Infectious Disease Pathology
Case 5 -
Jeannette Guarner, Emory University, Atlanta, GA
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54 year old woman that is being worked up for a lung mass that was seen in a chest X-Ray.
Coccidioides immitis is endemic in the San Joaquin valley in California. Coccidioides posadasii is
present in desertic regions of Northwest Mexico, Arizona and New Mexico, and desertic areas in Argentina
and Paraguay. However, very little differences have been found in morphology or clinical presentation
between the 2 species. There is a clear correlation between incidence of disease and environmental
factors; coccidioidomycosis increases when there are rainy summers followed by dry winters, following
earthquakes, or when humans settle and develop the previously mentioned desertic areas. In any of these
instances, Coccidioides arthrospores are released in higher numbers than the usual baseline.
Humans inhale the arthroconidia, which in the lung are transformed into multinucleated spherical
structures that contain hundreds of endospores. It is estimated that up to 60% of individuals exposed
have no symptoms while the remainder may present with what appears to be an acute community acquired
pneumonia. In those patients with acute pneumonia, the chest X rays show lobar infiltrates and
adenopathy. Several erythematous rashes (multiforme, nodosum or toxic) are reflections of immune
response to the acute infection. The majority of acute pulmonary infections resolve; however, in a
minority of patients the infection may become chronic progressive ("Valley fever") showing either a
cavity or nodule. Extrapulmonary disease can occur in certain risk groups including African Americans,
Asians, pregnant women, patients with diabetes, or those patients receiving corticosteroids for a variety
of conditions. The most common sites of dissemination include skin, lymph nodes, bones, and joints;
nonetheless the most feared complication is extension to the central nervous system. Besides acquiring
infection through the respiratory route, there are rare reports of direct inoculation of skin giving rise
to primary cutaneous lesions or acquisition through transplanted organs.
Pathological/Microscopic Findings and any Immunohistochemical or Other Studies:
The spherules of various sizes (10 to 100 microns) with multiple endospores (2 to 5 microns) are
characteristic of coccidioidomycosis and can be seen with routine H&E stain. The walls of some of
the spherules may appear ruptured and the endospores spill into surrounding tissues. Active lesions
contain multiple organisms while resolving or residual lesions usually show lower number of organisms.
GMS highlights spherule walls and endospores. In contrast, reactions with PAS vary with age of the
structures: young endospores and spherules stain strongly while staining fades as the organisms mature.
Mycelia can be observed in cavitary lung or skin lesions. Sensitivity for histopathologic detection of
Coccidioides is 84% and 75% for cytology.
Inflammatory reaction to endospores is predominantly neutrophilic while reaction to spherules is
granulomatous. Thus, early in the infection the lesions tend to show pyogranulomas because the
concentration of spherules and endospores is high. Lymphocytic clusters of B and T cells have been
described around well formed granulomas with necrosis and appear to be an important response to
coccidioidomycosis. Eosinophils can be abundant and can create the Splendore-Höeppli phenomenon (an
intense rim of eosinophilic material around the fungal elements). When coccidioidomycosis is suspected
it is important to look for spherules. This should be done by going deeper into the blocks or
submitting/ staining other blocks. It also needs to be remembered that in immunosuppressed patients,
more than one infection may coexist, thus in endemic areas Pneumocysitis and Coccidioides could be found
in the same specimen.
In the United States, Coccidioides spp. are select agents that are governed by specific rules related
to the possession, use, and transfer of these agents. This fungus grows easily in the laboratory (93%
sensitivity) and the arthroconidia can be easily aerosolized, thus all work with cultures must be
performed in a class II biological safety cabinet. Detection of antibodies to Coccidioides can be an
important diagnostic tool. Today, IgM and IgG are generally measured using EIA and/or immunodiffusion;
however, some laboratories continue to perform tube precipitin to measure IgM and complement fixation to
measure IgG antibodies. False negative serology has been seen in up to 38% of patients with hematogenous
infection and 46% of fatal cases. Detection of antigens in the urine using EIA has been shown to be
present in 71% of patients with coccidioidomycosis but shows cross-reaction in 10% of patients with other
Dimorphic fungi are defined as those that appear as yeast forms at 37°C but are molds when cultured at
colder temperatures (usually 25°C). Coccidioides is a dimorphic fungus and, with a few exceptions, most
of the fungi in the differential diagnosis are also dimorphic.
