CANDIDIASIS

SHARADA T RAJAN DEPT OF ORAL PATHOLOGY

Historical aspects
Hippocrates in 600 B.C & Galen in 130 A.D- aphthae in debilitating patients- included thrush. First observed microscopically by Leeuwenhoek in 17C. Langenbeck made the connection bet. Yeasts & oral candidosis- in a case of typhus.

Gruby classified the organism in the genus sporotrichum. Charles Robin- genus Oidium- egg shape of the yeast- Oidium albicans. Zopf in 1880- Monilia albicansMoniliasis Christine Berkhout in 1923- Candida.

Introduction
Study of fungi Mycology. Eukaryotic micro-organisms. Most important fungus of relevance to Dentistry. Oral commensal in about half the general population.

Morphology
Fungi exhibit 2 forms- yeast & mould forms. Dimorphic- exist as both forms at different times. Moulds- natural environment & lab culture. Yeasts- tissue.

Candida albicans in budding yeast form with daughter cell

Yeasts- unicellular with spherical or ovoid bodies. Moulds- multicellular. Hyphae are thread-like structures fungal cytoplasm & its organelles. Structural unit of the moulds. Hyphae divided into units by cross walls by septa. Mycelium- mass of hyphae that forms the mould colony

Candida
More than 50,000 sp of fungi exist but only 100-150 sp cause disease in humans. The largest portion of fungal micro biota is made up of the Candida sp. The genus Candida is a collection of 150 asporogenous yeast sp. & are classified among the fungi imperfecta( inability to form a sexual stage) in the class Deuteromycetes.

Within this class the distinguishing feature of Candida sp. is to form pseudohyphae except C.glabrata. The genus Candida are dimorphic predominantly in the yeast form & exhibit hyphae under certain

TEM of Candida albicans showing a blastospore and a daughter cell

Scanning electron micrograph of C.albicans

Candidal species of medical importance

Shepherd observed that 7 species of Candida are of major medical importance: C.albicans C.tropicalis C.glabrata C.parapsilosis C.stellatoidea C.guilliermondii C.krusei C.pseudotropicalis

Candidal species in the oral cavity

C.albicans 75% C.tropicalis 8% C.krusei 3-6% C.glabrata 2-6% Tongue C.albicans Root canal C.albicans, C.guilliermondii, C.glabrata Oropharynx C.glabrata

Candida albicans
Normal inhabitant of alimentary tract & mucocutaneous region. The yeast cells are globose, short ovoid(57), elongated(4-6x610m). All strains of C.albicans develop thick-walled chlamydospores, pseudohyphae & blastoconidia on cornmeal agar. All other sp. Do not dev. chlamydospores.

Gram stain of candida albicans

Demonstrates Braudes phenomenonserum incubated at 37C for 2 hrsC.albicans grows germ tubes. The colonies on SDA grow rapidly, creamy smooth, pasty within the 1st 3 days. In one month- glistening, waxy, soft, smooth to reticulated. C.dubliniensis isolated from the oral cavity of HIV+ patients is very similar to C.albicans- common oral inhabitant in health & disease.

Candida krusei
Growth on SDA appears flat, dull, dry, cream coloured after 3 days. After 1 month they become greenish yellow, dull, soft, smooth or wrinkled with a dense growth of mycelium. Microscopically they appear cylindrical or as ovoid cells. 3 to 5x 6 to 20m in size.

Candida glabrata

Most common yeast isolated from the skin infections. The colonies on SDA appears cream coloured, soft, glossy & smooth. Microscopically they appear as small round yeasts 2.5 to 4.5 x 4 to 6m. C.glabrata do not show pseudohyphae.

CHROMAGAR plate of C.glabrata

Candida tropicalis

Globose, short ovoid or long ovoid cells- 4 to 8x 5 to 11m. Growth on SDA appear creamy white, smooth colonies after 3 days. After 1 month white to cream coloured colonies become dull, soft, smooth, reticulated or wrinkled with overgrowth of mycelium.

Old cultures become hairy & tough. C.tropicalis may also appear to produce germ tubes in serum. They show a narrowing or constriction at the emergence from the yeast cell which is not seen in C.albicans.

