Sunday, 21 October 2012

Scedosporium prolificans

Scedosporium prolificans -Fungus (Previously known as Scedosporium inflatum)

Ecology: Scedosporium prolificans is a cosmopolitan fungus isolated primarily from soils (saprophyte), however its ecosystem and distribution may be more restricted than the other major species, Scedosporium apiospermum. Scedosporium prolificans has been isolated from hospital settings after the environment was disturbed during on-site construction. 
Macroscopic Morphology: S.prolificans exhibits moderately rapid spreading growth and will mature within 5 to 7 days at 25oC on sabouraud dextrose agar (SDA or SAB). Young suede-like to downy growth becomes increasingly cottony as it develops an olive-grey to a black colouration. Light coloured mycelial tufts may develop as the colony ages. Reverse is a dark brown to dark grey, almost black in appearance. 

Scedosporium prolificans SAB, 72 hrs,  30oC

Microscopic Morphology: Scedosporium prolificans produces hyaline septate hyphae from which conidiogenous cells are produced along the length. The conidiogenous cells are flask-shaped annellides which have a swollen base (inflated) from which extends an elongated 'neck'. Conidia are produced singly or in small groups at the apex of the annellide. The smooth walled single-celled conidia are hyaline to a pale brown in colour and are oviod to pyridaform in shape. These conidia (annelloconidia) have a truncated base where they were attached to the conidiogenous annellide. The conidia measure 2 - 5 μm to 3 - 13 μm (average 3.4 μm to 5.3 μm) in dimension. 

Note: All photographs which follow were taken using the Leica DMD-108 digital microimaging device. 

Scedosporium prolificans (LPCB, 250X) -First impression of a slide culture.

Scedosporium prolificans -hyphae with conida visible. Conidiogenous cells not all that distinct at this magnification,  (LPCB, 400X)

Scedosporium prolificans -another view of conidia along hyphae
(LPCB, 400X)

Scedosporium prolificans - conidia seen distributed singly and in clusters along the hyphae.  (LPCB, 400X) 

Scedosporium prolificans - At this magnification the conidiogenous cells are becoming more obvious.  Ovoid to pyridaform conidia can be seen attached at the apex of the annellide.  Note 100 micron bar at upper right. (LPCB, 400+10X)

Scedosporium prolificans - Here we can see a conidiogenous cell (annellide) extending from the hyphae at which four annelloconida can be seen at the apex. (LPCB, 1000X)

Scedosporium prolificans - hyphae with annellides bearing ovoid to pyridaform annelloconidia. (LPCB, 1000X)

Scedosporium prolificans - Here we can distinctly see the inflated, vase-like annellide conidiophore (arrows) from which are produced the annelloconidia.  (LPCB, 1000X)

Scedosporium prolificans - Here we can see the elongated 'neck' (1) at the apical end of the annellide and the truncated base or scar at the end of the annelloconidia where it was once attached (2).  The swollen or inflated vase like base is evident just right of (1) where the conidiogenous cell attaches to the hyphae.  (LPCB, 1000X)

Scedosporium prolificans - Here again we can see the elongated 'neck' (1) with an annelloconidia at the apex, the inflated annellide (2) and the annelloconidia, each with a flattened, truncated base of attachment (3).
(LPCB, 1000X)

Scedosporium prolificans - Measurement of annelloconidia - 7.0 μm to 7.4 μm in length, 4.7 μm in diamenter in this field. (LPCB, 1000+10X)

Scedosporium prolificans - small clusters of conidia (annelloconidia) at the apex of annellides (conidiogenous cells) extending from hyphae.
(LPCB, 1000+10X)

Scedosporium prolificans - one last photo showing branched septate hyphae with annelloconidia at apex of annellides.  (LPCB, 1000+10X)

Pathogenicity: Scedosporium prolificans is an emerging fungal pathogen can infect both immunocompetent and immunocompromised individuals. Noted risk factors for scedosporiosis have included malignancy, cystic fibrosis as well as solid organ transplantation. Onychomycosis and mycotic keratitis have been reported in the literature. Inhalation of the fungus has resulted in colonization of the paranasal sinus and the lungs. Penetrating injuries may result in localized infections of the joints resulting in septic arthritis as well as osteomyelitis. Disseminated have been reported in immunosuppressed patients such as those experiencing prolonged neutropenia and post-transplantation therapy. Accurate identification is imperitive as S.prolificans exhibits increased resistance to common antifungal medications and disseminated infections is often fatal even in immunocompetent patients. Asymptomatic colonization as also been reported.

