Diagnostic Cytopathology: Something for Everyone
Moderator: Dr. Celeste N. Powers
Case 5 -
Fungal infection: Pseudallescheria boydii
Dr. Ritu Nayar
Bronchioloalveolar lavage. A 62-year-old female presented with decreased appetite,
weight loss and left pleuritic chest pain. A lavage was performed.
Case 5 - Slide 1
Fungal infection: Pseudallescheria boydii
Clinical Categorization of Fungal Infections
Fungi are members of the plant kingdom that lack leaves, stems or roots. They are eukaryotes that
possess a nucleus, nuclear membrane, ER, Golgi apparatus and mitochondria. They also have a rigid cell
wall composed of chitin, mannans and occasionally cellulose. It is due to adsorption of several dyes by
these cell wall constituents that special stains can be used to identify fungal forms in wet mounts,
smears and tissue sections. A yeast is defined as a cell that reproduces by budding; they are generally
unicellular. Moulds are multicelled, filamentous forms of fungi, consisting of thread-like filaments
termed hyphae that interweave to form a mat-like structure termed mycelium. Reproduction is by means of
spores, and may be sexual or asexual; spores are derived from vegetative mycelium (arthrospores,
chlamydospores, blastospores) or from the surface of special aerial fruiting bodies. The morphology,
arrangement and mode by which spores are derived serve as the essential criteria by which genus and
species identification of fungi are made.
From a broad perspective, fungal infections may be superficial or deep-seated/ systemic; however clinically superficial infections can cause
disseminated disease. The term "opportunistic" is currently used to describe
"non-pathogenic" fungi that cause subcutaneous and disseminated infections, usually in immunocompromised
individuals or those with prosthetic / intravascular devices. Aspergillus,
Candida and Zygomycetes are the three fungal
groups that are classically categorized as opportunistic fungi. Phaeohyphomycosis is a generic term used for a subcutaneous or systemic disease
caused by a variety of black fungi that develop in tissue as dark hyphae and/or yeast like cells. Hyalohyphomycosis may also be subcutaneous or systemic, and is a generic term used
for saprophytic fungi that produce colorless-transparent (hyaline) hyphae in tissue.
Opportunistic fungi have emerged during the past decade as important causes of morbidity and mortality in
immunocompromised patients. Aspergillus species are the most common
infectious cause of pneumonic mortality in bone marrow/stem cell transplant recipients. Hyaline septated
filamentous fungi, such as Fusarium species, Acremonium species, Paecilomyces species, and Trichoderma species, are increasingly reported as causing invasive mycoses
refractory to conventional therapy. Dematiaceous septated filamentous fungi, such as Pseudallescheria boydii, Bipolaris species, and Cladophialophora bantiana cause pneumonia, sinusitis, and CNS infection
unresponsive to current therapy. As we enter the new millennium, we may anticipate that emergent fungal
infections will continue to develop in the settings of permissive environmental conditions, selective
antifungal pressure, and an expanding population of immunocompromised hosts.
Definition : A species of fungus. Synonym: Allescheria
boydiii. Scedosporium apiospermum (anamorphic or asexual form), also known as Pseudallescheria boydii
(sexual or telomorphic form) is the most commonly clinically encountered species in the group of
hyalohyphomycetes producing conidia singly. Scedosporium prolificans is a newly designated species
which also causes human infections.
Human infection with P. boydii can produce two distinct rare diseases:
mycetoma and pseudallescheriasis (scedosporiosis). Mycetoma is a chronic subcutaneous infection, whereas
pseudallescheriasis includes all other infections caused by P. boydii. The most common sites of the latter are lung, bone, joints, and CNS.
This fungus is found in soil, fresh / stagnant water, throughout the world. Disease acquisition is via
inhalation into lungs or sinuses or by traumatic inoculation into the skin. Colonization is more common
than infection; however an invasive pulmonary disease similar to invasive pulmonary aspergillosis is
seen, usually in immunocompromised hosts. Immunocompetent patients tend to have a chronic/subacute
course, while those in immunocompromised hosts are severe/ acute. P. boydii
can colonize bronchiectatic lungs or obstructed sinuses; masses of P. boydii hyphae (fungus balls) can be found in lung cavities. It is also a cause of
allergic bronchopulmonary disease. Keratitis, otitis, CNS, meningitis, endocarditis, osteomyelitis,
lymphadenitis and peritonitis have all been described.
