Yeasts

1. Yeasts . Yeasts are fungi that grow as unicellular cells producing daughter cells asexually either by budding or by binary fission or combination of them. Many yeasts reproduce sexually by developing ascospores or basidiospores Yeasts are characterized by a wide dispersion of natural habitats, common on plants (leaves and flowers), soil and salt water. It also found on skin surface, mouth, intestinal tract and vagina in man and animals

2. Classification of yeasts A. Ascomycetous yeasts 1- Candida 2- Blastoschizomycetes 3- Geotrichum B. Basidiomycetous yeasts 1- Cryptococcus. 2- Malassezia. 3- Rhodotorula. 4- Trichosporon.

3. II. Methods used for yeast identification: 1- Macromorphology Colonies are pasty and smooth, mucoid or rough . Their color varies from white, creamy, brown or have carotenoid pigments. 2- Micromorphology on corn meal agar: On corn meal agar yeasts shows blastoconidia pseudohyphae, chlamydoconidia or arthrospores

4. - 3- Physiological characteristics: a) Fermentation: Ascomycetes yeasts ferment sugar in varies mannar while Basidiomycetes yeasts does not ferment sugars. b) Assimilation: Manual methods: Many commercial kits are propagated for yeasts identification which read manually. The most important of them is AP20 cAux Automatic methods: A new series of methods adopted to read automatically through computers as Vitak 2Yst c- Urease Test d- Nitrate assimilation e- Cycloheximide

5. Identification of medical important yeast into genera

6. Genus Candida Contain more than 170 species The most medically important of them are 1- Candida albicans 2- Candida dubliniensis 3- Candida tropicalis 4- Candida parapsilosis 5- Candida krusei 6- Candida glabrata

7. Methods used for Candida species identification 1 -Macromorphology: - Apperance of colonies on SDA. - Color of colonies on chromogenic candida agar. 2 -Micromorphology: - Germ tube in serum. - Chlamydospores and pseudohyphae on corn meal agar or rice agar.

8. 3- Biochemcical and physiological charcters: - Growth on the presence of cycloheximide - Fermentation of sugars - Conventional assimilation - Commerical methods a)manual as API20 b) automatic as Vitak system.

9. Morphological characters of important Candida species

10. Physiological characters of important Candida species

11. Candidiasis in human 1-cutaneous : intertrigo, diaper(napken) and cutaneousgranuloma 2- Nail : paronychia and onychomycosis 3- Oral : thrush 4- Vaginal candidosis and male genital infection 5- Invasive : esophageal, pulmonary, gastrointestinal and candidemia

12. Candidiasis in animals Mycoticrumanitisin cattle and gastritis in calves (area of necrosis surrounded by congestion) Mycotic mastitis caused by different species Mycottic abortionin cattle, horses and sheep caused by C.albicans,C.tropicalis and C. glabrata Thrush in birds the lesion involve mouth,croppreventriculus and gizzard with whitish circular area slough to give superficial ulcer Cutaneous in dogs and cats and keratitis Systemic in cattle with pneumonia,nasal discharge and diarrhea

13. Laboratory diagnosis of Candidiasis I.Microscopic examination 1- KOH 20% or DMSO-KOH specially for skin and nails. 2- Gram stain for gram positive yeast cellsand pseudohyphae. 3- Calcufluor white 1% with KOH and examined under fluorscent microscope 4- periodic acid shiff specially for tissue specimens

14. II.Culture Clinical specimens can be cultured on Sabouraud dextrose agar, potato dextrose agar or brain heart infusion agar and incubated at 25°C and 37°C. The colonies identify by: 1- Microscopic examination after lactophenol cotton blue or Gram-stain. 2- Germ tube test to detect Candida albicans. 3- Microscopic examination after culture on corn-meal or rice agar for demonstration of pseudohyphae and chlamydospores. 4- CHROM agar Candida which give presumptive identification by color of the colonies. 5- Assimilation using commercial method as API20.