Differentiation: Scedosporium prolificans can be differentiated from Scedosporium apiospermum as the former has a distinctive swollen (inflated) base. This inflated base was the reason this fungus was previously known as Scedosporium inflatum. S.prolificans produces annellides that are flask-shaped, unlike the cylindrical conidiogenous cells of S.apiospermum. S.prolificans is inhibited by media containing cycloheximide.  Finally, S.prolificans has no sexual stage (telemorph) as does Scedosporium apiospermum/Pseudallescheria boydii.

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Friday, 19 October 2012

Aspergillus calidoustus

Aspergillus calidoustus - Fungus (Aspergillus ustus)

Note: A. calidoustus has previously been identified A.ustus.
A typical representative strain of Aspergillus ustus had been difficult to select as isolates exhibited a great deal deal of phenotypic variation, and more recently, demonstrable genotypic variation. As a result, recent taxonomic changes have shown there are currently eight separate species which comprise the Aspergillus section usti including the novel species A.calidoustus. Re-examination of clinical isolates previously identified as A.ustus has shown that a great number are actually attributed to A.calidoustus. As molecular analysis would be employed to speciate isolates with confidence, a procedure not routine to most clinical laboratories, I have included both names in this post.

Ecology: One source i states that Aspergillus ustus is among the most ubiquitous soil species, found more frequently in tropical and subtropical areas. For Aspergillus calidoustus in particular, another source states that it is present in low numbers in soilii.

Macroscopic Morphology: Aspergillus calidoustus exhibits rapid growth and will mature in about three days. Colonies may show variation in surface colour from a yellowish brown to a drab olive with possible greys. They may show a lighter coloured outer edge and droplets of purple exudate may appear on the surface of the maturing colony. The reverse is a yellowish brown colour. Texture was even and rather velvety.

Aspergillus calidoustus on SAB, ~5 days at 30o C.

Microscopic Morphology: Aspergillus calidoustus produces septate hyphae from which smooth-walled conidiophore stipes extend. Conidiophores may develop a brownish colour as they mature. Stipes are rather short (130 µm to 300 µm), though the ones measured on the isolate discussed here did not exceed about 150 µm in length. Vesicles, subspherical in shape, also were rather small (~7 µm to 15 µm dia.) From the vesicles, biseriate conidiogenous cells extend with the metulae being slightly shorter than the phialides. These conidiogenous cells cover the upper half to three quarters of the vesicle and produce round conidia (3.0 µm to 4.5 µm dia.) bearing a noticeably rough wall. A distinguishing feature is the irregular shaped and elongated Hülle cells this fungus is capable of producing, however, the production of Hülle cells may be photosensitive and some searching may be required.

Note:  All photos which follow were taken from a slide culture of the fungus using the DMD-108 Microscope/camera.

Aspergillus calidoustus (LPCB X250)
Hyphae with stipes bearing fruiting structures

Aspergillus calidoustus (LPCB X400)
Two Stipes (Conidiophores) bearing fruiting body

Aspergillus calidoustus - a closer look (X400+10, LPCB)
At this magnification the vesicle, conidiogenous cells & conidia are becoming more distinct.

Aspergillus calidoustus - yet closer (X1000+10, LPCB)
Aspergillus calidoustus - Biseriate structue of the conidiogenous cells (metulae & phialides) are visible. (X1000+10, LPCB)

Aspergillus calidoustus - more of the same.  The rough surface of the conidia is evident in this and some of the previous photos.
(X1000+10, LPCB)

Aspergillus calidoustus - one more photo showing the rather small vesicle, the biseriate conidiogenous cells and rough conidia.
(X1000+10, LPCB)

Aspergillus calidoustus - stepping back a bit, here we see the full length of the stipe (conidiophore) and can judge the size in comparison to the 100 micron bar at the upper right of the photo.  A 'foot cell' appears at the base with the fruiting structure at the apex.  (X250, LPCB)