Invasive pulmonary pseudallescheriasis is usually seen in patients with prolonged neutropenia, those
on high dose steroid therapy, or status post allogenic bone marrow transplantation, AIDs patients and
after solid organ transplants. Pulmonary disease manifests as fever, pleuritic pain and hemoptysis.
Chest films may show nodularity, alveolar infiltrates or cavitation. Co-infection with Mycobacterium avium complex has been described.
Isolation of P. boydii from sterile sites is diagnostic. Growth of this
organism from sputum, bronchoalveolar lavage, draining wounds or sinus aspirates, is less convincing of
disease unless accompanied by hyphae on smears or biopsy. Microscopically P.
boydii resembles Aspergillus, with dichotomously branching septate
hyphae. In neutropenic patients blood vessel invasion and thrombosis can occur.
The fungus grows well in culture and in a few days produces characteristic house-mouse gray, silky
colonies on which water droplets tend to aggregate. Dark pigment may extend to the reverse side of the
colony, suggesting a dematiaceous fungus. Microscopically, the hyphae are hyaline and septate. A
characteristic feature is the production of smooth, thin walled, oval conidia, ranging in diameter from
3-5 microns; that arise from unbranched, irregularly spaced conidiophores (lollipops). As the conidia
mature, they take on a dark pigmentation. The sporulating structures are quite different from those of
Aspergillus. Cultures that produce asexual conidia but do not produce the
sexual reproductive structure, the cleisthothecia, after 2-3 weeks are designated by the amorph name
On cytologic preparations and tissue sections, the hyphae of P. boydii
are septate, branching, non-pigmented, varying in width from 2-5 microns. In contrast to the regular
dichotomous branching pattern of Aspergillus spp., P.
boydii shows an "irregular" branching pattern. Practically, it is often impossible to distinguish
Pseudallescheria hyphae from those of Aspergillus spp., Fusarium spp., non- pigmented Alternaria spp. or Bipolaris in cytologic and
histologic preparations. Stains such as GMS, PAS do not help. Although the branching pattern of P
boydii may be helpful, more reliable identification requires visualization of characteristic conidia. In
the asexual stage, septate hyaline hyphae, conidiophores, and (annello) conidia are produced. Conidia
(4-7 x 5-12 micron) are unicellular and oval in shape, and typically truncate at their base.
Cleistothecia, asci and acospores are visualized in the sexual stage. The conidia of Scedosporium apiospermum are often formed singly on the conidiophores, while those
of Graphium eumorphum are arranged in clusters at the apices of each
synnema. Scedosporium, Graphium or both forms may be present in the same
P. boydii tends to
be refractory to amphotericin therapy but has shown response to azoles, voriconazole is best for CNS
Aspergillus species are widely distributed in nature; within soil,
vegetation and organic matter. Inhalation of dust contaminated by spores is the most common mode of
infection in humans. Aspergillus may cause various clinical forms of
colonization and infection, including (1) Incidental: asymptomatic colonization (2) Immunologic:
extrinsic allergic alveolitis/ sinusitis, allergic bronchopulmonary aspergillosis (3) Non-invasive
mycetoma (4) Invasive Disease: angioinvasive aspergillosis and chronic necrotizing pulmonary
Aspergillus fumigatus, A. flavus, A. niger, A. nidulans,
and A. terreus are the species that may be encountered in the clinical
laboratory; the majority of serious infections are caused by A. fumigatus.
