Seminars in Diagnostic Pathology

Seminars in Diagnostic Pathology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Review article

Histopathology of fungal diseases of the lung

Anja C. Roden n, Audrey N. SchuetzDepartment of Laboratory Medicine & Pathology; Mayo Clinic Rochester, 55905 MN, USA

a r t i c l e i n f o

Keywords:Fungal infectionAspergillusHistoplasmaPneumocystisGranulomaImmunocompromised host

x.doi.org/10.1053/j.semdp.2017.06.00270/& 2017 Elsevier Inc. All rights reserved.

espondence to: Department of Laboratory Mochester Hilton 11 200 First St SW, Rochesterail address: [email protected] (A.C. Roden

e cite this article as: Roden AC, Schu

a b s t r a c t

Fungal pneumonias can be a diagnostic problem. However, their recognition is important as they can pose asignificant health risk, especially in the immunocompromised host. While many of these infections are ac-companied by necrotizing or non-necrotizing granulomas, some might be characterized by cellular interstitialpneumonia, intra-alveolar frothy material or only minimal inflammatory change. Much of the tissue reaction isdependent on the immune status of the patient and the type of fungal organism. While many of the fungi canbe identified in tissue, especially if using histochemical stains such as Grocott's Methenamine Silver (GMS) stainand/or Periodic Acid Schiff (PAS) stain, in some cases, these stains are negative and the organisms can only beidentified in cultures or using special techniques such as PCR or fungal serology. Some fungi can be accuratelyidentified in tissue based on morphologic features; others require culture for exact classification. Knowledgeabout immune status, geographic region and social history of the patient are helpful in identifying the fungusand, therefore, detailed clinical and travel histories are important. In this manuscript we aim to describe themost common fungal infections that occur in the lung, their morphologic features, and differential diagnoses.

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Contents

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1Altered immune defenses in the immunocompromised host . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Diagnosis of fungal infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Pneumocystis jirovecii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Aspergillus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Cryptococcus neoformans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9Histoplasma capsulatum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Coccidioides spp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Blastomyces dermatitidis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Candida spp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11Mucorales . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

Sporothrix schenckii complex . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Introduction

Fungal infections are an important consideration in the differ-ential diagnosis of necrotizing and non-necrotizing granulomatous

edicine & Pathology MayoMN, 55905.).

etz AN. (2017), http://dx.do

inflammation in the lung. However, fungal infections are not al-ways associated with granulomatous disease, especially in theimmunocompromised host, or in small biopsies, where granulo-mas might not have been sampled. Therefore, a high level ofsuspicion for such an infection is necessary for its diagnosis,especially in the immunocompromised host, if the biopsy showsan acute lung injury and/or there is clinical concern of such apossibility.

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A.C. Roden, A.N. Schuetz / Seminars in Diagnostic Pathology ∎ (∎∎∎∎) ∎∎∎–∎∎∎2

Identification of the fungus with which the lung of a patient isinfected depends upon several variables: (i) the geographic regionin which the patient resides and/or travels (for instance, whileHistoplasma capsulatum is common in the Mississippi and OhioRiver Valleys, Coccidioides spp. is endemic in the southwestern U.S.); therefore, obtaining a detailed travel history of the patient isimportant; (ii) the immune status of the patient as fungal organ-isms differ in pathogenicity between immunocompromised andimmunocompetent patients (for instance, Pneumocystis jirovecii,Aspergillus spp. and Cryptococcus neoformans are usually seen inthe immunocompromised host while Histoplasma capsulatum andCoccidioides spp. can infect the immunocompetent host); a medi-cal history is therefore crucial; (iii) patient's social and travel his-tory (for instance, Histoplasma capsulatum is found in the soilusually from bird and bat droppings; therefore, cave spelunkingincreases the possibility of an infection with that organism); avidgardeners are more likely to be exposed to Sporothrix schenckiicomplex.

Some radiologic features might be indicative of the possibility ofa pulmonary fungal infection. Radiographic signs and patterns, theirspecific imaging features and possible association with certainpulmonary fungal infections are described in Table 1.1 Consolida-tion is one of the more common radiologic manifestations of apulmonary infection. However, none of these patterns are specificfor fungal infections in general or for any particular fungus and canbe seen in other diseases. For instance, while the “crazy-paving”pattern might be seen with Pneumocystis infection, it is also theclassic radiologic finding in pulmonary alveolar proteinosis.

While many of the fungi can be grown in tissue or bronch-oalveolar lavage (BAL) culture, some fungal species such as Pneu-mocystis jirovecii do not grow in culture. Furthermore, not un-commonly, tissue is not sent for culture as clinically the lesion wasbelieved at the time of biopsy to represent a neoplasm and aninfectious process was not considered. For certain fungal infectionssuch as Histoplasma capsulatum, Coccidioides spp. and Blastomycesdermatitidis, serology might also be helpful in guiding diagnosis.While culture will often lead to a diagnosis, some fungi (especiallyfungal yeasts in the lung) can be identified to genus or specieslevel based on morphologic features (e.g., size of the organism,shape, pattern of budding, branching of hyphae). However, there isconsiderable morphologic and clinical overlap between some

Table 1Radiologic findings suggestive of (but not specific for) pulmonary fungal infections.1

Radiologic sign Imaging features

Consolidation Areas that are radio-opaque compared to normally air-filled lungAir bronchogram Air-filled bronchi that become visible when surrounded by den

consolidated lung parenchymaCavitation Abnormal lucency within an area of consolidation with or with

associated air-fluid levelReverse halo sign Peripheral consolidation surrounding a central area of ground-g

opacityBird's nest sign Associated irregular and intersecting areas of stranding or irreg

lines present within the area of ground-glass opacityHalo sign Peripheral rim of ground-glass opacity surrounding a pulmonar

dule or massAir crescent sign Crescentic collection of air that separates a nodule or mass from

wall of a surrounding cavityFinger-in-glove sign Tubular and branching opacities that appear to emanate from th

hilaCrazy-paving sign Combination of ground-glass opacity and smooth interlobular s

thickeningGrape-skin sign Very thin-walled cavitary lesion that develops in lung parenchy

previously affected by consolidation, or lung granulomas that hundergone central necrosis

Miliary pattern Multiple small pulmonary nodules of similar size randomly distrthroughout both lungs

Please cite this article as: Roden AC, Schuetz AN. (2017), http://dx.do

fungi (especially fungal hyphae in the lung), and therefore exactidentification cannot always be established on a tissue specimen.In these cases a differential diagnosis might be given in the pa-thology report with the suggestion to correlate with culture andserology results.

