Saturday, 23 February 2013

Arthrographis species

Arthrographis species (Mould)

Ecology:  Arthrographis is widespread, found in compost, decaying plant material and in the soil.

Pathology:  Arthrographis is generally considered to be a contaminant. Arthrographis kalrae has possibly been implicated in eye and central nervous system infections as well as onychomycosis (nail infections).  A case of ethmoid sinusitis has also been reported.  Arthrographis cuboidea* has been isolated from bronchial wash specimens however its pathogenicity remains unsubstantiated.  Other recognized species are A.lignicola, A.pinicola, A.sulphurea and A.alba however they have yet to be implicated as human pathogens.

*Recent molecular study appears to have reclassified this fungus, now known as Scytalidium cuboideum.  I will use the previous name throughout this post as I have found but a few references to this change.

Physiology:  Arthrographis cuboidea exhibits relatively fast growth at 25 – 30oC, maturing in about 3 to 5 days.    It grows equally well at 37oC at which temperature it may develop a pink to lavender pigment on prolonged incubation, however it fails to grow at 45oC  A.cuboidea possesses strong cellulolytic activity which can cause a pink coloured ‘spalting’ in many species of hardwoods.
 Arthrographis kalrae exhibits slow growth between 25 - 30oC, maturing within 1 to 3 weeks. Growth may be enhanced at 37oC, perhaps giving it some advantage as a human pathogen.   A.kalrae is capable of growing at 45oC which helps to distinguish it from A.cuboidea.  A.kalrae is resistant to cycloheximide.

Macroscopic Morphology:  (Species dependant)
A.cuboidea – rather fast growing as discussed above.  Colonies are generally described as cottony to granular.  Radial ridges or folds may develop.  Colouration is white to pale yellow with a yellow reverse.  A pink to lavender pigment may develop and diffuse into the media on prolonged incubation.
A.kalrae – is initially glabrous, smooth and yeast-like.  Colonies are slightly raised and become velvety or powdery as they develop.  Colour is described as a pale yellow (cream) to yellow-buff to tan.  The reverse is pale yellow to tan in colour.

 Arthrographis species - SAB, 72 hrs at 30oC

 Arthrographis species - Note no noticeable pink or lavender pigment on prolonged incubation.

Microscopic Morphology:  Both species produce hyaline, septate hyphae.  Arthrographis species develop conidiophores which differentiates this species from Geotrichum & Scytalidium[i].  Conidiophores are generally short and can be branched or unbranched.  The arthroconidia which are produced from the conidiophores are smooth single celled and hyaline.  Arthroconidia produced from the conidiophores are rectangular to cylindrical in shape and are usually produced in chains.  With A.cuboidea, arthroconidia formed from undifferentiated hyphae are generally square or rectangular in shape.  A. kalrae may also produce lateral sessile (blastoconidia/aleuroconidia) which may be submerged in the agar and difficult to discern.  Intercalary arthroconidia may arise from undifferentiated hyphae and are generally longer and narrower than those produced from the conidiophores.  Arthroconidia separate by fission through double septa.

Note:  All photos which appear below were taken with the DMD-108 digital microscope.

 Arthrographis species - hyphae bearing conidiophores from which chains of arthroconidia extend.
(LPCB, X400)

Arthrographis species - as above but a closer look.  The larger or somewhat 'swollen' structures (arrows), point to the conidiophores.  Chains of arthroconidia are seen extending from the conidiophores.  (LPCB, X400+10, DMD-108)

Arthrographis species - another look at the septate hyphae, conidiophors and chains of conidia.  Note the 100 µm bar in upper right for scale.
(LPCB, X400)

 Arthrographis species - Branched hyphae with conidiophores and chains of arthroconidia
(LPCB, X400+10)

Arthrographis species - intercalary conidophores,  Chains of arthroconidia have collapsed around the tip.  No, this is not a mixed culture. (LPCB, X400)

 Arthrographis species - long chain of cylindrical or barrel shaped arthroconidia.  Smaller, narrower conidia (intercalary?) seen bunched may be those produced from an undifferentiated hyphae discussed above.  (LPCB, X 400+10)

Arthrographis species - Conidiophores bearing arthroconidia extending from hyphae.
(LPCB, 1000+10)

Arthrographis species - the two types of conidia as discussed above are shown in this photo.
(LPCB X400)

Arthrographis species - An older culture.  Septate hyphae with conidiophores bearing chains of arthroconidia.  (LPCB, X1000)

Arthrographis species - as above
(LPCB, X1000)
Arthrographis species - Again, an older culture - branched conidiophores extend from a hyphae with arthrospores collapsed around tip. 
(LPCB, X1000)