Candida parapsilosis
Short ovoid to long ovoid cells. Growth on SDA appear as smooth, soft, white or sometimes lacy colonies. After 1 month they appear cream to yellow, glistening, smooth or wrinkled.

Candida guilliermondii
Short to ovoid yeast cells with a few cylindrical cells. Colonies on SDA appear as thin, flat, glossy, cream to pinkish. After 1 month they become yellowish to pink glistening, smooth or dull wrinkled.

Candida kefyr

Previously it was known as C. pseudotropicalis They appear as short ovoid yeast cells with a few elongate cells. Pseudomycelium is abundant in most strains. The cells are very elongate, they fall apart & lie parallel to each other like logs in a stream. Colonies are creamy, smooth on SDA, after prolonged incubation become cream to yellow, dull, soft & smooth.

Cornmeal tween 80 plate

Laboratory diagnosis of candida

Smear: Direct examination with Gram stain show characteristic rounded or oval budding cells of yeast blastospores with/ without the hyphal phase. presence of large amounts of hyphaesuggestive of clinical infection.

Swabs
Rub sterile cotton tipped swabs over the lesional area. C.albicans can survive for 24hrs on a moist swab without loss of viability.

KOH & PAS stain

A 10-20% soln of KOH is useful in detecting the fungal elements in the smear. It is mixed in equal proportion with the specimen material on the slide& a cover slip is placed & is heated gently. This prep. clears the debris without damaging the fungal elements. This process req only 10 min after which the slide can be examined.

KOH stain demonstrating the fungal elements

Only a tentative diagnosis can be obtained since the hyphae & the pseudohyphae are indistinguishable from the other dermatophytes. The PAS stain is a special stain used to study the fungal elements. The aldehydes produced by the oxidation of the fungal polysaccharide react with the periodic acid to produce the magenta colour.

PAS stain X 40 x demonstrating candidal hyphae

Grocott methenamine silver stain

Special stain used to detect fungal elements in histologic sections. Deparaffinize the section, rehydrate, oxidize in 5% aqueous chromic acid, water wash. Rinse in Na. metabisulphate.

Water wash - 5 min Distilled water wash place in preheated (56C) silver incubating solution in a dark place upto 1 hr Rinse in distilled water Place in 3% Na thiosulphate 5 min Counterstain with Arzacs stain 15 to 30 sec Blot, dehydrate, clear, mount.

This procedure results in the brownish black colouration of all forms of viable & non-viable fungal cells in a pale green background. The GMS stain is the best special stain for fungal screening. However there is no substitute for microbiological culture.

GMS stain seen under high power demonstrating the candidal hyphae

Culture Sabourauds dextrose agar:

species C.albicans C.krusei 3 days Rapid, creamy smooth, pasty Flat, dull, dry, cream colour Cream colour, glossy Creamy white, smooth 1 month Glistening, waxy, smooth, soft Greenish yellow, dull, smooth, dense mycelium _ Dull, soft, mycelial overgrowth

C.glabrata C.tropicalis C.parapsilosis C.guilliermondii C.kefyr

Soft, smooth, lacy Yellow, glistening white Thin, flat, pink Creamy, smooth Yellow, smooth, dull Yellow, dull, soft

Subcultures on SDA showing pure isolates of C. albicans, C. tropicalis, C. parapsilosis

Germ tube test

Rapid screening procedure for diff. C.albicans from other species. 1st rep. by Reynolds & Braude in1956. Filamentous, cylindrical outgrowth from the yeast cell with no constriction at the base. All strains of C.albicans form germ tubes after incubation at 37c for 2 hrs. C.tropicalis form germ tubes in serum showing a constriction at the emergence of the yeast cell.

Candida albicans showing germ tube formation

Germ tube formation in C.tropicalis

Cornmeal agar growth

This agar without Tween 80 produces chlamydospores in 72 hrs. With Tween 80 they appeared in 48 hrs. Chlamydospores are characteristic of C.albicans. Thick walled asexual spore formed by the rounding of a cell/cells. Dormant forms produced during nutritionally depleted conditions.

