Wednesday 30 October 2013

Exserohilum rostratum

Exserohilum rostratum (Mould)

Exserohilum species are dematiaceous fungi (ie black mould), widely distributed in nature.  They are cosmopolitan, commonly found on many plants and grasses and can also be isolated from soils and water.

Exserohilum species are unlikely human pathogens.  Exserohilum species have been implicated in phaeohyphomycosis[i]. Most commonly they are isolated from nasal sinuses (sinusitis) and the eye (keratitis) after a scratch or traumatic injury.

In 2012, Exserohilum rostratum was implicated as the primary pathogen isolated from injectable methylprednisolone. Numerous patients receiving steroid therapy primarily for degenerative lumbar-disk and joint disease in the U.S., developed meningitis after injection. While normally not invasive, the fungus will cause illness when directly injected into the body. While sources vary in statistics, injectable methylprednisolone contaminated with Exserohilum rostratum appears to have been responsible for sickening upwards of 700 people, 30 of which died.  Many others continue to have ongoing neurological problems associated the infection or subsequent therapy.

Macroscopic Morphology:
E.rostratum exhibits rapid growth and matures within 4 to 5 days.
Surface growth is grey in colour, quickly darkening with the production of melanin, eventually developing shades from olive to brown to black.
The texture is woolly or cottony in appearance.

Exserohilum rotratum SAB 30oC, 72 Hours

Exserohilum rostratum SAB 30oC, 1 Week

Microscopic Morphology:
Hyphae are septate and darken with the development of melanin.  Conidiophores are rather long (up to 200 - 230 µm in length, 5 – 8 µm wide) and are also septate.  Conidiophores exhibit sympodial geniculate growth, where conidia are produced at bends (geniculate) as the conidiophore extends.  This gives the conidiophore a knobby, zigzag appearance where the conidia attach.  The mature conidia (ave. 14 X 80 µm or greater) are straight to slightly curved and are fusiform or ellipsoidal in shape with rather smooth walls.  Conidia are compartmentalized with between 7 to 11 septa and has a distinctive protruding dark hilum (scar) at the base where once attached.
More specifically, the conidia are ‘poroconidia’, a distinction where the conidia are produced through the extrusion or extension of the inner walls of the conidiogenous cells through a pore or channel.

 Exserohilum rostratum -First look.  Free conidia.  Tape mount at 250X (LPCB, DMD-108)

Exserohilum rostratum -Conidia attached to septate hyphae (400X, LPCB, Nikon)

Exserohilum rostratum - Large, compartmentalized conida which are somewhat fusiform in appearance and may appear slightly bent or curved.  (400X, LPCB, DMD-108)

Exserohilum rostratum - as above.  Showing variation in size and shape of conidia.  Brown pigmentation due to the production and accumulation of melanin. (400X, LPCB, DMD-108)

Exserohilum rostratum - Conidia attached to conidiophore.
(400X, LPCB, DMD-108)

 Exserohilum rostratum - as above, conidia attached to conidophore seen extending through the camera's plane of focus.  (400X, LPCB, DMD-108)

Exserohilum rostratum - a closer look at the septate conidiophore and the attachment of the conidia to the conidiophore.  Note the bent or zig-zag location on the conidiophore at the point of conidial attachment.  This appearance us referred to as geniculate growth.  (400X, LPCB, Nikon)

Exserohilum rostratum - Pigmented, fusiform shaped conidia, usually containing between7 to 11 internal septa. Note the prominent projection or hilum (arrow) which remains at the point of the attachment to the conidiophore. (400+10X, LPCB, DMD-108)

Exserohilum roatratum - single conidium with hilum visble on right side.  Length reads 76.01
µm. (400+10X, LPCB, DMD-108)

Exserohilum rostratum - another view of the geniculate growth / attachment of the conidia to the conidiophore. (400X, LPCB, DMD-108)

Exserohilum rostratum - Single conidium attached to conidiophore.
(1000X, LPCB, DMD-108)

Exserohilum rostratum - okay, I like photos!  Another loose conidium which is slightly bent.  This one has seven, possibly eight compartments.  The conidium is smooth walled and again, the hilum is clearly visible at one end.  (1000X, LPCB, DMD-108)

Exserohilum rostratum - More photos, just for the beauty of this organism.
(1000X, LPCB, DMD-108)

Exserohilum rostratum

Exserohilum rostatum - geniculate (zig-zag) conidiophore after the conidia have dispersed.
(400X, LPCB, DMD-108)
Exserohilum species may be confused with  Bipolaris and Drechslera species however Exserohilum has the protuberant hilum.