1. Rhinosporidium seeberi, a mesomycetozoan parasite that causes palate and nasopharyngeal polyps, is
in the differential diagnosis of coccidioidomycosis because it produces large sporangia (some can be seen
with the naked eye) with multiple internal endospores. R. seeberi has very similar morphology but its
sporangia and endospores are bigger than Coccidioides spherules, and its inner sporangial wall stains
with mucicarmine. It is important to remember that endospores outside spherules or young spherules
without endospores can be confused with Blastomyces, Histoplasma, Emmonsia, Candida, Pneumocystis and
2. Blastomyces dermatitidis in tissue appear as yeasts that measure 8 to 15 microns in diameter, have
thick refractile cell walls, and may show a single, broad-based bud. The yeasts can be observed in a
variety of specimens including sputum, bronchoalveolar lavage, fine needle aspirates from lung, skin or
other lesions, cerebrospinal fluid and surgical resections. The thick refractile cell wall of this
organism gives the appearance of a space between the fungal cell contents and the surrounding tissue when
hematoxylin and eosin (H&E) stain is used. Inside the cell wall, the multiple nuclei of the yeasts
stain with hematoxylin. Occasionally, B. dermatitidis can show smaller yeast forms, the so called
microforms. In addition, B. dermatitidis can be seen with a variety of routinely used preparations and
stains such as KOH and Papanicolau. The contour of the yeast is best highlighted by staining the cell
wall with fungal silver stains such as GMS or PAS. The inflammatory reaction accompanying the yeasts is
primarily granulomatous with varying degrees of neutrophilic infiltrate, thus it has been described as
pyogranulomatous inflammation. It needs to be remembered that blastomycosis can be concomitantly present
with neoplasias and tuberculosis.
Few studies have systematically compared the presence of broad-based budding yeasts in histopathologic
or cytologic specimens with culture or other diagnostic methods that would confirm the diagnosis of
blastomycosis. A retrospective study of 53 patients showed that Coccidioides immitis, Candida albicans
or Aspergillus were recovered from 4 (10%) pathologic specimens demonstrating broad-based budding yeasts
in direct histopathologic examination. An earlier study of patients with blastomycosis commented that a
high percent of their cultures were overgrown with Candida. This suggests that not all broad
based-budding yeasts in the 8 to 15 micron size range are Blastomyces. Since histopathologic or
cytologic results can usually be provided before the culture is available, there is pressure to use these
results to guide treatment, particularly because B. dermatitidis can take up to 3 weeks to grow or may
not grow at all from these specimens. Sensitivity of culture varies depending on the sample that was
obtained, and may range from 62% to 100%. Diagnostic yield of histopathology will depend on the
expertise present in the center where the patient is seen. Because of the possibility of histopathologic
false positive results, pathologists should describe the yeast and budding pattern that is observed in
the tissue specimen, and should list the yeasts that can have this morphology in the report comment
field. In addition, alternative tests (urine or serum antigen detection or presence of antibodies in
serum) may need to be menitioned in the comment, especially in cases from non-endemic areas or when the
clinical picture is not typical.
3. Histoplasma capsulatum var. capsulatum in tissue is an oval 2 to 4 micron yeast that may show
narrow-based buds. With H&E stain, the basophilic yeast cytoplasm is separated from the surrounding
tissue by a clear zone corresponding to the cell wall. GMS and PAS stains highlight the cell wall.
Because the yeasts are initially ingested by macrophages, they appear to be clustered and some authors
have suggested this is an important diagnostic feature. This clustering within histiocytes and
occasionally within neutrophils is the presentation of Histoplasma in fluids stained with Papanicolaou
stain or blood smears stained with Giemsa. H. capsulatum var. duboisii is larger (8 to 15 microns in
diameter) than var. capsulatum, and has similar morphology but is pigmented.
