IDENTIFICATION OF ORGANISMS 306
GENERAL FEATURES OF INFECTIOUS GRANULOMAS 307
HISTOPLASMOSIS 308
Clinical features 309
Pathologic features 310
Staining techniques 311
COCCIDIOIDOMYCOSIS 312
Clinical features 312
Histopathologic features 312
CRYPTOCOCCOSIS 313
Clinical and radiographic features 313
Histologic features 314
NORTH AMERICAN BLASTOMYCOSIS 317
Clinical features 317
Histologic features 317
Differential diagnosis 318
MISCELLANEOUS FUNGAL INFECTIONS 319
TUBERCULOSIS 319
Clinical features 319
Pathologic features 320
NONTUBERCULOUS MYCOBACTERIAL INFECTIONS 320
Hot tub lung 321
M. bovis infection 322
DIROFILARIASIS 322
Clinical features 323
Pathologic features 323
The evaluation of granulomatous lung inflammation
is a common task for the surgical pathologist. Most
necrotizing granulomas are caused by infection, usually
fungal or mycobacterial, and the responsible organism
is usually demonstrable in the tissue. A number of
important noninfectious lesions, however, especially
various pulmonary vasculitides and sarcoidosis, can
also cause pulmonary granulomas. Although ideally all
specimens should be cultured, in practice this procedure
is not always possible, since the tissue may be immersed
in formalin before being received by the pathology
laboratory. Furthermore, in acutely ill patients, a
diagnosis may be needed before the results of cultures
are available. The pathologist, therefore, rather than the
microbiologist, must assume primary responsibility for
identifying infectious organisms in lung granulomas.6
Helpful guidelines for evaluating necrotizing granu-
lomas are summarized in Table 11–1. Not only can the
pathologist be expected to document the presence or
absence of organisms in granulomas, but also he or she
11
INFECTION II. GRANULOMATOUS
INFECTIONS
305
Table 11–1 Guidelines for Identifying Organisms in
Necrotizing Granulomas
• Use the H and E in addition to special stains to identify
fungi (both the tissue reaction and organism
morphology are important).
• Begin the search for organisms in the center of necrotic
zones rather than in the surrounding viable
inflammatory areas.
• Perform special stains (AFB, GMS) on at least two
blocks that contain active, necrotic granulomatous
areas.
• Remember that organisms may not be identifiable in up
to one-third of solitary necrotizing granulomas. Absence
of organisms does not by itself imply a noninfectious
condition.
W0041-Ch11.qxd 2/4/06 12:14 PM Page 305
can, with good accuracy, specifically identify many
fungi (Table 11–2).
IDENTIFICATION OF ORGANISMS
Special stains for acid-fast bacilli and fungi should be
examined in all cases.2,3,5,6 We prefer the Ziehl–Neelsen
(AFB) stain for mycobacteria and Grocott–Gomori
methenamine silver (GMS) stain for fungi, although the
auramine–rhodamine and PAS–Gridley stains are also
satisfactory. Use of the periodic acid–Schiff (PAS) stain
without a counterstain is not recommended when
searching for fungi in necrotizing granulomas, because
it fails to adequately differentiate the organisms from
the background necrosis and debris. However, it can
be helpful in delineating the internal structural detail
of a fungus that already has been located by other
staining methods. Other stains that are useful include
the Fontana–Masson (FM) stain and the combined
FM–alcian blue, FM–mucicarmine, and alcian blue–PAS
stains, especially for identifying cryptococci.35,36,40
Rarely, ordinary bacteria have been reported to cause
necrotizing, usually suppurative, granulomatous
reactions.1,4 Examples include Burkholderia pseudomallei
(melioidosis), Brucella suis (tularemia), and other rare
organisms (Burkholderia cepacia, Pseudomonas andersonii).
Gram stains or silver impregnation techniques (Warthin
Starry stain) can sometimes outline the organisms, but
more often the diagnosis depends on cultures.
The importance of an ordinary hematoxylin and
eosin (H and E) stain cannot be overemphasized in
evaluating necrotizing granulomas. Several fungi,
including blastomyces, cryptococcus, and coccidioides,
for example, are easily visualized with H and E and
show distinct morphologic features in this stain. The
combination of the H and E appearance and the special
stain findings can greatly facilitate identification.