In this review we will highlight characteristic morphologicfindings of common and less common fungal infections that mightbe encountered in the lung. Moreover, we will emphasize themorphologic differences between various fungi as not all fungi willgrow in culture and/or cultures or other diagnostic assays such asmolecular testing might not be available. Finally suggestions onhow to report some of these cases will be provided.

Altered immune defenses in the immunocompromised host

As alluded to earlier, certain fungal infections have a predilectionfor the immunocompromised host, including pneumocystosis,cryptococcosis and aspergillosis, especially invasive Aspergillus in-fections. The morphologic features that these fungi elicit will bediscussed in the following paragraphs. In the immunocompromisedhost, certain cellular immune deficiencies are associated with cer-tain fungal infections. For instance, T-cell deficiencies are associatedwith Pneumocystis jirovecii infection (therefore, its common asso-ciation with HIV-infected patients and therapies targeting T-cellactivation). Furthermore, B-cell deficiencies have also been asso-ciated with Pneumocystis jirovecii infections. For instance, recentcase reports of patients treated with anti-CD20 antibodies, a treat-ment used for non-Hodgkin lymphoma or certain autoimmunediseases, suggest that Pneumocystis infection might be more com-mon in this patient population than originally believed.2,3 As CD20is expressed during most developmental stages of B cells, treatmentwith anti-CD20 antibody leads to B-cell deficiency. Although theassociation between anti-CD20 treatment and Pneumocystis infec-tion is still debated given that patients who are treated with anti-CD20 antibodies usually also receive other immunosuppressiveagents, a recent study showed that anti-CD20 treatment alone ispermissive for Pneumocystis infection.4 The study demonstratedthat anti-CD20 impairs components of type II immunity, such asproduction of interleukin-4 (IL-4), IL-5, and IL-13 by whole-lungcells, in response to Pneumocystis murina, which causes diseaseequivalent to pneumocystosis in humans. Moreover, this study

Possible fungal infection

tissue Any pulmonary fungal infectionse,

out an Angioinvasive fungal infections (e.g., aspergillosis and mucormycosis)

lass

ular

y no- Angioinvasive aspergillosis in a patient who has fever and neutropeniaAlso, Candida infection and mucormycosis

the Angioinvasive aspergillosis or mycetoma (Monad sign)

e lung Allergic bronchopulmonary aspergillosis

eptal Pneumocystis infection

maave

Chronic coccidioidomycosis

ibuted Hematogenous dissemination of fungal disease; can be seen with histo-plasmosis or coccidioidomycosis especially in the immunocompromisedhost

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A.C. Roden, A.N. Schuetz / Seminars in Diagnostic Pathology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ 3

showed that CD20-positive B-cells are important for generatingprotective CD4-positive T-cell immune responses against thisorganism.

Recent evidence suggests that eosinophils also play a role in thedefense of the lung infected by Aspergillus fumigatus.5 In addition,eosinophils might contribute to the early clearance of Pneumo-cystis murina infections and there might be an early role of CD4-positive T cells to recruit eosinophils to the lung.6 Polymorpho-nuclear neutrophils are important for the control of invasive as-pergillosis as it has been shown that neutrophils employ oxidativeand non-oxidative mechanisms for clearance of Aspergillus fumi-gatus infections.7

Diagnosis of fungal infection

As some fungal organisms are not readily identifiable on he-matoxylin eosin (H&E)-stained tissue sections, a Grocott Methe-namine Silver (GMS) stain and/or a Periodic Acid-Schiff (PAS) stainshould be performed when there is morphological and/or clinicalsuspicion of a fungal infection. GMS is preferable to PAS foridentification of Histoplasma, especially in necrotizing granulomasof the lung. These stains will highlight most fungal organisms intissue and facilitate their identification. If microorganisms arepresent, they are usually found within granulomas, specificallywithin the necrotic area or the histiocytic rim of a necrotizinggranuloma. In Pneumocystis jirovecii infection the best location toidentify the organisms is within the intraalveolar frothy material.In suspected Aspergillus infection it is important to inspect care-fully the walls of pulmonary arteries and veins to identify invasiveaspergillosis. Another location where fungal organisms (especiallyAspergillus) might be found is within a mycetoma in bronchiectaticor other preformed cavities. In allergic bronchopulmonary asper-gillosis (ABPA), usually rare, fragmented Aspergillus hyphae mightbe seen either within allergic mucin in the lumen of airways or inthe airway walls. However, in ABPA, it is not uncommon that nofungal organisms are identifiable.

Geographic location and social history of the patient should bestrongly considered when evaluating pulmonary lesions. In a largeretrospective study of 500 pulmonary granulomas identified inbiopsy cases from seven countries, fungal infections accounted for38/200 (19%) of the lesions in the U.S., while only 13/300 (4%) oflesions outside the U.S. were fungal in origin. Additionally, in thisstudy, although fungi were most often detected by histology alone,concomitant culture was often positive in cases of coccidioido-mycosis, aspergillosis and cryptococcosis. Cultures were notcommonly positive in cases of histoplasmosis diagnosed by biop-sy; these were mainly resected necrotizing granulomas, wherecultures are well known to be negative. However, microbiologiccultures continue to play a large role in the diagnosis of pulmonaryfungal infections overall.8

Fungal infections usually lead to necrotizing granulomatousinflammation; however, the pathologist needs to be cognizant ofthe patient's clinical history, as some fungal infections of the lungmay present with non-necrotizing granulomatous inflammation,cellular interstitial pneumonia, intra-alveolar frothy material oralmost normal lung parenchyma. Tissue reaction to the fungus, inpart, also depends on the immune status of the patient and isoften blunted in the immunocompromised host.