Few lung tissue samples of cases with acute pulmonary histoplasmosis have been studied and have shown
nodular areas of parenchymal and vascular necrosis associated with lympho-histiocytic vasculitis. The
histopathologic picture resembles lymphomatoid granulomatosis but scattered small granulomas with small
yeasts in the parenchyma should suggest the diagnosis of histoplasmosis. Chronic lung infections that
radiographically appear as coin lesions show typical granulomatous inflammation with central necrosis and
calcified material. Yeasts are usually found in this necrotic calcified material, which can be lost
during processing and cutting of the tissue. In immunosuppressed patients, sheets of macrophages filled
with yeasts characterize disseminated disease. The collections of macrophages distort the organ
architecture and produce necrotic areas. Because the morphology of H. capsulatum is not specific, it is
important to perform clinico-epidemiological correlation.
Several fungi can be confused with H. capsulatum var. capsulatum when studying tissue sections: The
small variant of B. dermatitidis (in these cases the presence of broad-based budding and seeking larger
forms can be helpful in making the diagnosis of blastomycosis), capsule deficient cryptococci (in these
cases size variation and looking for weakly positive mucicarmine-staining yeasts may suggest the
diagnosis of cryptococcosis), endospores of Coccidioides spp. (looking for remnants of a ruptured
spherule or an intact spherule is paramount for diagnosis of coccidioidomycosis), Pneumocystis jirovecii
(these organisms lack budding and have a silver staining dot), Penicillium marneffei (show formation of a
transverse septum rather than budding), lastly, Candida glabrata (may show more size variability than
histoplasmosis). In addition, several protozoa can also show intracellular organisms of similar size
including the agents of leishmaniasis, toxoplasmosis, and Chagas disease, that should be differentiated
from histoplasmosis: The histopathologic difference between these organisms and Histoplasma is that
H&E stains the entire protozoan and none show the halo produced by the fungal cell wall.
Kinetoplasts (a distinct hematoxylin-stained bar to the side of the nucleus) should be observed if the
patient has leishmaniasis or Chagas disease. Infected cells in toxoplasmosis and Chagas disease are
somatic (cardiomyocytes, neurons) rather than histiocytes. In summary, definitive diagnosis of
histoplasmosis can be difficult in tissue sections and, if cultures were not obtained in the tissue
specimen, alternative testing should be considered. Pathologist should describe the yeast in the
diagnosis field and in a comment suggest the different fungi that can have this morphology.
4. Another unusual fungus that is in the histopathologic differential diagnosis of coccidioidomycosis
is Emmonsia crescens. This dimorphic fungus is inhaled or inoculated into the skin when humans are in
close contact with rodent burrows and aerosolized conidia. The amount of conidia inoculated will
determine the presentation: Small inocula are asymptomatic and present as walled off granulomas, while
larger inocula can result in acute severe pulmonary disease. The disease is known as adiaspiromycosis or
haplomycosis, due to the presence in tissue of adiaspores, large thick-walled structures formed by
transformation of hyphae. Adiaspores are thick, double-walled spherules that measure 20 to 400 micron or
more and are empty or contain eosinophilic hyaline globules. When stained with GMS the entire wall
thickness stains and shows fenestrations. Adiaspores usually elicit a granulomatous inflammatory
reaction and the differential diagnosis is frequently with helmiths.
5. In tissues Candida organisms appear as mats of yeasts measuring 3 to 5 microns in diameter
intermingled with pseudohyphae also referred to as filaments. The filaments may show periodic
constrictions. The organisms can be seen with H&E, GMS and PAS. The only Candida species that does
not produce filaments is C. glabrata. Because of its inherent resistance to fluconazole, C. glabrata
needs to be recognized. C. glabrata is the Candida spp. that can be confused with Histoplasma,
Blastomyces, endospores of Coccidioides and Cryptococcus.