Aspergillus calidoustus has rather short stipes.  Published sources state that the length varies between 130 µm to 300 µm.  My measurements in the above photo were somewhat shorter, between 70 µm and 120 µm in this field. (X400, LPCB)

Aspergillus calidoustus - at the apex of the conidiophore there is a rather small vesicle (~6.7 µm here) from which extend the metulae and phialide structures which comprise the biseriate conidiogenous cells that produce the conidia.
(X400+10, LPCB)

Aspergillus calidoustus - Biseriate structure bearing rough walled conidia are seen in this photo.  Note the rather small vesicle from which the conidiogenous cells extend.  (X1000+10, LPCB)

Aspergillus calidoustus - yet another view (X1000+10, LPCB)

Pathogenicity: Aspergillus calidoustus, though not highly pathogenic, is opportunistic and emerging as an infectious agent particularly in immunocompromised patients. A.calidoustus is capable of colonizing water distribution systems and has been isolated from heated water installations. This may be of concern in health care facilities where colonized water systems may be a source of infection for susceptible patients. Sites of infection range from pulmonary, heart, cutaneous (particularly burns) to isolation from otitis media. A.calidoustus may exhibit increased resistance to some antifungal drugs however that discussion is beyond the scope of this post.
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ii Medically Important Fungi (5th ed.) -A Guide To Identification: Davise H. Larone, ASM Press, 2011
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Friday, 5 October 2012

Cladophialophora species

Cladophialophora species  (Cladosporium species?) Fungus

Note:  This fungus was isolated as a plate contaminant and initially thought to be of the Cladosporium species however certain features discussed within this post left me to question the initial identification.  Recently, several of the more pathogenic species of Cladosporium have been reclassified under the genus Cladophialophora which this isolate appears to more closely resemble.  One of the more pathogenic species, Cladosporium bantiana was reclassified as Xylohypha bantiana and most recently as Cladophialophora bantiana.  Because of the serious pathogenicity of this particular species, most textbooks advise not making slide cultures of this fungus.  Unfortunately you really can’t identify what you have until you examine it by adhesive tape preparation or slide culture.  All preparations were carried out in a laminar flow biological safety cabinet and the organism was suspended in Lactophenol Cotton Blue of which the phenol component is lethal to fungi.  Utmost safety was observed in growing, working with and disposing of this and all isolates.

I remain uncertain of the genus/species of this fungus and certainly would welcome comments on the organism discussed below.
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Ecology: Both Cladosporium species and Cladophialophora species are cosmopolitan fungi, widely distributed in soils and decaying plant material.  Cladosporium species are often found as laboratory media contaminants as was this probable Cladophialophora species.

Macroscopic:  The colony was very slow growing at 30oC and was approximately 2 cm in diameter after 14 days of growth.  The colony was had a velvety texture and was brownish-black in colouration.  The reverse was a rather nondescript black.  An important feature was that this isolate also grew well at 42oC.

 Cladophialophora species on SAB grown at 30oC for 14 days

Microscopic: Septate hyphae were observed which had a brown to black appearance.  Conidiophores did not appear significantly differentiated from the hyphae.  Moderately long chains of conidia extended from the conidiophores which also showed moderately frequent branching.  Both of these features are important in distinguishing the highly pathogenic Cladophialophora bantiana, other Cladophialophora species and the relatively non-pathogenic Cladosporium species.  Cladophialophora bantiana is described as having rather long, wavy and sparsely branched chains of conidia while other species (eg. Cladophialophora carrionii) show moderate branching with shorter conidial chain length.  Cladosporium species show the greatest amount of branching and have shorter conidial chain length. In this regard, the isolate shown here best fits the description of a Cladophialophora species other than bantiana.  The chains of Cladosporium are said to be rather fragile, easily liberating individual conidia.  Chains on the isolate presented in this post did not disarticulate easily but remained intact during manipulation.

