spread from adjacent structures, are typically fibrotic and solitary. These are more likely to cause single space-occupying lesions or a skull base syndrome. Granulomas contain variable proportions of lymphocytes, plasma cells, epithelioid macrophages, collagen, and/or multinucleated giant cells.

Aspergillus is readily visible on H&E stain by its regular, radially-arrayed and dichotomously-branching septate hyphae which are approximately 4 to12 micrometers in width (Image E and Image F). The hyphae often are seen as silhouette-like unstained structures (Image D). The branches depart at acute angles from the main body (45 degrees, “tuning fork”). Hyphae can be seen infiltrating the blood vessels and are frequently visible within the lumen, wall, and adjacent tissue of blood vessels of varying caliber (Image G). Aspergillus can cause extensive infarcts; the hyphae are of large enough caliber to obstruct arterioles, and aggregated hyphae may promote occlusive thrombus formation in larger vessels. Sputum or bronchoalveolar lavage fungal stains can be performed using calcofluor white stain and 10% potassium hydroxide. GMS or PAS stains may be helpful to identify organisms on cytology and fixed tissue.

IHC may be used as an ancillary technique in the diagnosis of Aspergillus spp. IHC with anti-Aspergillus antibody is available and may be particularly useful when distinguishing aspergillosis from mucormycosis or Fusarium. In a study by Jung et al, IHC showed 87% sensitivity and 100% specificity for culture-proven aspergillosis cases and 100% sensitivity and specificity for culture-proven mucormycosis cases. Therefore, IHC may be a useful adjunct, particularly if culture or PCR are unavailable.

Drug choice is critical and depends on the extent of CNS penetration and the spectrum of activity. Empiric therapy is often implemented prior to definitive diagnosis. Amphotericin B has limited distribution in CSF but is recommended if combined with flucytosine for cryptococcal meningoencephalitis, CNS candidiasis, or mucormycosis. Voriconazole has good CNS penetration and is considered first line therapy for CNS aspergillosis. Other triazoles have negligible concentrations in the CSF except for the novel drug isavuconazole, a drug with clinical data showing efficacy for treatment of some fungal CNS infections. Despite the available treatment regimens, CNS aspergillosis is associated with a mortality rate of 90% to 100% in immunosuppressed patients.

• CNS fungal infections are often deadly and are most commonly encountered in immunosuppressed patients, particularly organ transplant recipients. Aspergillus spp. are the most common causative organisms.
• Aspergillus typically spreads hematogenously to the brain from a remote site, usually the lung, due to its inherent ability to invade blood vessels. If hematogenous, lesions are likely multifocal and hemorrhagic. If contiguous invasion, lesions are usually singular and granulomatous.
• Morphologically, Aspergillus is characterized by septate hyphae that branch at acute angles.
• Fungal culture remains the gold standard for diagnosis, speciation, and therapy selection. However, laboratory studies such as PCR on CSF samples may be useful in situations where obtaining a tissue sample is not feasible.

1. Challa S, Uppin SG, Uppin MS, Pamidimukkala U, Vemu L. Diagnosis of filamentous fungi on tissue sections by immunohistochemistry using anti-aspergillus antibody. Medl Mycol. 2015;53(5):470-6.
2. Ellison D, Love S, Chimelli LMC, et al, eds. Neuropathology. 3rd ed. Oxford, England: Mosby; 2013.
3. Góralska K, Blaszkowska J, Dzikowiec M. Neuroinfections caused by fungi. Infection. 2018;46(4):443-59.
4. Jung J, Park YS, Sung H, et al. Using immunohistochemistry to assess the accuracy of histomorphologic diagnosis of aspergillosis and mucormycosis. Clin Infect Dis. 2015;61(11):1664-70.
5. Perry A, Brat DJ. Practical Surgical Neuropathology: A Diagnostic Approach. 2nd ed. Philadelphia, PA: Elsevier; 2018.