Case 5 -

Fungal infection: Pseudallescheria boydii Dr. Ritu Nayar

Clinical History 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 Click to view with ImageScope Click to view with a Web-Based Viewer Diagnosis: 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. Pseudallescheria boydii 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 Scedosporium apiospermum. 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 isolate. Clinical implications: P. boydii tends to be refractory to amphotericin therapy but has shown response to azoles, voriconazole is best for CNS infections. Aspergillus Species 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 aspergillosis 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). References Walts A. Pseudallescheria: an underdiagnosed fungus? Diagn Cytopathol; 2001 Sept: 25:153-7. Tadros TS, Workowski KA, Seigel RJ, et al. Pathology of hyalohyphomycosis caused by Scedosporium apiospermum (P. boydii): an emerging mycosis. Hum Pathol 1998;29(11):1179-80 Stanely MW, Deike M, Knoedler J, et al. Pulmonary mycetomas in immunocompetent patients: diagnosis by fine needle aspiration. Diagn Cytopathol 1992; 8: 577-579. McGinis MR, Nunn GB. Sequence based fungal identification and classification.Chapter 42. In Molecular microbiology: diagnostic principles & practice.Eds.Persing DH et al. ASM Press 2004. Larone, D. H. 1995. Medically Important Fungi - A Guide to Identification, 3rd ed. ASM Press, Washington, D.C. Hayden RT, Isotalo PA, Parrett T, et al. In situ hybridization for the differentiation of Aspergillus, Fusarium, and Pseudallescheria species in tissue sections. Diagn Mol Pathol 2003: 12(1): 21-26. Laboratory approach to the diagnosis of fungal infections. In Koneman's Color atlas and textbook of medical microbiology, 2006 Miller MA, Greenberger PA, Amerian R, et al. 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