Fungal organisms can cause significant granulomatous lungdisease. Localized disease is typical in the patient with normalimmunity, where fungal organisms generally produce nodulargranulomatous lung lesions, characteristically with necrosis. Dif-ferential diagnostic considerations in the setting of necrotizinggranulomatous inflammation in the lung most often include in-fections produced by Histoplasma capsulatum, Cryptococcus neo-formans, Coccidioides spp., Blastomyces dermatitidis, and Sporothrix


schenckii. In a case series undertaken in the Northeast of the U.S. ofcore needle lung biopsy findings of benign lung masses or nodules,Histoplasma was the most common organism identified on tissuesections (10/15 organisms identified in total).9 The most commonspecific benign diagnosis in this series was necrotizing granulo-matous inflammation (45/159 biopsies, 28%), with organisms seenin one-third of the biopsies with necrotizing granulomas (15/45).Diffuse and/or disseminated fungal pneumonias more commonlyoccur in patients with compromised immunity and may be life-threatening. Granulomatous inflammation can be recognized oncytologic examination by epithelioid histiocytes forming looseclusters. Intact granulomas might also be appreciated on BALspecimens.

Although microbiologic cultures and/or molecular assays aretraditionally considered to be the gold standard for identificationof microorganisms, sometimes the histopathologic features offungi are sufficiently distinctive to permit a definitive diagnosis.For instance, Blastomyces encountered in surgical specimens/cy-topathology can usually be reliably diagnosed by morphologicalexamination allowing for prompt treatment.10 Moreover, someorganisms are only seen in tissues while cultures are negative.11 Inaddition, Pneumocystis jirovecii cannot be grown in culture and canonly be identified morphologically in tissue, by PCR or by smear.

Patients following lung transplantation are especially prone toinfection given the close interface of the lung with airborne mi-croorganisms traveling to human tissues. This, combined withimmunosuppressive therapy of patients with allografts, makesthese patients especially vulnerable to fungal infections. However,fungal infections also can involve the native lung in single lungtransplantation cases, especially in patients with chronic ob-structive pulmonary disease.

The histopathologic characteristics of the most common fungalinfections of the lung are summarized in Table 2.

Pneumocystis jiroveciiOriginally thought to be a parasite, Pneumocystis jirovecii is

currently classified taxonomically as a fungus. Pneumocystis jir-ovecii is pathogenic in immunocompromised hosts such as trans-plant recipients, HIV-infected patients and patients with hema-tologic malignancies. Fortunately, currently, this disease appears tobe less common due to frequent use of prophylaxis. Similarly,patients who have undergone lung transplantation have an in-creased susceptibility to P. jirovecii infection, but prophylaxis withtrimethoprim-sulfamethoxazole is effective in preventing theinfection.

The natural reservoir of Pneumocystis is currently not known.Imaging studies often show symmetric, bilateral, patchy or

diffuse pulmonary infiltrates. However, atypical imaging findingsmight occur such as pulmonary nodules, cavitary lesions or air-space consolidation or reticulation.12

Morphologic features of P. jirovecii infection can be highly vari-able. The classic finding is the frothy intra-alveolar pink material(Fig. 1) in which the microorganisms usually will be identified usinga GMS stain. However, the frothy material might be focal or notsampled for instance in small biopsies or can be absent altogetherespecially in patients with moderate immunocompromise. Other,less common morphologic manifestations of Pneumocystis infectioninclude cellular interstitial pneumonia (interstitial thickening due tochronic inflammatory cells), organizing pneumonia and diffuse al-veolar damage. Granulomas are rare in Pneumocystis infection, buthave been well described (Figs. 2–4). In some cases of Pneumocystispneumonia, the lung parenchyma appears normal, requiring a highlevel of suspicion for Pneumocystis infection, especially in im-munocompromised hosts. Therefore, GMS should be routinelyperformed in all cases with morphologic findings of an acute lunginjury pattern, especially in specimens from immunocompromised

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Table 2Distinguishing histopathologic characteristics of common fungal infections of the lung.

Characteristic Pneumocystis jirovecii Aspergillus spp. Cryptococcusneoformans

Histoplasmacapsulatum

Coccidioides spp. Blastomyces dermatitidis Mucormycosis

Size in μm 4–6 N/A 2–10 2–4 20–200 (spherules) 8–20 5–25Budding of yeasts No N/A Narrow-based Narrow-based No Broad-based N/APattern ofinflammation

� Frothy intraalveolar material� Various patterns, including

cellular interstitial pneumo-nia, diffuse alveolar damage,or granulomas

� Inflammation may be absent

� Aspergilloma� ABPA with bronchiectasis, mucus

impaction, allergic mucin, Charcot-Leyden crystals

� Invasive Aspergillus (tissue and vas-cular invasion)

Necrotizinggranulomas

Necrotizing ornon-necrotizinggranulomas

Necrotizing granulomas Necrotizing or non-ne-crotizing granulomas

Invasive, especially inblood vessels. Vascularthrombosis and infarctsGranulomatous vasculitis

Immuno-compro-mised host

Yes No/Yes Yes/No No/Yes No/Yes No/Yes Yes

Route of infection Unknown Inhalation of conidia Inhalation of smallyeast cells

Inhalation ofconidia

Inhalation ofarthrospores

Inhalation of conidia Inhalation of sporesPercutaneous routesIngestion

Histopathology offungus

Oval, central dot Hyphae with parallel walls, regularlyspaced septations and dichotomously45° branching þ/- Calcium oxalatecrystals þ/- Conidial heads