Cornmeal agar growth

Cornmeal agar with Tween80 streaked with C.albicans

C.albicans demonstrating chlamydospore growth

Biochemical tests
Carbohydrate assimilation test is a useful test for diff. candida species. Measures the ability of the yeast to utilize a specific CHO as the sole source of carbon in the presence of oxygen. Introduced by Wickerham & Burton in 1948.

-CHO fermentation test

species C.albicans fermentation Glu,L,Ga-acid & gas prod. Su-acid prod.La- not fermented

C.krusei
C.glabrata C.tropicalis C.parapsilosis C.guilliermondii C.kefyr

Glu-gas prod.
Glu-gas prod. Glu, mal, & Su- gas prod.La not fermented Glu- acid & gas Glu & Su- gas prod. Mal & La not fermented Glu, Su & Mal

-CHO assimilation test

Species C.albicans Assimilation Glu, mal, gal assimilated. La not assimilated

C.krusei
C.glabrata C.tropicalis C.parapsilosis C.guilliermondii C.kefyr

Glu only assimilated

Glu, tre only assimilated Glu, mal, tre, ga assimilated. La not assimilated Glu, mal, su, ga assimilated. La not assimilated Glu, mal, su, ga assimilated. Glu, su, la, ga assimilated

CHROMagar candida culture

Distinguishes the species by colour as a result of biochemical reactions. Basis: strongly contrasted colony colours produced reactions of species specific enzymes with a proprietary chromogenic substrate. Colony readings are made after 48 hrs.The colour of the colonies deepened after 72 hrs.

CHROMagar growth characteristics

Candida species C.albicans C.parapsilosis C.tropicalis C.krusei C.glabrata Colony colour Green Cream Blue Pink Pink to Purple

CHROMAGAR

Green colonies c albicans, cream colony c. parapsilosis

Blue colony C tropicalis

CHROMagar plate culture of the candidal species

Candida - commensal
Present in the oral cavity, GIT, anus, groin, vaginal canal & vulva of healthy people. In the oral cavity C.albicans is the dominant species. Tongue is a major reservoir. C.glabrata, C.krusei, C.tropicalis, C.parapsilosis. Opportunistic pathogens- cause disease when host defenses are

Immature or low immunity Reduced salivary output Hypothyroidism Hypoparathyroidism Adrenal insufficiency Iron deficiency Vitamin, zinc deficiency Broad- spectrum antibiotics

Candidiasis
In the past it was considered to be an opportunistic infection affecting the debilitating individuals but now it is recognized that it may also develop in people who are otherwise healthy. The infection may range from mild, superficial mucosal involvement to fatal, disseminated disease in severely immuno-compromised patients.

Pathogenesis
Adherence of the organism to the host surface virulence of the organism. Complex host organism interaction. Terminal sugar in the glycoprotein of the human epithelial cells interact with a lectin-like compound adhesin in the yeast wall van der waals, electrostatic forces. ALS family of gene plays a critical role in candidal adhesion.

14(4):253-267 (2003)

Crit Rev Oral Biol Me

Presence of candidal hyphae contributes to the induction of the infection. The tips of these hyphae secrete proteinases that help in maintaining an acidic environment growth of candida. This association demonstrated by straid et al;1965. This also contributed to the adherence

14(4):253-267 (2003)

Crit Rev Oral Biol Me

It was also demonstrated that these proteinases & lipases were concentrated in the hyphal elements & very negligible in the blastospores. Also the amount of proteinases were more in the invading front of the lesion. These proteinases also acted as keratinases thereby it invades the keratinised mucosa.

14(4):253-267 (2003)

Crit Rev Oral Biol M

Scanning electron micrograph of candidal hyphae penetrating the tissue

Pseudomembranous candidiasis

Also known as the thrush. Presence of adherent white plaques- cottage cheese or curdled milk appearance on the oral mucosa. Composed of tangled masses of hyphae, yeasts, desquamated epithelial cells & debris. The underlying mucosa is erythematous.