Colonies of Aspergillus spp. are suspected in culture if the isolate grows
rapidly (3-5 days), has a distinct outer margin, often with a white border at the advancing edge of
growth. New colonies are cottony in consistency but as they mature, the surface becomes powdery/ sugary
as the conidia are produced. Microscopically, Aspergillus spp. produce
uniform, 4-6 micron in diameter, hyaline, septate hyphae with parallel walls. A 45 degree angle
dichotomous branching is characteristic of the invasive mycelium in tissue, but is not seen as commonly
in mounts made from culture plates. A hyphal segment called the foot cell is the base of origin for the
conidiophore. The conidiophore terminate in a swollen vesicle from whose surface are borne uniserate
(one) or biserate( two) rows of philades, giving rise to chains of conidia. The length and width of
conidiophores, size/shape of vesicle; arrangement of the philades and the color/size and length of
conidia are used to make the species distinctions.
In tissue sections, the hyphae do not stain well on H&E, but are clearly outlined by GMS and PAS
stains. Initial tissue reaction in immunocompetent persons may be purulent, followed by granuloma
formation. More commonly, the hyphal invasion is not accompanied by a host response, and only necrosis
may be seen. Aspergillus has a predilection for angioinvasion, resulting in
hemorrhage and thrombosis. Some strains produce calcium oxalate and birefringent crystals may be
observed in association with this fungus. Growth of the fungus in a pre-existing cavity such as a lung
cyst or nasal sinus, is called a "fungus ball" In this situation, the hyphae appear amorphous and pale
staining. Fruiting heads and chains of conidia can be seen within cavities exposed to air.
Difficulties in classification cannot always be resolved by morphology alone and correlation with
culture characteristics and biochemical testing may be necessary for accurate speciation. In some cases
one organism can be morphologically very similar to another. As a case in point, Pseudallescheria boydii can look very similar to Aspergillus, but it does not respond to amphotericin therapy. Fusarium is also morphologically very similar to Aspergillus. However, their differentiation can have significant clinical
implications, as the latter two groups are often resistant to commonly used antifungal agents. Such
circumstances have lead to the use of more generic terms such as "Fungal elements, morphologically
consistent with Aspergillus species".
Other Diagnostic modalities
(1) Sequence based fungal identification and classification. The use of
gene sequence information for the identification of fungi grown in culture and for direct identification
of fungi in blood and tissue is becoming increasing important for fungal disease diagnosis. To determine
rRNA sequence of a fungal isolate, organisms are grown and nucleic acid is extracted. Commercial
sequencing kits such as the MicroSeq D2 LSU rDNA fungal sequencing kit (Applied Biosystems, CA),
consisting of a PCR module and sequencing module can be used to amplify the D2 region. MicroSeq
microbial identification and analysis software provides a semiautomated interface for rapid assembly,
proofreading, alignment and phylogenetic analysis of the fungal fragments collated from the
electropherograms for each organism.
(2) In situ hybridization (ISH). ISH may assist in rapidly
distinguishing these organisms in the absence of available culture. In a study reported by Hayden et al.
(reference 6) oligonucleotide DNA probes were directed against the 5S, 18S, or 28S rRNA sequences of
three groups of fungi with a high degree of specificity for each. Probes were tested on 26
formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these
organisms: Fusarium species, n = 12; Pseudallescheria
boydii, n = 5; Aspergillus species, n = 9 (probe set validated in an
earlier study). Accuracy of both ISH and morphology was compared with culture. Morphologic examination
(GMS and PAS) showed a greater sensitivity in detecting fungi (100%) as compared with ISH (84.6%). When
detected, however, DNA probes allowed definitive identification of organisms. While there was no ability
to distinguish between the three groups of organisms by morphologic features, ISH probes showed 100%
positive predictive value (PPV, 19/19 organisms identified correctly). No cross-reactivity was observed
when the probes were tested against other genera (100% specificity). Furthermore, the use of ISH allowed
the detection of mixed fungal infections involving multiple organism types in two cases, demonstrating
another advantage over morphology. In situ hybridization, directed against rRNA sequences, provides a
rapid and accurate technique for distinguishing commonly encountered, non-pigmented filamentous fungi in
histologic sections. While less sensitive than morphology, ISH is highly accurate and may help to
distinguish between organisms that have similar morphologic features by light microscopy
(3) The Platelia Aspergillus test system. This is a sandwich ELISA for
the detection of Aspergillus circulating galactomannan in the blood early in
the course of progressive disease (Bio-Rad, Richmond, WA).
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