Round, clear haloon H&E, variablysized

Oval, uniform Large thick-walledspherules which may ormay not containendospores

Round, uniform, thickwalled

Hyphae with thin walls,often twisted and folded

þ/- Splendore-Hoeppli phenomenonIrregular branching, 90°branching occurs.Septation may beinconspicuous

Mucin Negative Negative Positively stainingcapsule

Negative Negative Occasionally the outsiderim of yeast forms maystain faintly positive bymucin

Negative

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Fig. 1. Classic Pneumocystis jiroveci A. At low power the surgical lung biopsy shows an alveolar filling process. B. The alveoli are filled with pink material. C. On highmagnification the frothy pink intraalveolar material is classic for Pneumocystis jirovecii infection. The inset shows typical oval, crescent and helmet-shaped, non-buddingPneumocystis organisms. Magnification �20 (A), 100 (B), �600 (C, C inset). (For interpretation of the references to color in this figure legend, the reader is referred to theweb version of this article.)

Fig. 2. Pneumocystis jirovecii infection associated with necrotizing granulomas. A. At low magnification, there is a nodular structure within lung parenchyma. B. At highermagnification pink frothy material similar to the material seen in Fig. 1A-C is surrounded by epithelioid histiocytes and chronic inflammatory cells to form a necrotizinggranuloma (C). D. Many non-budding small organisms, some of which have intracystic bodies consistent with Pneumocystis jirovecii. Magnification �20 (A), 100 (B), �600(C), �1000 oil immersion (D). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


Please cite this article as: Roden AC, Schuetz AN. (2017), http://dx.doi.org/10.1053/j.semdp.2017.06.002i




Fig. 3. Pneumocystis jirovecii infection associated with granulomatous reaction. A. In this low power view geographic necrosis is seen, which is comprised of frothy pinkmaterial surrounded by an extensive lymphohistiocytic reaction (B). Occasional Pneumocystis organisms are identified on GMS stain (C). Magnification �20 (A), 100 (B),�600 (C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. Pneumocystis jirovecii infection associated with necrotizing granulomas. A. This case shows an ill-defined lesion in the lung parenchyma. B. On higher power the lesionis comprised of a necrotizing granuloma with necrosis as seen in other infections. Scattered small non-budding organisms are present on GMS (C). Magnification �40 (A),�200 (B), �400 (C).


patients. Sometimes, frothy material might be seen on cytologysamples, especially on Wright Giemsa staining.

Pneumocystis organisms are characteristically oval, cup-, cres-cent- or helmet-shaped. Often, a dot is identified in the center of


an organism (intracytoplasmic). Importantly, budding is notidentified. The size of the organisms ranges from 4 to 6 mm.

Pneumocystis jirovecii cannot be cultured as it will not growunder culture conditions. Therefore, histologic or cytologic

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Fig. 5. Aspergilloma. A. A transbronchial biopsy shows detached clusters of fungal organisms containing yellow conidial heads (B), morphologically consistent with As-pergillus niger. C. In areas of pink material, clusters of hyphae with parallel walls and narrow angle branching hyphae are identified, consistent with a fungus ball. Mag-nification �4 (A), �600 (B, C). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


findings can be critical for confirming the diagnosis, and carefulinspection of the GMS stain is imperative. Furthermore, if a GMSstain is negative but suspicion of Pneumocystis infection is high,PCR can be performed on BAL fluid (this is routinely done for lungtransplant recipients in some centers). Alternatively, other mi-crobiologic testing such as Pneumocystis smear or beta-D-glucantesting of BAL can aid in the diagnosis.

In immunocompromised hosts without Pneumocystis prophy-laxis, co-infection with CMV is not uncommon, requiring a carefulsearch for viral inclusions if Pneumocystis is identified.

The histopathologic differential diagnosis of P. jirovecii includesHistoplasma capsulatum and Coccidioides spp. While H. capsulatumorganisms are small (2–4 mm) and oval, they also show narrow-based budding and lack the central dot and grooving of Pneumo-cystis. Coccidioides spp., in general, are not difficult to distinguishfrom Pneumocystis given the large thick-walled spherules andendospores. However, rarely, in small biopsies, spherules mightnot be sampled and only endospores may be present. Similar toPneumocystis, Coccidioides organisms do not bud. However, en-dospores might have some size variation. In the absence of thediagnostic large spherules, a definitive diagnosis might not bepossible. Small biopsies might be reported as follows:

“A GMS stain reveals small oval fungal organisms. Budding isnot identified. See comment.

Comment: Although budding is not identified in this smallspecimen, this might represent Histoplasma capsulatum. How-ever, Coccidioides spp. cannot be completely excluded eventhough spherules are not appreciated. Correlation with tissueculture, clinical history and serology would be recommended.”

AspergillusAspergillus infection (a.k.a., aspergillosis) is another important

infection of immunocompromised hosts. Aspergillus is a ubiquitousdimorphic soil fungus. Infection occurs through the inhalation of


spores. Aspergillus infection in lung transplant patients is mostcommonly characterized by local invasion of necrotic bronchialanastomoses (i.e., ulcerative tracheobronchitis), which typicallyoccurs within 4 months of transplantation.

In the lung, aspergillosis can occur in three different patternsincluding (i) colonization (aspergilloma), (ii) hypersensitivity re-action (ABPA, eosinophilic pneumonia, mucoid impaction, hy-persensitivity pneumonitis), and (iii) invasive.