Arthrographis species - longer, narrower intercalary conidia extending from undifferentiated hyphae.
(LPCB, X1000)

Arthrographis species - as above (LPCB, X1000+10)

Arthrographis species - one feature I haven't found mentioned in any of my resource material is the small lateral structure at the tip of the conidiophore.  Any ideas?
 (LPCB, X1000)

Arthrographis species - the curious structure found at the tip of the conidiophore.
(LPCB, X1000)

Arthrographis species - One last view of the curious lateral structure found at the tip of the conidophores of this particular species.  I have not found mention of this structure in any of my resource material.  (LPCB, X1000+10)

Note:  I won’t venture a guess at what specific species I’ve isolated here.  Growth was quite rapid and produced a pale yellow pigment favouring C.cuboidea.  I did not attempt temperature studies.  While the species survived on Dermasil™ media containing cycloheximide, growth was weak and stunted.  Only molecular studies would provide the definitive identification.

[i] Recent molecular study appears to have reclassified this fungus, now known as Scytalidium cuboideum.  I have not found reference to this reclassification nor how the presence or absence of conidiophores is reconciled.

Saturday, 2 February 2013

Helicobacter heilmanii

Helicobacter heilmanii  (Gastrospirillum hominis) -Bacteria

What the #&$*! is this?
While reading gastric biopsy gram preparations for Helicobacter pylori, I came across this interesting gram negative helical bacterium. It differed from the typical 'gull-winged' or wavy appearance characteristic of Helicobacter pylori by having up to eight tightly wound spirals.  Roughly double the size of Helicobacter pylori, it ranges from about 3.5 to 8.0 μm in length and the spirals reach an amplitude of about 1.0 μm.  In my career, this was only my third encounter with this organism, however morphology alone immediately suggested the organism most likely was Helicobacter heilmanii.

Some Background:
Helicobacter heilmanii was previously known as Gastrospirillum hominis after first being described in 1987[i]. Though figures in published literature vary, Helicobacter heilmanii appears to be responsible for less than 1% of infections of the gastric mucosa.  While Helicobacter pylori is transmitted human to human, evidence suggests that Helicobacter heilmanii has an animal reservoir and is probably acquired by close association with household pets or farm animals.  Children also may be prone to infection by H.heilmanii due to closer contact with family pets. This apparent 'zoonosis' also occurs more frequently in poorer socio-economic settings.

Symptoms & Pathology:
Symptoms of Helicobacter heilmanii infection vary from epigastric pain, nausea, vomiting, decreased appetite dyspepsia and chronic gastritis.  Infection with H.heilmanii has been associated with gastric and duodenal ulcers in adult patients. Cellular dysplasia, metaplasia and mucosal atrophy may possibly predispose the patient to gastric carcinoma.

Challenges in Detection and Identification:
Organisms which invade the gastric mucosa usually possess the enzyme urease which can split the urea molecule into Ammonia (NH3) and Carbon Dioxide (CO2).  Tests have been developed which take advantage of this specific reaction.  With the CLOtest (Campylobacter Like Organism test), a very small biopsy tissue sample is place on the CLO media and allowed to react.  If organisms possessing the urease enzyme are present in the tissue, they will degrade the urea molecule as outlined above.  The free CO2 dissipates however the remaining ammonia will raise the media's pH with a resulting colour change; with a negative, no colour change occurs.  The alternative test is the C13 or C14 “Breath Test” which utilizes urea labeled with one of the two isotopes of Carbon. (Carbon-13 is preferential as it is non-radioactive).  A base-line breath level is taken after which the patient ingests a urea drink.  If bacteria are present which possess the urease enzyme, the urea molecule is split into ammonia and carbon dioxide, however now the carbon dioxide is labeled with the C13 isotope.  It can be distinguished from ambient carbon dioxide and the level obtained can be compared to the base line.

Sensitivity and specificity of the CLOtest varies significantly, with the product manufacturer claiming values of 98% & 97% respectively (product insert), to another evaluation having determined values of 77% & 96% respectively[ii]. Our own in-house evaluation generated even less optimistic values.
The C13 Breath Test claims 95% sensitivity and 96% specificity[iii].
Regardless, both tests rely on the bacterium's ability to metabolize the urea as well as the proper administration and interpretation of the test[iv].  It is unclear as to whether H.heilmanii is as consistent and proficient as H.pylori in metabolizing urea and these tests cannot distinguish between the two organisms.
Serological and immunohistochemical tests for H.pylori are available however there is cross-reactivity with H.heilmanii.  As such, current antibody tests also cannot distinguish between the two species and there is no serological test specific to either.
Unlike H.pylori, H.heilmanii cannot be cultured in the routine laboratory setting and no doubt would be time consuming if a viable option.