 Cladophialophora species: First impression - edge of slide culture at low power
(~ 72hrs, LPCB, 250X, DMD-108)

Cladophialophora species -septate hyphae with branching.
(LPCB, Nikon 400X)

 Cladophialophora species (?) -Fairly extensive branching which is a feature of Cladosporium species.
(LPCB, 400X, DMD-108)

Cladophialophora species - Hyphae with poorly differentiated conidiophores bearing chains of eliptical (oval) conidia.  Note the smaller conidia at the tips of several chains.  The conidia are produced 'acropetally' (with the youngest conidia produced at the apex (tip), as opposed to being produced and extended at the base. Dark pigment showing through the LPCB stain on some of the chains of conidia.        (Note 100 µm bar at top right of photo -LPCB, 400X, DMD-108)

Unicellular conidia vary in size from about 2-5 µm to 4.5- 10 µm depending on the species.  The conidia of Cladosporium species are ellipsoid to round in shape.  Conidia of Cladophialophora bantiana and Cladophialophora carrionii are ellipsoid in shape while Cladophialophora emmonsii have a ‘bent’ appearance and Cladophialophora boppii are fairly round in shape.

  Cladophialophora species showing branched chains of oval conida extending from main hyphae.  Only a few free conidia are present (inset) and these show a hilus (H) where the conidia once were attached.  Darker pigment visible in the older released conidia.
(LPCB, 400X, DMD-108)

Cladosporium species also exhibit ‘shield cells’, named for their appearance (as in sword & ‘shield’- see diagram which follows below).  Conidia formed by Cladosporium species show a prominent black scar (hilum) at their point of attachment.  Both features are absent or at best questionable on the isolate discussed on this post.

Cladophialophora species - branched chains of oval conidia
(LPCB, 1000X, DMD-108)

Cladophialophora species - branched chains of elongated oval conida extending from hyphae.  Smaller conida at apex as the conidia develop acropetally (youngest at tip).  Convincing 'shield cells'  are not seen in any of the photos presented in this post which is evidence against it being a Cladosporium species.  Very few free conida are noted suggesting the chains are not easily disrupted, suggesting again that the isolate is a Cladophialophora rather than Cladosporium species.
(LPCB, 1000X, DMD-108)

Cladophialophora species or Cladosporium species? - the same isolate but here is the only cell I found in all my photos (too many to post) that somewhat resembles a shield cell (arrow).  The moderate to extensive branching does favour the Cladosporium species identification. 
(LPCB, 1000+10X, DMD-108

*Variable29% grow up to 40oC
**Table borrowed & modified from Larone, 1989
About 86% of Cladosporium species can liquefy gelatin as well as grow in the presence of 15% NaCl which distinguishes this fungus from Cladophialophora species.

Cladophialophora bantiana tolerates cycloheximide, assimilates nitrate and produces urease.  The unknown isolate discussed here was not tested against cycloheximide.

Pathogenicity:  Cladosporium species are generally considered to be non-pathogenic, however, as with most organisms, immunocompromised individuals may be at greater risk at developing an infection.
Many species previously grouped under the Cladosporium genus have been recently reclassified as Cladophialophora species.  Cladophialophora now appears to encompass the pathogenic species.

1.      Cladosporium sp.– generally non-pathogenic
2.      Cladophialophora carionii – causes chromoblastomycosis
3.      Cladophialophora bantiana – causes cerebral phaeohyphomycosis (neurotropic, causing brain abscess.)  Infection is frequently fatal.  Has been isolated from pulmonary sources.  Highly infectious even in immunocompetent hosts.
4.      Cladophialophora emmonsii – rare cause of subcutaneous phaeohyphomycosis
?       Cladophialophora species (post isolate) – found as contaminant, pathogenicity unknown but interestingly this isolate grew well at 42oC as does the pathogenic Cladophialophora bantiana.

In summary, the isolate presented here shows moderate to extensive branching which leans towards Cladosporium species as the identification.  However, the rather delicate structure with the absence of shield cells points more towards Cladophialophora species. Chains of conidia are rather long and not so easily disrupted which again adds evidence favouring Cladophialophora species. The clincher for me was that this isolate grew with ease at 42oC, a temperature at which Cladosporium and most other Cladophialophora species are inhibited.  As such this isolate appears to resemble Cladophialophora species more than it does Cladosporium species and possibly the highly pathogenic Cladophialophora bantiana as it is the only one I'm familiar with that grows at 42oC.