An aspergilloma occurs when Aspergillus organisms colonizepreformed cavities including those caused by healed tuberculosis,emphysema, sarcoidosis, bronchiectasis, infarcts, and cavitatingtumors such as squamous cell carcinoma, among others. In thesecavities, Aspergillus usually forms a saprophytic, non-invasivefungus ball (e.g., mycetoma) that is comprised of mycelial ele-ments, fibrin, mucus and debris (Figs. 5 and 6). Clinically, patientswith aspergilloma might be asymptomatic and the lesion may befound incidentally; they can also present with hemoptysis, feverand weight loss. An aspergilloma might also present as a masslesion or nodule that is clinically suspicious for a neoplasm andonly resection will reveal the nature of this lesion. Sometimes,imaging studies show a ball-like structure within a cavity. The“ball” will roll around if the position of the patient is changed.

ABPA is a hypersensitivity reaction to fungal antigens, particu-larly to Aspergillus fumigatus antigens, and is not an infection. Di-agnostic criteria include history of asthma, pulmonary infiltrates,central bronchiectasis, eosinophilia, increased IgE serology, elevatedspecific IgE and IgG, a sputum culture that is positive for Aspergillusand a positive immediate skin test.13 On gross examination,bronchiectasis with mucoid impaction of bronchi is commonlyseen. On microscopy, intrabronchial and intrabronchiolar allergicmucin becomes apparent. Allergic mucin is typically layered andcontains eosinophilic cell debris (primarily derived from eosino-phils) with brightly eosinophilic Charcot-Leyden crystals (Fig. 7).Findings of eosinophilic pneumonia might also occur. Granulomasmight be identified in the vicinity of the airways. Although this is an

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Fig. 6. Aspergilloma. A. Lung parenchyma with bronchiectasis and associated fibrosis. B. Within bronchiectatic airways there is crystalline material containing degeneratedfungal elements, consistent with a fungus ball. C. GMS confirms hyphae with parallel walls and narrow angle branching consistent with Aspergillus spp. While the culture attime of biopsy was negative, a previous culture had revealed Aspergillus fumigatus. Magnification �12.5 (A), �200 (B), �600 (B insert, C).

Fig. 7. Allergic mucin. A. Low power view shows layered pink material which contains eosinophils, eosinophilic debris and Charcot-Leyden crystals (B, arrow). Magnification�12.5 (A), �400 (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


allergic reaction, usually to Aspergillus, Aspergillus hyphae are oftennot seen even on GMS stains. Sometimes, fragments of fungal hy-phae might be noted, particularly within allergic mucin. Again, ahigh level of suspicion is necessary to reach such a diagnosis andallergic mucin should always raise suspicion for ABPA.

Invasive aspergillosis is often a life-threatening disease and needsto be recognized on pathology. Invasive aspergillosis occurs largelyonly in the immunocompromised host and might lead to pulmonaryinfarction and hemorrhage. It is characterized by tissue and/or vascularinvasion (Fig. 8). Acute invasive aspergillosis (or any other invasivefungus) can present as a “target lesion”. Microscopically, this is anodular pulmonary infarct associated with vascular invasion by fungalhyphae. On gross examination, these lesions usually have a centralpale necrotic zone surrounded by a hemorrhagic rim. They are oftenmultiple and are usually peripherally located. Microscopically, fungalhyphae occlude the lumen of the pulmonary artery that is associatedwith the infarcted area. Miliary microabscesses might occur in a smallpercentage of cases. Aspergillosis can also lead to chronic necrotizing


granulomatous inflammation, which is a “semi-invasive” form of as-pergillosis. Fungal hyphae are usually identified within the areas oftissue necrosis and parenchymal invasion is apparent, but vascularinvasion is not a feature.

The most important Aspergillus species in the lung are Asper-gillus fumigatus, A. niger and A. flavus. Aspergillus organisms arecharacterized by hyphae that have parallel walls, regularly spacedseptations and dichotomously branching hyphae that branch atnarrow angles (45°). Birefringent calcium oxalate crystals can beassociated with Aspergillus spp., especially Aspergillus niger. Con-idial heads (fruiting heads, fruiting bodies) might occur in lesionsin which fungal organisms are exposed to air, such as cavities as-sociated with aspergilloma (Fig. 5). In chronic necrotizing asper-gillosis (granulomatous lesions) the hyphae may be surrounded bythe Splendore-Hoeppli phenomenon, characterized by radiatingeosinophilic material.

On histologic examination, Aspergillus organisms cannot bereliably differentiated from other fungi with similar morphology

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Fig. 8. Invasive aspergillosis. A, B. Lung biopsy reveals a nodular process. C. The nodule is mainly comprised of necrotic debris. D. Within the necrosis there are hyphae withparallel walls and narrow branching (arrows) consistent with Aspergillus spp. Magnification, �12.5 (A), �200 (B), �400 (C), �600 (D).


such as Scedosporium (Pseudallescheria) spp. and Fusarium, unlessthe typical conidial heads of Aspergillus are seen.14 Therefore, de-finitive confirmation of Aspergillus should usually be based onmicrobiologic cultures. Provision of an inclusive differential diag-nosis in specimens with evidence of septate hyphal elements isparticularly important in the immunocompromised population,since these patients are prone to infections with Fusarium andother multidrug-resistant organisms, and because these emergingorganisms often require antifungal therapy which differs from orextends beyond typical antifungal therapy. It is also important torecognize that dematiaceous fungi (brown or black molds whichcontain melanin in their cell walls) can appear histologically si-milar to Aspergillus spp. Helpful clues to their histologic identifi-cation are that they are often pleomorphic and contain brownpigment on H&E-stained slides due to melanin production. Fur-thermore, the hyphae of Aspergillus may appear distorted andbroad if the patient has received antifungal therapy which wouldbe considered active against Aspergillus. Here is an example howthe finding of hyphae might be reported:

"Non-pigmented narrow branching hyphae present (hyalohy-phomycosis). See comment. Comment: The differential diag-nosis includes Aspergillus spp, Fusarium, and Pseudoallescheriaspp. Correlation with clinical presentation and culture in-formation is recommended."