The appearance of the tissue reaction can provide addi-
tional clues to the organism. For example, suppurative
granulomas are characteristic of blastomycosis and
coccidioidomycosis, non-necrotizing granulomas are
common in cryptococcosis, and eosinophilia may be
prominent in coccidioidomycosis. In some cases,
immunofluorescence or immunoperoxidase techniques
using specific antibodies can help in identifying the
organism. More sophisticated methods utilizing the
polymerase chain reaction (PCR) have been utilized
in tuberculosis cases.73,81,83,87,89
It should be remembered that organisms are almost
always located in the central necrotic zones of caseating
granulomas rather than in surrounding viable tissue or
non-necrotizing granulomas.6,92 In the study by Ulbright
and Katzenstein,6 acid-fast bacilli and histoplasma were
306 INFECTION II. GRANULOMATOUS INFECTIONS
Table 11–2 Contrasting Morphologic Features of Common Fungi Causing Granulomatous Inflammation in the Lung
Histoplasma Coccidioides Cryptococcus Blastomyces
Average size 3 μm (range, 1 to 5 μm) 30 to 60 μm (spherules); 4 to 7 μm (range, 8 to 15 μm
2 to 5 μm (endospores) 2 to 15 μm) (range, 2 to
30 μm)
Morphology Oval, budding yeast; Spherules, endospores; Round, budding yeast; Round, budding
uniform in size, buds no budding forms variation in size, yeast; uniform
uncommon fragmentation common in size
Distinguishing Single nucleus, perinuclear Thick wall, central Pale, thin cell wall; Thick cell wall,
structural features clear zone (intracellular basophilic endospores extracellular clear zone basophilic
(H and E) organisms only)* (spherule only) (halo) protoplasm,
multiple nuclei
Mucicarmine staining Negative Negative Usually positive Negative
Type of granulomas Necrotizing Necrotizing; early lesions Necrotizing, Necrotizing with
suppurative; eosinophils non-necrotizing suppuration
common
*Intracellular organisms are seen only in disseminated histoplasmosis. Histoplasma cannot be visualized within caseous
necrosis in lung granulomas without special stains.
W0041-Ch11.qxd 2/4/06 12:14 PM Page 306
found almost exclusively within such an area. Although
cryptococcus and coccidioides occasionally were found
in surrounding viable histiocytes, they were mainly
present in necrotic zones. To conserve time and energy,
therefore, the pathologist should begin his or her search
for organisms in the central, most necrotic portion of
the granuloma.
In most cases, the examination of sections from two
tissue blocks is sufficient to identify an infectious agent,
provided that the blocks include areas containing
the most active and preferably most necrotic granulo-
matous inflammation. With this technique, an infec-
tious agent will be identifiable in most necrotizing
granulomas. It should be remembered, however, that
a small but significant proportion of otherwise typical
necrotizing granulomas (one-third in Ulbright and
Katzenstein’s study6 of radiographically solitary lesions)
will be negative for organisms by special stains and
sometimes cultures. These cases likely represent
infectious granulomas in which organisms have been
removed by the inflammatory process, and the diagnosis
of other diseases, especially pulmonary vasculitides,
should not be considered unless specific diagnostic
features are present (see Chapter 8).
GENERAL FEATURES OF INFECTIOUS
GRANULOMAS
Infectious granulomas share some features with the
pulmonary vasculitides (see Chapter 8), which may make
diagnosis difficult.5,6 Irregular, ‘geographic’-shaped
necrosis, a characteristic finding in Wegener’s granulo-
matosis, occasionally occurs in infections (Fig. 11–1a).
Remnants of inflamed blood vessels and ghosts of
alveolar septa are sometimes seen in the necrotic zones
(so-called infarct-like necrosis, Fig. 11–1b). Vasculitis,
which is a hallmark of Wegener’s granulomatosis and
other vasculitides, is common in both fungal and
mycobacterial infections (Fig. 11–2). The vasculitis of
infection is not necrotizing, however, and is generally
characterized by a mural infiltrate of lymphocytes and
307GENERAL FEATURES OF INFECTIOUS GRANULOMAS
Figure 11–1 Infectious granulomas. (a) Irregular, geographic-shaped necrosis is seen in this example of tuberculosis.
Inflammation in blood vessels is also prominent. (b) Infarct-like necrosis is prominent in this example of histoplasmosis. Note the
remnants of alveolar septa in the necrotic zone. An adjacent artery is also inflamed (left).
(a) (b)
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plasma cells. The intima may be expanded by the
cellular infiltrate, and, sometimes, epithelioid histiocytes,
and even non-necrotizing granulomas, accompany the
other chronic inflammatory cells. A transmural neutro-
phil infiltrate with necrosis that is characteristic of
Wegener’s granulomatosis, however, is not a feature of
infection-related vasculitis. Bronchocentric granulomas,
the characteristic finding in bronchocentric granulo-
matosis (see Chapter 6), are common in infections.