[i] A particular presentation of a fungal infection in tissue caused by certain dematiaceous fungi.  This presentation may be an initial clue as to the particular fungus responsible for the infection.  'Google' the term for a better definition.

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Saturday 19 October 2013

Trichophyton terrestre

Trichphyton terrestre (Fungus/Mould)

Trichphyton terrestre is a cosmopolitan (found everywhere), geophilic (soil loving) fungus.
It may also be recovered as a saprobe (living on dead organic matter) on the fur of small animals, presumably picked up from soils.

Trichophyton terrestre fails to grow once 35ºC to 37ºC is reached
As it fails to grow at human body temperature, there have been no reports of human infection by this fungus, nor has it been implicated in animal infections.
T.terrestre may occasionally be encountered as a laboratory contaminant.
Correct identification is important so as to not confuse it with pathogenic dermatophytes.

Macroscopic Morphology:
  • Trichophyton terrestre exhibits moderate growth at 25ºC, maturing in about 8 days.
  • Colonies expanded in diameter rather slowly.
  • Colonies were off-white to light in colour.
  • Reverse appeared yellowish to ochraceous, or even slightly reddish in colour.
  • Texture was felty to powdery
The isolate presented here developed a pale to golden yellow exudate on prolonged incubation which was not reported in other sources.

 Trichophyton terrestre on SAB, 15 days incubation at 30˚C

Trichophyton terrestre on SAB,  25 days incubation at 30˚C

Microscopic Morphology:
  • Trichphyton terrestre produces hyaline (clear, non-pigmented), septate hyphae.
  • Microconidia (4 – 7 µm by 1 -5 µm) are tear-drop to slightly club-shaped.
  • They are borne directly from the vegetative hyphae or are found on pedicles (stalk).
  • Macroconidia (4 – 5 µm by 8 – 50 µm) have smooth, thin walls and usually contain between 2 to 6 cells or divisions internally.
  • Macroconidia are cylindrical (parallel sides) or slightly clavate (club) shaped.
There may not be an obvious distinction between what may be called micro or macro conidia.  (ie. The two are not clearly differentiated.)
Free micro (& macro conidia) exhibit a truncate base or basal scar at what was their point of attachment.

Trichophyton terrestre - Adhesive tape preparation, 250X, LPCB (Nikon)

Trichophyton terrestre - Branching with development of Macro & Micro Conidia.
 LPCB 400X (DMD-108)

Trichophyton terrestre -as above
 LPCB 400X (DMD-108)

 Trichophyton terrestre - free macro & micro conidia
400+10X, LPCB (DMD-108)

Trichophyton terrestre - Conidia stain darker with the LPCB than the hyphae usually do
1000X, LPCB (DMD-108)

Trichophyton terrestre - Here again, the conidia at the tips of the hyphae (conidiophore) can be seen as staining a darker blue than the hyphae themselves.  Measurements shown (inset) are for the conidia and hyphe.  I regret that I didn't just measure the length of the conidia alone.
1000X, LPCB (DMD-108)

Trichophyton terrestre -extensive branching at near right angles.
1000X, LPCB (DMD-108)

Trichophyton terrestre -more of the same.  Darker blue conidia are seen at the end of the hyphae, branching at near right angles.
1000X, LPCB (DMD-108)

Trichophyton terrestre - divisions can be seen in some of the developing conidia (macroconidium)
1000X, LPCB (DMD-108)

Trichophyton terrestre -conidia staining a darker blue with the LPCB stain.  Divisions can be seen in the conidia.  The one on the lower left of the photo clearly has two.
1000X, LPCB (DMD-108)

Trichophyton terrestre -free macroconidium (4 septations or 5 compartments)
1000X, LPCB Nikon (appears larger due to cropping of photo)

Physiological Characteristics:
  • Hair perforation test is POSITIVE
  • Urease is POSITIVE
  • BCPCG Media reaction is POSITIVE
  • No growth at 35ºC to 37ºC
Trichophyton Agars:
Good growth on all Trichophyton agars.  No special growth requirements are required for growth.
Caution: on early growth, the fungus may somewhat resemble a Chrysosporium species.  Chrysosporium’s conidia generally do not exceed two cells in length.