"Pigmented hyphae present (dematiaceous fungus). See com-ment. Comment: Definite species identification through cultureis recommended."

Cryptococcus neoformansCryptococcus neoformans is encountered worldwide. Crypto-

coccosis is acquired by inhalation of small yeast cells. It is an op-portunistic fungus. Pulmonary Cryptococcus neoformans infectionis almost always seen in an immunocompromised host. Althoughthe infection is well reported in immunocompetent individuals,the consequences of such infection are generally less serious. The


reservoir of the fungus is the bird (particularly the pigeon) and thesoil. Besides pneumonia, these patients may also present withmeningitis. Cryptococcus may disseminate.

Histologically, Cryptococcus is usually associated with non-ne-crotizing or necrotizing granulomas, especially in the im-munocompetent host (Fig. 9). In immunocompromised patientsthe organism might proliferate unhindered by granulomas. Theorganisms vary in size (2–20 mm) within a single lesion, shownarrow-based budding and typically possess a mucin capsulewhich is often seen on H&E. A large number of giant cells – somecontaining organisms – may be appreciated. A mucicarmine staincan be helpful in highlighting the mucin-rich capsule. Sometimes,mucicarmine is negative because the capsule is absent (so-calledcapsule-deficient forms), but organisms can still be recognized byhistology by the characteristic combination of light staining, sizevariation and narrow-based budding. Occasionally, histologicallyconfirmed cases are culture-negative.8,15 The major differentialdiagnosis is Histoplasma and Blastomyces spp. The presence ofnarrow-necked budding differentiates Cryptococcus from Blas-tomyces dermatitidis. The variability of the size of Cryptococcus is aconsistent finding and can aid in differentiation from Histoplasmaand Blastomyces which are uniform in size. Mucicarmine staining,when present, also helps to exclude these organisms.

Histoplasma capsulatumHistoplasma capsulatum can lead to an infection in im-

munocompetent hosts. In the United States, histoplasmosis is en-demic in the Mississippi and Ohio River valleys and focally in manyMideastern states. However, Histoplasma exists worldwide; there-fore travel history is important.16 Histoplasma capsulatum is a di-morphic soil fungus. Sources are bird and bat droppings andtherefore caves are common places of infection. In fact a history ofcave spelunking is suggestive of the possibility of histoplasmosis.Conidia are usually inhaled. 80% of infected patients are asympto-matic or present with flu-like symptoms. The presentation of theimmunocompetent host is usually subacute and characterized by

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Fig. 9. Cryptococcus neoformans infection. A. A necrotizing granuloma is comprised of a large zone of central necrosis rimmed by epithelioid histiocytes, multinucleated giantcells and chronic inflammatory cells (B). C. On H&E, within the necrosis, there are clusters of fungal organisms that have pale-staining walls. The organisms are alsohighlighted on GMS stain (D). They vary in size, show narrow-based budding (arrows) and have a thick mucinous wall as highlighted by a mucicarmine stain (D inset),characteristic of Cryptococcus neoformans. Magnification �20 (A), �200 (B), �600 (C, D, D inset).


non-specific symptoms. The disease might be focal or diffuse and isoften characterized by a nodular infiltrate of the lung althoughdisseminated disease can occur. Often, granulomas associated withHistoplasma are incidentally found in lymph nodes for lung cancerstaging. The clinical manifestations of histoplasmosis are varied andinclude primary histoplasmosis (acute pulmonary histoplasmosis),dissemination (disseminated histoplasmosis), chronic cavitary his-toplasmosis, broncholithiasis, fibrosing mediastinitis, and hyaliniz-ing pulmonary nodule. “Histoplasmomas,” or solitary Histoplasmanodules, can mimic lung cancer because of increasing size over timeand the presence of cavitation, spiculation, lack of calcification andhigh uptake on positron emission tomography (PET).17,18 The diag-nosis might be established either by GMS stain in tissue sections,culture and/or serology. In cases of solitary pulmonary histo-plasmoma, serology is negative in a high proportion of cases.11 Aretrospective case review of surgically-resected pulmonary necro-tizing granulomatous lesions for which initial histologic examina-tion was unexplained revealed upon re-examination of histology inconcert with ancillary microbiologic and non-microbiologic testing,that the majority of initially overlooked organisms wereHistoplasma.11 Furthermore, culture for Histoplasma was generallynegative, particularly for histoplasmomas.

H. capsulatum var. capsulatum and H. capsulatum var. duboisiiare the only two forms that are pathogenic to humans. The yeastform is commonly identified inside (in disseminated histo-plasmosis) and outside of macrophages, the latter being a commonfinding in pulmonary histoplasmomas. The yeast is 2–4 mm, oval,uniform in size, and shows narrow-based budding (Fig. 10).

In the lungs, acute histoplasmosis is characterized by lymphohis-tiocytic infiltrates within airspaces and the interstitium along withlarge areas of necrosis and/or small necrotizing granulomas.19 Chronicpulmonary histoplasmosis is also characterized by necrotizing granu-lomas, although granulomas often are hyalinized and may even becalcified; the pathology of this clinically defined form of the disease


has not been described in detail in the literature. In “histoplasmomas”,organisms are usually non-viable and cultures are virtually alwaysnegative.8,11,20 For this reason, histology is the only method by whichorganisms might be identified.8,11,17,21 Classically, the yeasts of Histo-plasma are not well visualized on H&E-stained sections within ne-crotizing granulomas in localized pulmonary disease, but they aretypically readily identifiable within histiocytes on H&E in cases ofdisseminated histoplasmosis.22,23 A thorough search with a GMS stainshould be performed within the areas of necrosis for Histoplasma, asthese organisms can occasionally be pale-staining, few in number, anddifficult to visualize.