In these lesions, bronchiolar mucosa is partially or
completely replaced by palisading epithelioid histiocytes
(Fig. 11–3). The former instance is easy to recognize
because the necrotizing granuloma is partly surrounded
by bronchiolar epithelium. When there is no residual
epithelium, the bronchocentric location of the granulo-
matous inflammation is inferred by the presence of
a nearby pulmonary artery (see Fig. 6–20). These
observations underscore the need to carefully exclude
an infectious etiology by means of special stains and
cultures before considering a diagnosis of noninfectious
granulomatous diseases. The differential diagnosis
of granulomatous infections and the noninfectious
pulmonary vasculitides is discussed in more detail in
Chapter 8.
The following sections concentrate mainly on fungal
organisms that are common in the United States.
Histologic features that aid in their recognition and
differentiation are summarized in Table 11–2. Dirofilarial
infections are also discussed, since they are an impor-
tant cause of solitary lung nodules in certain areas
of the United States, and aspects of tuberculosis and
nontuberculous mycobacteria that are relevant to the
surgical pathologist are briefly reviewed.
HISTOPLASMOSIS
Histoplasma capsulatum is a widespread fungus whose
natural habitat is the soil. It exists in the mycelial
form in nature but assumes the yeast phase at body
temperature. The organism is endemic primarily in the
central United States, but cases of histoplasmosis may
occur well outside of this area.
308 INFECTION II. GRANULOMATOUS INFECTIONS
Figure 11–2 Vasculitis in infectious granulomas. (a) A dense chronic inflammatory cell infiltrate is present in the wall of this
artery and narrows the lumen. The artery is located on the edge of a necrotizing granuloma (right), in which histoplasma were
identified. (b) This artery from a case of tuberculosis shows granulomatous inflammation in its wall. The necrotic center of the
granuloma is on the right.
(a) (b)
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Clinical Features
The vast majority of histoplasma infections in humans
do not produce symptoms. Their existence is docu-
mented by the subsequent development of skin or
serologic reactivity to histoplasma-related antigens
or by the presence of characteristic radiographic calcifi-
cations. Several clinical syndromes occur, however,
including acute pulmonary histoplasmosis, disseminated
histoplasmosis, and chronic histoplasmosis.7,8 Histo-
plasmoma is an important complication of acute pulmo-
nary histoplasmosis which may be encountered by
the surgical pathologist.
Acute Pulmonary Histoplasmosis
Acute pulmonary histoplasmosis encompasses the forms
of the disease previously termed primary histoplasmosis
and acute histoplasmosis.7,8 Primary histoplasmosis is
considered a misnomer since, unlike primary tuber-
culosis, which is followed by life-long tuberculin
hypersensitivity, histoplasmosis is not necessarily
associated with lasting histoplasmin sensitivity, and,
therefore, infection and reinfection are common in
endemic areas. Clinically, most patients are asympto-
matic. The uncommon symptomatic form of the disease
occurs predominantly in infants or children, and fever
and cough are the usual clinical manifestations. Hilar
and mediastinal lymph node enlargement and a patchy
parenchymal infiltrate are seen radiographically. The
course is usually self-limited, although sometimes
dissemination occurs (see text following). Occasionally,
inhalation of a large number of spores causes sympto-
matic disease, a form of infection previously termed
acute histoplasmosis. These patients present with
influenza-like symptoms accompanied by patchy, soft
infiltrates or nodules that can be seen on chest radio-
graphs. This form of the disease can represent a type of
primary infection, or it can occur in previously infected
individuals (reinfection type). It is usually self-limited,
and the diagnosis is based on clinical, laboratory,
and radiographic findings. Occasionally, in acutely ill
patients in whom the diagnosis is unsuspected clinically,
a lung biopsy will be performed.
Disseminated Histoplasmosis
This entity usually occurs in patients with abnormal
immune systems, although otherwise apparently healthy
individuals can be affected as well.7,10,12 Approximately
a third of the cases involve infants younger than 1 year
old. This form of the disease is characterized by wide-
spread parasitization of macrophages by the organisms,
and multiple organs, especially reticuloendothelial, are
affected. Interstitial lung infiltrates are often seen
radiographically. The diagnosis is usually established
from urine or blood cultures or by liver or bone marrow
biopsy. Bronchoalveolar lavage specimens and lung
biopsy specimens may be used in some cases.
Chronic Histoplasmosis
This type of histoplasmosis usually occurs in patients
with emphysema or other chronic lung disease.9 White
males are affected more frequently than women or
blacks, and the symptoms resemble those of tuber-
culosis. Pneumonic infiltrates and cavitary lesions are
seen radiographically. The diagnosis is usually based
on recovery of the organisms from sputum cultures,
although lung biopsy occasionally may be necessary.