6. Atypical inflammatory reactions to Pneumocystis have been documented including interstitial
pulmonary fiborisis, granulomas, hyaline membranes, and interstitial lung infiltrates. In these
instances, it is difficult to suspect that Pneumocystis is present in the lesion. The usual
histopathology of Pneumocystis in lung tissue sections stained with H&E is as foamy intraalveolar
eosinophilic exudates with minimal inflammatory infiltrate. In Papanicolaou stained respiratory cytology
specimens, the organisms blend into the mucous blue green background. GMS stain demonstrates that the
foamy material in tissue sections or cytologic specimens corresponds to multiple organisms which are
thin-walled spheres of 2 to 5 microns that have a large capsular dot. Collapsed organisms are usually
found intermingled with intact organisms.
Fungal infections are becoming more frequent because of expansion of at-risk populations and use of
treatment modalities that permit longer survival of these patients. Some of the changes in endemic
fungal infections can be attributed to climate changes, extension of human habitats, ease of travel, and
shifting populations. At-risk populations for opportunistic fungal infections or disseminated endemic
fungal infections include patients who have received transplants, those prescribed immunosuppressive and
chemotherapeutic agents, HIV-infected patients, premature infants, the elderly, and patients undergoing
major surgery. Thus, a shift in the mycoses encountered in the healthcare setting has occurred with
fungi previously considered non-pathogenic, including mucoraceous genera (formerly zygomycetes) and a
variety of both hyaline and dematiaceous molds, now being commonly seen in immunocompromised patients.
Diagnosis of infections outside the usual geographic or epidemiologic setting can be challenging.
Furthermore, advances in diagnostic radiology have allowed greater ability to pursue specific diagnoses
by collecting tissue biopsies from body sites formerly not available for histopathologic examination. In
addition, the therapeutic armamentarium now available and the presence of resistance to different drugs
by these fungi have compounded the diagnostic challenges. Nowadays, the use of fungal GMS cannot solve
alone these challenges and newer diagnostic techniques may be required. However, histopathology
continues to be a rapid and cost- effective means of providing a presumptive or definitive diagnosis of
an invasive fungal infection while waiting for fungal culture results. Histopathology may be the only
material available when no culture growth occurs or cultures were not ordered. Thus, microbiologists,
pathologists and clinicians need to be aware of the limitations of tissue diagnosis, the pitfalls of
morphologic diagnosis, and the tests that can be performed in tissue and other samples to make
Retrospective studies that correlate culture results with histopathology and cytology showed that
overall accuracy for microscopic morphologic techniques can vary from 20 to 80%. The lowest correlation
has been reported for invasive septate molds. Even though GMS and PAS stains were used more frequently
in the cases correctly diagnosed than those misclassified, special stains did not significantly improve
pathologists' diagnostic capabilities. Misclassification of cases occurs when the pathologist has a
false sense of ability to categorize fungal organisms by genus based on microscopic morphology alone,
when inappropriate terminology is used such that other potential molds within a particular category are
not included in the differential diagnosis, or when there is lack of knowledge of morphologic mimics of
yeasts and hyphal forms. The misclassifications with greater potential for adverse consequences occurred
when there were few, folded, fragmented and/or necrotic fungal elements in the specimen and the
structures in hyphae could not be adequately categorized as septate versus pauciseptate hyaline molds.
Therefore, clinicians need to be aware that misclassifications in histopathologic examination occur in at
least 20% of cases, and pathologists need to give as much information as possible without overextending
their diagnostic capabilities.
In order to avoid misclassifications, pathologists should describe the fungal elements observed in the
sample and refrain from trying to offer a specific diagnosis. Pathologists need to remember that there
are very few instances where morphologic characteristics are specific. Some groups have suggested the
use of templates or synoptic reporting for the diagnosis of fungal infections. In these templates the
diagnosis line includes a description of the yeast or mold, the presence or absence of fungal invasion of
tissues and vessels, and the host reaction to the fungus (inflammation, necrosis, hemorrhage). In a
second comment field, the pathologist should clearly state the most frequent fungi associated with that
morphology as well as other possible organisms (fungi and parasites) that should be considered in the
differential diagnosis. All pathology reports should also include a statement in the comment regarding
the importance of correlating clinico- epidemiologic features and results of cultures and other
laboratory tests that could aid in the diagnosis.
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