The differential diagnosis of Histoplasma includes Pneumocystisjirovecii, endospores of Coccidioides spp., Leishmania (which con-tains kinetoplasts which may be very difficult to appreciate onH&E) and Candida glabrata. However, the clinical manifestations ofthe latter two are different from that of histoplasmosis. The his-tologic reaction is also a key differentiating feature in lung speci-mens, since the latter organisms have not been reported in ne-crotizing granulomas of the lung.

Coccidioides spp

Coccidioides spp. is also a dimorphic soil fungus. This organismis endemic to the Southwest of the United States, the north ofMexico and some areas in Brazil and Argentina. Arthrospores areinhaled. 60% of patients infected are asymptomatic. Patients mightpresent with fever, chills, malaise, cough, dyspnea, arthralgias,rash and persistent/progressive pneumonia. Solitary pulmonarynodules or thin walled cavities might be identified on imagingstudies. “Coccidioidomas” (localized residual coccidioidal disease)are often discovered incidentally and can be PET-positive in ser-onegative patients.24 In the immunocompromised host, Cocci-dioides may disseminate. The organism can be identified either onhistology, culture and/or serology.

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Fig. 10. Histoplasma capsulatum infection. A. A necrotizing granuloma with a large area of central necrosis shows vague layering and is rimmed by hyalinized fibrosis andchronic inflammation (B). C. Within the necrosis there are clusters of small oval fungal yeasts, some of which show narrow-based budding (arrows). The morphology ischaracteristic of Histoplasma capsulatum. Magnification �12.5 (A), �100 (B), �600 (C).


On microscopic examination the thick-walled spherules ofCoccidioides are typically seen on H&E stains (Fig. 11).23 However, alarge case series of pleural coccidioidomycosis (an uncommonclinical presentation of coccidioidomycosis) in an endemic regionin Arizona revealed that typical spherules of Coccidioides in thisform of disease is uncommon.25 Spherules are large (20–200 mm),and some are filled with endospores which can be highlighted ona GMS stain. Eventually, endospores will be expelled leaving be-hind an empty spherule and many small endospores. An eosino-philic tissue reaction may be seen but is not invariably present.These endospores do not show budding which helps to distinguishthem from Histoplasma. In contrast to “histoplasmomas”, organ-isms often remain viable within “coccidioidomas”, and cultures oflesional tissue may be positive.26

Blastomyces dermatitidisBlastomyces is a dimorphic soil fungus that grows in warm,

moist soil of wooded areas rich in organic debris. Aerosolizedspores are inhaled and can lead to pulmonary disease. Beaverdams and rivers are possible sources of the organism. Patientsinhale conidia. Endemic areas include states adjacent to the Mis-sissippi and Ohio River basin in the South, Southeast, and North-central states in the U.S. and Canadian territories adjacent to theGreat Lakes. Additionally, a small area in New York State and Ca-nada along the St. Lawrence River and the Chicago area are con-sidered endemic.

Patients may be asymptomatic and the disease might be self-limited, although this is less likely as compared to other fungi.However, symptomatic pneumonia and progressive respiratoryfailure, such as acute respiratory distress syndrome (ARDS) canoccur. The disease may involve skin, bone, epididymis, testes andprostate in humans. Dogs also carry disease and may show pul-monary symptoms. The diagnosis can be made on histology, cul-ture and/or serology. The organism might be also identified in thesputum of the patient.


In the immunocompetent host, Blastomyces pulmonary infec-tion is characterized by necrotizing granulomas, which are histo-logically distinctive in that their necrotic centers are almost in-variably suppurative, containing numerous neutrophils (Fig. 12).Blastomyces yeasts are usually identified within the suppurativecenter and the granulomatous rim. The organisms have thick, re-fractile cell walls, are 8–20 mm in diameter and show broad-basedbudding. The nuclei of Blastomyces may be apparent on H&E assmall dots within the yeast cells. The daughter cell approaches thesize of the mother cell before detachment.

The differential diagnosis includes Cryptococcus neoformans.However, the latter shows variation in size of the yeast and nar-row-based budding. Often, a mucin capsule is identified in Cryp-tococcus which is not a feature of Blastomyces. Culture continues toplay an important role in the diagnosis of blastomycosis. A retro-spective study over 9 years of microbiologic culture-confirmedcases of blastomycosis at a Chicago medical center showed that 4/35 (11%) cases for which tissue was submitted to surgical pathol-ogy were negative on tissue stains for blastomycosis.10

Candida spp

Pulmonary infections due to Candida are rare, and growth ofCandida from the respiratory tract almost always indicates coloni-zation rather than infection. A definitive diagnosis of Candida pneu-monia requires histopathologic evidence of invasive disease. Candidais part of the normal flora of the mouth, skin and gastrointestinaltract. In these areas it is the most common fungus to cause infection(thrush). It is commonly found in warm and moist surfaces. It mightmanifest as pneumonia in the immunocompromised host especiallyif neutropenia is present. It can disseminate and also infect the kid-ney and lead to endocarditis. In HIV-infected patients chronic mu-cocutaneous candidiasis might occur.

Candidiasis in the lung is exceedingly rare and may be asso-ciated with vascular invasion culminating in infarct and

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Fig. 11. Coccidioides spp. infection. A. A necrotizing granuloma is rimmed by epithelioid histiocytes (B). C. High magnification reveals multiple large spherules (H&E stain). D.GMS stain shows scattered large and small microorganisms within the necrosis. E. These microorganisms are characterized by large spherules, some of which containendospores (GMS stain). Within the necrosis there are also smaller microorganisms which are expelled endospores. They do not show budding. F. This spherule has rupturedand is releasing endospores. These morphologic features are characteristic of Coccidioides spp. Magnification �12.5 (A), �200 (B), �600 (C, E, F), �100 (D).


hemorrhage. Factors that predispose patients to Candida pneu-monia include hematogenous spread from non-pulmonary sour-ces, such as an indwelling intravenous catheter, extensive ab-dominal surgery, intravenous drug abuse, or total parenteral nu-trition. Other risk factors include neutropenia, extensive steroiduse, intensive antibiotic therapy, diabetes mellitus, colonizationwith Candida at multiple sites, renal insufficiency, primary or as-piration pneumonia and congenital pneumonias.12

Non-pulmonary candidiasis is a common infection in im-munocompromised patients.27 Lung involvement may manifest aseither endobronchial growth or multiple abscesses containingyeast forms and pseudohyphae centered in small vessels and air-ways. Candida infection can also occur as septic thrombi in largearteries, a pattern occasionally mimicking Aspergillus, or as gran-ulomatous infection that mimics tuberculosis.