Elevated complement-fixation titers can also help to
establish the diagnosis in some cases. Skin tests are
not useful, because false-negative results can occur, and
positive results are found in the majority of healthy
persons in endemic areas.7
Histoplasmomas
Histoplasmomas are thought to develop around a healing
or healed focus of acute pulmonary histoplasmosis.7
They appear radiographically as well-circumscribed
309HISTOPLASMOSIS
Figure 11–3 Bronchocentric granuloma in tuberculosis. A
necrotizing granuloma partially replaces the bronchiole in the
center.
W0041-Ch11.qxd 2/4/06 12:14 PM Page 309
masses, and they may slowly enlarge. Patients are
usually asymptomatic, the lesions being found on a
routine chest radiograph. This is the form of histo-
plasmosis most frequently encountered by the surgical
pathologist, since the nodules are often excised.
Pathologic Features
The gross appearance of most forms of pulmonary
histoplasmosis resembles that of any other necrotizing
granulomatous process. Pulmonary histoplasmomas
tend to be distinct, however, because their caseous
central portion is often composed of multiple concentric
lamellae, resembling the growth rings of a tree
(Fig. 11–4). Although this lamellated appearance is
characteristic of a histoplasmoma, it is not patho-
gnomonic and may also be produced by other organisms.
Disseminated histoplasmosis differs grossly in that
necrotizing granulomas are usually not seen.
Microscopically, histoplasmomas and other forms of
histoplasmosis (except disseminated histoplasmosis, see
text following) are indistinguishable from necrotizing
granulomas caused by other fungi and mycobacteria.6
Usually, the granulomas are well circumscribed and
often contain a layer of laminated acellular collagen
that is external to a rim of active granulomatous inflam-
mation (Fig. 11–4b). Central calcification is common.
In occasional cases, the granulomas are poorly circum-
scribed, and the inflammatory process may invade
and destroy the pulmonary parenchyma in an irregular
pattern. The frequent occurrence of infarct-like necrosis
and vasculitis (see Fig. 11–1b) in such cases initially
may suggest Wegener’s granulomatosis. The correct
diagnosis is established, however, when organisms are
demonstrated in special stains or cultures. GMS is the
most useful stain for identifying histoplasma in lung
granulomas. Numerous organisms can usually be found
within the central necrotic zones, although they may be
sparse in the poorly circumscribed lesions (Fig. 11–5).6
They appear as small, uniform, oval-shaped yeasts
ranging in size from 1 to 5 μm (average, 3 μm). Budding
forms can be found but usually are not numerous.
The organisms cannot be seen in routine H and E stains
unless they are present within histiocytes, a finding
usually observed only in disseminated histoplasmosis
(see text following). Rarely, hyphae have been described
310 INFECTION II. GRANULOMATOUS INFECTIONS
Figure 11–4 Histoplasmoma. (a) Gross appearance of a necrotizing granuloma due to histoplasma. Note the lamellated, tree-
ring appearance of the necrotic zone. This appearance, although characteristic of histoplasmoma, is not specific and can be seen
in other fungal as well as mycobacterial granulomas. (b) Microscopic appearance of necrotizing granuloma, showing the
characteristic epithelioid histiocytes surrounding central necrosis.
(a) (b)
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in tissue, most commonly in cases of endocarditis with
intravascular growth.11
In one case of acute histoplasmosis biopsied early in
its course, granulomas were said to be absent, and the
main finding was an intraalveolar mononuclear inflam-
matory cell exudate, containing organisms.14 That
patient, however, was an intravenous drug user and
may have had underlying immune compromise (AIDS)
that altered the inflammatory response. In our experi-
ence, active granulomatous inflammation is seen even
in early acute histoplasmosis, although the granulomas
may have purulent centers.
Disseminated histoplasmosis differs microscopically
from other forms of histoplasmosis in that well-formed,
necrotizing granulomas are usually absent, and the
organisms are present extensively within histiocytes.10,13
In the lung, the interstitium is the main site of involve-
ment, although histiocytes accumulate within alveolar
spaces as well (Fig. 11–6). At low magnification, the
alveolar septa and peribronchiolar tissue appear
expanded by sheets of plump histiocytes with granular
cytoplasm, and organism-filled macrophages may pack
alveolar spaces. The typical morphology of the intra-
cellular organisms is readily appreciated when the slides
are examined under higher magnification. In contrast
to the granulomatous forms of the disease, in which
the organism usually cannot be visualized in H and E
stains, the intracellular yeasts in disseminated histo-
plasmosis are readily visible in routine H and E stains.
They appear as small, ovoid bodies with a single, often
eccentric nucleus and a characteristic perinuclear
clear zone resulting from shrinkage of protoplasm from
the cell walls (Fig. 11–6, inset).
Staining Techniques
Sometimes, histoplasma stains only faintly with GMS,
even when a control slide using another fungus (usually
candida or aspergillus) stains strongly.6 For this reason,
a control slide conta