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Entamoeba polecki

Entamoeba polecki

This amoeba most likely has worldwide distribution and can be found as an intestinal parasite in pigs and monkeys.  It is generally considered to be non-pathogenic for humans but has occasionally been recovered from patients with loose stools and intestinal discomfort.  Several sources have stated that in Papua New Guinea, E.polecki has been recovered as the most common intestinal parasite from the inhabitants.  Living closely with these animals my increase the chances of carrying the parasite.  E.polecki makes its home in the large intestine of man.

E.polecki trophs have an average size of 12 µm to 18 µm but can range from 10 µm to as large as 25 µm.  Its cytoplasm can be vacuolated and may contain ingested bacteria and yeast.  The size and cytoplasmic appearance may cause it to be confused with Entamoeba coli.  The karyosome may not be visible which has the nucleus appear empty.  When present, the karyosome is usually minute and centrally located, or nearly so.  It however may occasionally appear diffuse.  Peripheral chromatin can vary from being fine and evenly distributed as in E.histolytica, or coarse and irregularly distributed as in E.coli.  This variability may make diagnosis a challenge.

E.polecki cysts are about 9 µm to 15 µm but can be as large as 24 µm and usually contain only one nucleus (usually E.histolytica has 4, E.coli has 8).  It has rarely been reported as being binucleate.  The karyosome again can be minute can compact or it may be larger and diffuse – it may be centrally located within the nucleus or somewhat eccentric.  Peripheral chromatin may be delicate to coarse but is evenly distributed on the nuclear membrane.  Chromatoidal material is usually abundant but highly variable in shape and size.  It may be present as larger rods with rounded or splintered ends and they may be arranged parallel to each other in the cyst.  Unique to these cysts may be an inclusion body of variable size.  It stains a monochromatic grayish-purple with Iron hematoxylin stain or greenish with the trichrome stain.  In an iodine stained preparation it usually appears light brown in contrast to the intensely dark brown staining of glycogen vacuoles.  The nature of these inclusions is not known but does not appear to be glycogen.

Differentiating the E.polecki trophozoites from E.histolytica and E.coli may be difficult as the size ranges overlap and both the cytoplasmic and nuclear appearance can mimic both.  If examining a specimen that has amoeba showing characteristic of both E.histolytica & E.coli, the presence of E.polecki should be considered. Chromatoid bodies are usually are more numerous and show greater pleomorphism in E.polecki than E.histolytica.  E.polecki cytoplasm often stains very darkly making it difficult to see the nucleus, inclusion mass and chromatoid bodies in the same plane of focus

Note: The photographs which follow were of E.polecki found in a fecal specimen obtained from a patient with gastrointestinal discomfort.  Only trophozoites were present in the sample.  I’m rather unhappy with the quality of the photos I took and present here.  The features lack resolution and the amoeba appears darker in the photos than they did when viewed through the light microscope.  The features appear to be over saturated in colour and “bleed” together, obscuring details.  I was unable to correct for this satisfactorily with photo software post exposure.  This specimen also contained Entamobea hartmanni trophs.  Identification was confirmed by our provincial public health laboratory. Here they are for what they’re worth.

All photos below were taken using the Nikon 'Coolpix' camera at 1000X magnification and are from an Iron-Hematoxylin stained preparation.

E.polecki trophozoite exhibiting a coarse cytoplasm.  Peripheral chromatin in the nucleus is rather evenly distributed.  Karyosome is central but not distinct.

E.polecki trophozoite showing rather evenly distributed peripheral chromatin in the nucleus and as above the karyosome is rather diffuse and slightly eccentric.

E.polecki trophozoite showing a rather coarse cytoplasm with ingested material (bacteria).  The karyosome shows even, dense peripheral chromatin but the karyosome appears to be absent in all planes of focus. Above the E.hartmanni troph may be a somewhat distorted Blastocystis hominis.

E.polecki troph as above.  "Dirty" cytoplasm, even dense peripheral chromatin in the nucleus with a somewhat diffuse karyosome, slightly eccentric.

This specimen contained both E.polecki trophs and E.hartmanni trophs.  Note the size difference.

E.hartmanni troph and a second cell with too little detail to identify confidently.

E.policki troph (inset left) to demonstrate the size difference between it and the E.hartmanni troph.

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