Microscopic visualization remains the definitive test for detection and identification of Helicobacter species in biopsy specimens.

While concomitant infections with H.pylori and H.heilmanii have been noted, they are exceedingly rare leading to unlikely speculation than one may perhaps exclude the other.

Treatment for H.heilmanii is generally accepted to be the same as for H.pylori; antibiotics and a proton pump inhibitor.  I will not discuss specifics as I will leave therapy to the physicians.  My intent with this post, as with all others, is simply to lay some groundwork for a few pretty pictures.

 Helicobacter heilmanii - Seen here in a gastric biopsy specimen (arrows).  The spiral appearance is much more evident in the enlarged insert.  The other large cells are gastric mucosal cells.  As we are only examining the biopsy for Helicobacter (pylori), which is a gram negative organism, there is no advantage to doing a full gram stain.  A small segment of the gastric mucosa, showing evidence of ulceration, was removed during an endoscopic procedure.  This tissue was mashed up onto a glass microscope slide and stained with carbol fuchsin.  (carbol fuchsin appears to stain these organism more intensely than gram safranin).  My experience has been that these gastric organisms seem to occur in 'pockets' in that you may search a slide for quite some time and not see a single organism, then suddenly, bang! several or even many in one area.  Pass over it and nothing once again.
(Nikon, Carbol Fuchsin Stain, X1000)

Heilicobacter heilmanii - from the same specimen as above.  The resolution is better in this photo with the tight wavey spirals (upwards of 8 in number) quite evident.  Organisms appear larger than above due to my cropping of the photograph.
 (Nikon, Carbol Fuchsin Stain, X1000) 

Helicobacter pylori - Compare the organism in the previous two photographs to Helicobacter pylori in this photo.  The arrow in the inset photo points to just one H.pylori organism showing the curved "S" shape or "gull-wing" shape of the bacillus.  Look carefully, the entire photo is teaming with H.pylori cells.
(Nikon, Carbol Fuchsin Stain, X1000)

Campylobacter jejuni - Compare the Helicobacter species in the previous photographs to Campylobacter sp in the one immediately above.  Campylobacter also exhibits a curved or spiral (helical) morphology.  In fact, Helicobacter pylori was initially called Campylobacter pylori.  Here we can see a greater number of twists or spirals but there are not as many, nor are they as tightly wound as with H.heilmanii.  While C.jejuni is usually isolated from fecal specimens, this photo shows the organism in a blood culture.
(Nikon, Carbol Fuchsin Stain, X1000)

To read more about Campylobacter pylori, check out my Blog post specific to that organism by clicking here to redirect.

[i] Lancet 1987; 2:96
Dent, JC, McNulty CAM, Uff JC. Wilkinson, SP Gear MWL.
Spiral organisms in the gastric antrum.

[ii] Am J Gastroenterol. 1997 Aug;92(8):1310-5
Prospective, multivariate evaluation of CLOtest performance.
Weston AP, Campbell DR, Hassanein RS, Cherian R, Dixon A, McGregor DH.
Department of Veterans Affairs Medical Center, Kansas City, Missouri 64128-2226, USA

[iii] Rev Esp Enferm Dig. 1996 Mar;88(3):202-8.
C13 urea breath test in the diagnosis of Helicobacter pylori infection in the gastric mucosa. Validation of the method.
Pérez García JI, Pajares García JM, Jiménez Alonso I.

[iv] Eur J Gastroenterol Hepatol. 1999 Nov;11(11):1251-4.
The CLO test in the UK: inappropriate reading and missed results.
Prince MI, Osborne JS, Ingoe L, Jones DE, Cobden I, Barton JR.
University of Newcastle Regional School of Medicine, North Tyneside Hospital, North Shields, U

v Practical Gastroenterology, February 2006; 47-50
Clinical Significance of Helicobacter heilmanii Colonizing Human Gastric Antrum
Anup Hazra, Carlos Ricart and Januz J. Godyn
Dept of Pathology & Laboratory Medicine & Dentistry, New Jersey; Robert Wood Johnson Medical School, New Brunswick, New Jersey

vi Tzu Chi Med J, 2004; 16:  No1 59-62
Helicobacter heilmanii of the Stomach – A Case Report
Jeh-En Tzeng, Ying-Lung Lin, Yi-Tsui Chu, Sue-Mei Chung
Department of Pathology, Family Medicine, Buddhist Dalin Tzu Chi General Hospital, Chiayi, Taiwan
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