Thursday, 3 July 2014
Malassezia furfur complex
Malassezia furfur complex (Yeast)
Note: While reading a gram stain taken as the swab of the ear canal, I noticed a number of yeast cells which appeared to show broad-based budding. A Sabouraud-Dextrose media plate (SAB) was added to the routine culture to better isolate any yeast present. When no yeast grew after 24 hours incubation, my suspicion of Malassezia furfur complex was confirmed. Culture of the yeast is not necessary for confirmation M.furfur complex as its appearance (broad based mono-polar budding), location of isolation (lipid-rich ear canal), and lack of growth on basic mycological media is generally considered sufficient evidence. Never having tried the traditional olive oil overlay technique, I thought I’d shot on this specimen just for the fun of it. The photos that follow are the result.
History: Malassezia yeast was first identified by the French scientist Louis-Charles Malassez in the late 19th century. Currently, the Malassezia furfur complex is thought to be comprised of 14 Malassezia species, eight of which have been associated with humans. Of the eight, M.furfur, M.sympodialis, M.globosa and M.restricta are the most common. Another Malassezia species, not part of the complex, is Malassezia pachydermatis. M.pachydermatis is not lipophilic and while it can on occasion be isolated from human skin, it is most often associated with the ears of canines (dogs).
Ecology: Malassezia furfur is a lipophilic yeast (need lipids/fatty acids) to grow, therefore habitats are limited to where exogenous source of lipids are available. Malasszia furfur can be found as a saprobe, living on the excretions of normal skin flora on over 90% of healthy adults. Usually acquired at an early age, it prefers the oilier parts of the skin such as the scalp and ear canals.
Pathogenicity: While Malassezia furfur complex can be found as part of the normal human skin flora, members are implicated in a variety of diseases. Hyperhydrotic individuals (excessive sweating) may develop a disorder known as pityriasis versicolor[i] (tinea versicolor) and Pityriasis folliculitis[ii]. It has been implicated in seborrheic hyperkeratosis[iii] , and more recently as a causative agent of seborrhoeic dermatitis[iv]. So, under certain, poorly understood conditions, Malassezia furfur complex may cause or accentuate various skin conditions.
Catheter associated infections are commonly seen in neonates and adults receiving prolonged intravenous lipid supplements. Immunocompromised individuals and those with other underlying conditions may find themselves at increased risk to Malassezia furfur complex infection.
Colonial Morphology: Malassezia grows fairly rapidly, maturing in about 5 days at 30 -35˚C. It has a rather narrow temperature growth range as it grows poorly at 25˚C and some species will not grow above 37˚C. Colonies are cream to yellowish-brown in colour. They appear smooth and pasty, often becoming brittle and wrinkled as they age. The Margin can be entire or lobed. As already mentioned, lipid supplement is required for growth and in culture this can be as simple as adding an olive-oil overlay to the Sabouraud-Dextrose surface. Specialized media, specifically for the isolation and growth of Malassezia species is commercially available. Malassezia is resistant to cycolohexmide and therefore will grow on selective media such as Mycosel™ & Dermasel™. Urea test is positive.
Malassezia furfur growing on a SAB plate which was overlaid with a thin layer of olive oil. The olive oil is rich in long chain fatty acids which in nature is supplied by the host environment. The SAB agar is prepared using water which repels oil. However carefully you spread the oil, it will separate into individual droplets. The M.furfur will grow and produce colonies around or near the oil droplets where it can obtain the nutrient. (other techniques are possible)
(Mycosel™ Agar, 30˚C, 72 hrs, Nikon)
Malassezia furfur inoculated onto SAB media with olive oil supplement (left) and without olive oil supplement (right). As is evident, growth only occurs where the olive oil supplies the long-chain fatty acids. The slight haze (growth?) may be explained by the yeast continuing to grow for a short period of time on the reserve of fatty acids carried over from the original source. Once depleted, growth stops. The first photograph was taken of the yeast growing on Mycosel™ agar which shows that M.furfur is resistant to cycloheximide. In comparison between these two photos, there appears to be better growth on the Mycosel than the SAB. This may simply be due to the inoculation load, that is a heavier inoculum placed on one than the other.
(SAB, 30˚C, 72 hrs, Nikon)
Microscopic Morphology: On initial examination of material taken from the patient (skin swab or scrapings), both yeast and hyphal forms may be present. This appearance is often referred to as “spaghetti & meatballs” when both round & linear elements are seen mixed together. Hyphal elements are usually absent on culture but rudimentary forms may occasionally be seen.
The yeast-like cells (1.5 µm – 4.5 µm X 3 µm – 7 µm) are actually phialides and may show small collarettes, though they may be difficult to discern with the light microscope. The cells are referred to as being unipolar, with one end round and the other somewhat blunt, where bud-like structures form singly on a broad base. (Relative size of the budding base differs between M.furfur complex species).
Note: While I cannot say which species of Malassezia furfur complex this is, it has to be one of the eight species associated with disease in humans. From this point on, I will refer to this yeast as M.furfur for simplicity, however the reader must realize I am referring to M.furfur complex.
M.furfur complex as seen in the original gram of a child's ear swab. Arrows point to the individual yeast cells amongst some cellular debris. The 'B' points to one yeast cell which is exhibiting broad based budding. "Broad" in that the daughter cell may appear to be attached to the parent cell by a connection, perhaps by up to half the width of the cell. Other yeast such as Candida albicans may exhibit a narrow, almost point like area of attachment before the daughter cell is 'pinched off'.
(Direct gram Stain, 1000X, DMD-108)
M.furfur direct gram stain as above but at a higher magnification. Without the arrows, can you pick out the yeast cells? (Direct gram stain, 1000+10X, DMD-108)
M.furfur seen in a potassium hydroxide suspension. The appearance is that of typical yeast.
(KOH, 400X, Nikon)
M.furfur -arrows point to cells with broad-based budding
(KOH, 400+10X, DMD-108)
M.furfur - Broad based budding evident between daughter and parent yeast cell.
(LPCB, 400X, Nikon)
M.furfur - Broad based budding again seen
(Gram from culture, 1000X, Nikon)
M.furfur -daughter cell about to split from mother cell.
(Gram from culture, 1000X [cropped], Nikon)
Note: I applied a drop of olive oil to a Sabouraud-Dextrose agar plate (SAB) and spread it as evenly as possible over the surface. This is most effectively accomplished using a 'hockey stick' (Canadian, eh?), which is just a plastic or glass rod bent in the shape of an ''L". The moisture in the agar media repels the olive oil to some degree as it can be seen beading up in the plate photos. Sufficient oil remains distributed to allow growth and growth may be seen greatest around the globules of oil. An alternative method is to soak some sterile filter-paper discs or strips in olive oil and lay them on the agar surface. Growth will be observed closest to the impregnated disc edge where the lipid is available. As an afterthought, I was going to try this for photographic purposes but regretfully, I had already discarded the isolate. Perhaps I’ll try this next time.
[i] Pityriasis versicolor –a condition characterized by a rash on the trunk and proximal extremities, usually caused by M.globosa of the M.furfur complex, and occasionally by M.furfur itself. May be more prominent in hot, humid environments. Results in pale, dark-tan or pink patchy skin.
[ii] Pityriasis folliculitis – Proliferation of Malassezia yeast within hair follicles resulting in acne like eruptions particularly on the trunk.
[iii] Seborrheic hyperkeratosis – a pigmented waxy or scaly raised growth which may resemble skin cancer, however it is benign. It occurs more frequently in older adults.