Candida spp. are oval yeasts that measure 2–6 mm and aresometimes accompanied by mycelial forms with pseudohyphaeand occasionally true hyphae. The pseudohyphae of Candida maybe difficult to distinguish from Aspergillus on GMS stains of tissuesections. In this setting, microbiological culture is required for aspecific diagnosis. In contrast to true hyphae, pseudohyphae are


elongated and are pinched at the point of attachment. They haveconstrictions at the attachments between adjacent cells. The tissueresponse depends on the route of infection and the immune statusof the patient. Primary Candida pneumonia is characterized his-tologically by bronchopneumonia, hemorrhage, and necrosis. He-matogenous dissemination might result in bilateral, symmetrical,miliary, circumscribed, centrally necrotic nodules that are he-morrhagic at the periphery. These lesions consist of colonies oforganisms with associated abscesses oriented around blood ves-sels. Mycotic emboli may cause large hemorrhagic infarcts.

The differential diagnosis of Candida includes Aspergillus whichis characterized by true hyphae and acute or 45° angle branching.Trichosporon also needs to be distinguished which also consists ofyeast-like cells and hyphal elements. However the average dia-meter of Trichosporon is 5 mm rather than 2.5 mm and the histo-pathologic appearance is more pleomorphic.

Mucorales

Mucormycosis (previously known as zygomycosis) is less commonthan aspergillosis and occurs almost exclusively in either diabetic or

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Fig. 12. Blastomyces dermatitidis infection. A. At low magnification, the lung contains a multinodular lesion. B. At high magnification, there are ill-defined necrotizinggranulomas with central necrosis composed of neutrophils and neutrophilic debris (C), consistent with suppurative granulomas. D. On H&E, high magnification revealsrelatively large fungal microorganisms (arrows), some of which show broad-based budding. E, F. These microorganisms are highlighted by a GMS stain and are characteristicof Blastomyces dermatitidis. Magnification �20 (A), �200 (B), �400 (C), �600 (D-F).


immunocompromised patients. Like Aspergillus in the im-munocompromised host, infectionwith Mucorales oragnisms can leadto septic thrombi with infarction. Mucormycosis is often a fatal in-fection. Mucorales that are linked to humans most commonly includeRhizopus spp., Mucor, Rhizomucor, Absidia, and Apophysomyces. Thespores are inhaled or reach the lungs via percutaneous routes, or byingestion. Pulmonary infections are most commonly caused by Rhi-zopus spp. There is a strong predilection for vascular invasion anddissemination in immunocompromised patients.

Mucorales are characterized by broad hyphae that rangefrom 5 to 25 mm and have thin walls (Fig. 13). They are oftentwisted and folded, branching is irregular, and 90° branchingoccurs. While septation is usually not seen it may be incon-spicuous. Genus identification of Mucorales cannot be per-formed on histology alone and requires culture. Sometimes,the GMS stain might not stain (or only faintly stains) Mucor-ales; in such cases, the organisms might be best seen on H&E.Pulmonary mucormycosis is characterized by invasive growth,especially in blood vessels. This is typically associated withvascular thrombosis and infarcts, and granulomatous vasculitismight be apparent.


The differential diagnosis includes Aspergillus, which usuallycan be distinguished by its parallel walls, thinner appearance, 45°branching and clear-cut septation.

Sporothrix schenckii complexSporotrichosis (a.k.a. “rose gardener's disease”) is a rare in-

fection caused by Sporothrix. This fungus lives throughout theworld in soil and on plant matter such as sphagnum moss, rosebushes, and hay. Sporotrichosis is obtained by coming in contactwith the fungal spores in the environment. Cutaneous (skin)infection is the most common form of the infection. Some casesof sporotrichosis have been associated with scratches or bitesfrom animals, particularly cats. Sporotrichosis is very rare in thelung but it can occur following inhalation of fungal spores fromthe environment. Sporotrichosis can disseminate to bones,joints, or the central nervous system, usually in the im-munocompromised host.

The yeast cells might be difficult to identify in tissue as theyare sparse and do not stain well on H&E. They are round, oval orcigar-shaped, and measure 2–6 mm. Buds appear elongated or“teardrop” in shape and show narrow-based attachments with

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Fig. 13. Mucorales infection. A. This lung shows a well demarcated infarct (left) caused by occlusion of a pulmonary artery by invasive fungus (arrow, B). The hyphae haveirregular walls and are barely visible on GMS (C). Cultures were positive for Absidia. Magnification �12.5 (A), �600 (B, C).


a pipestem appearance. The organisms may be surrounded byan eosinophilic Splendore-Hoeppli phenomenon.

Summary

Although not very common, fungal infections of the lung areimportant to recognize as they can lead to life-threatening situa-tions especially in the immunocompromised host. While manyfungal organisms can be identified on tissue sections using H&E,GMS and/or PAS stains, several have overlapping features on his-topathology and require cultures or other ancillary diagnosticmethods for definitive diagnosis. Pathologists should gather asmuch information as possible regarding the immune status, travelhistory and social history of the patient to aid in the diagnosis.Importantly, the pathologist needs to relay results to clinicians assoon as possible as patients may require immediate antifungaltreatment, especially if they are immunocompromised.

Funding

No disclosures.

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