Initiated by exposure to antibiotics, impaired immune function. Infants are affected due to their underdeveloped immunity. Symptoms are burning sensation of the oral cavity, unpleasant taste- salty or bitter. The common sites are buccal mucosa, tongue, palate.

Antibiotic sore mouth

Type of erythematous candidiasis. Also called as acute atrophic candidiasis. The white plaque is not a prominent feature. Typically follows a course of broad spectrum of antibiotics. Pt. complains that their mouth feels as if a hot beverage has scalded it. There is diffuse loss of filiform papillae.

Central papillary atrophy of the tongue

One other form of erythematous candidiasis is the central papillary atrophy of the tonguemedian rhomboid glossitis. Earlier it was thought to be a dev. defect. Appears as a welldemarcated erythematous zone affecting the midline, posterior dorsal tongue, asymptomatic, symmetrical. Erythema due to loss of filiform papillae. Resolves with antifungal therapy.

Chronic multifocal candidiasis

Type of erythematous type. Tongue, angles of the mouth, junction of the hard & soft palate. The palatal lesion is an erythematous area which contacts the tongue at rest kissing lesion.

chronic multifocal candidiasis

Multifocal candidiasis with central papillary atrophy of the tongue

Same pt. with kissings lesion in the hard palate

Angular chelitis

Also called as perleche. Erythema, fissuring & scaling of the angles of the mouth. Typically seen in older persons with reduced vertical dimension of occlusion. It is caused by C.albicans sometimes with Staph.aureus

cheliocandidiasis

Candidal infection more extensively involving the perioral skin, usually secondary to actions such as thumbsucking, liplicking etc.cheliocandidiasis

Chronic atrophic candidiasis

Denture stomatitis form of erythematous candidiasis. Erythema, petechial hemorrhage. The patient will admit to wearing the denture continuously. DD: denture base allergy, improper design, inadequate curing of the acrylic.

Denture stomatitis

Chronic hyperplastic candidiasis

Also called candidal leukoplakia. White patch cannot be removed on scraping. Asymptomatic. Common in the anterior buccal mucosa. Some authors feel that it represents candidal infection superimposed on a preexisting leukoplakic lesion. Diagnosis confirmed by the presence of candidal hyphae & complete resolution after anti fungal therapy.

Mucocutaneous candidiasis

White plaques some of which are removable. Commonly occurs in the tongue, buccal mucosa & palate. Rare seen in conjunction with immune dysfunction. The immune problem becomes evident during the 1st few yrs of the life. Pt. begins to dev. candidal infection in the mouth, nails, skin & other mucosal surfaces.

Chronic mucocutaneous candidiasis

J Oral Pathol Med (2007) 36: 5283

Chronic mucocutaneous candidiasis

J Oral Pathol Med (2007) 36: 5283

H & E stain

J Oral Pathol Med (2007) 36: 5283

PAS stain ( 100x)

J Oral Pathol Med (2007) 36: 5283

Sometimes candidal infection is associated with endocrine disturbancesendocrine candidiasis syndrome. Hypothyroidism Hypoparathyroidis m Addisons disease Diabetes

Section of chr. Mucocut. Candidiasis demonstrating candida yeast & pseudohyphae PAS stain

The endocrine abnormality dev. months to yrs after the onset of the candidal infection. The infection remains superficial. The lesions are well controlled by anti fungal therapy.

Histopathology
Organism can be seen in exfoliative cytologic preparation & in tissue sections PAS stain. H/P: increased thickness of parakeratin on the surface of the lesion, elongation of the epithelial reteridges, chronic inflammatory infiltrate adjacent to the epithelium. Small coll. of neutrophilsmicroabscesses seen in the parakeratin layer. Hyphae embedded in the parakeratin layer.

Parakeratosis, neutrophilic microabscess, thickened spinous layer, chronic inflammation

Diagnosis of candidiasis

PAS stain in high power

Tubular hyphae of C. albicans embedded in the parakeratin layer, PAS stain

Treatment & prognosis

Several anti fungal drugs have developed for the management of candidiasis. In most cases oral candidiasis is an annoying superficial infection that is easily resolved by antifungal therapy. If a re-infection occurs, a thorough investigation of potential factors including immunosuppression should be made.