15. III. Detection of antibodies In case of invasive candidosis detection of antibodies by slide agglutination test, immunoprecipitation or haemagglutination inhibition test (Fumoze) IV. Detection of antigens Detection of Candida species antigens can done by - The Cand-Tec latex agglutination (Ramco lab). - Platelia Candida antigen, the double sandwich enzyme immunoassay (Biorad France).

16. V.Detection of metabolites of Candida D-arabinital as metabolites of Candida species can be detected in sera of patient VI.Molecular methods Numerous polymerase chain reaction (PCR) based methods have been developed for the detection of Candida DNA

17. Treatment of candidiasis in human 1- Oral candidosis Nystatin oral suspension in enfants and fluconazole in adult 2-Vaginal candidosis Topical by nystatin, clotrimazole or miconazole passeries or oral by fluconazole or itraconazole 4- Skin and nails Oral Fluconazole or itraconazol 5- Systemic candidosis 1)Fluconazole 2) Combination of Amphotericin and fluconazole 100 mg/kg/day 3) Caspufugin intravenously alone or with combination with azoles.

18. Treatment of candidiasis in animals Topical by nystatin,gentian violet or 2% azoles in oral and cutaneous infections Amphotericin I/v or itraconazole oral in mucocutaneous and generlized infections

19. Genus Cryptococcus Genus Cryptococcus contain 38 species, the only pathogenic is C.neoformans C.neoformans is an encapsulated yeast. Mucopolysaccharide antigen of capsule divided it into 4 serotypes A, B, Cand D Cryptococcus neoformans varieties 1- C. neoformans var grubii (serotype A). 2- C.neoformans var gattii (serotypes B and C). 3- C. neoformans var neoformans (serotype D).

20. The varieties differ in their natural habitat C.neoformans var grubii and C. neoformans var neoformans isolated most frequently from soil contaminated with pigeon or other birds droppings. C. neoformans var gatii is associated with eucalyptus trees

21. Filobasidialla neoformans is the teleophase (perfect state) of Cryptococcus neoformans var neoformans and Filobasidialla bacillispora is the teleophase of neoformans var gattii.

22. Methods for identification of C.neoformans Culture on SDA showed white to creamy mucoid colonies Microscopy after India ink capsule appear as clear zone around the cell mostly more than 50% of its diameter. On corn meal agar round or elliptical blastospores, no hyphae, absence of pseudohyphyae. On caffeic medium brown color effect (BCE).

23. Fermentation of sugars negative. Assimilation of sugars positive for glucose, galactose, sucrose and maltose Assimilation of nitrate negative. Urease production positive. Hydrolysis of inositol positive. Cyclohexamide resistance negative.

24. Differentiation of C. neoformans varieties 1 canavanine glycine bromothymol blue agar (CGB) : C. n.var gattii turn the medium from yellow to blue color while in case of C. n. var grubii and C.n. varneoformans the medium remain yellow. 3) Creatinine dextrose bromothymol blue thymine medium (CDBT) : C.n.var gattii grow with blue green color and C. n. var neoformans with orange color while C. n. var grubii fail to grow in it.

25. Human cryptococcosis 1-Pulmonary form Benign with asymptomatic form or symptomatic shows cough, chest pain or mild fever 2-Central nervous system form (cerebral or meningeal form) follow pulmonary infections, the commonest signs are headache, mild fever and signs of increased intracranial pressure as mental changes and ocular diseases.

26. 3-Cutaneous form Secondary or a primary infections. Face, scalp, trunk or limps showed single or multiple nodule turn to ulcer 4- Other forms Osteomylitis with bone lesion, ocular infections with visualless or asymptomatic infection of prostate.

27. Laboratory diagnosis of cryptococcosis I.Microscopy Capsule of C. neoformans can be detected in specimens of CSF or other host fluid by India ink or nigrosin and in tissue biopsy by by periodic acid Schiff or Mayer’s mucicarmin silver stain. II.Culture Cryptococcus neoformans can isolated from CSF, blood, sputum prostate fluid and other specimen by culturing on SDA +C with The mucoid cream to buff colonies identify by:

28. 1 - Brown effect on caffeic medium. 2- Not ferment sugar while assimilate of g,ga,s ,m 3- Hydrolysis of urea and inositol. 4- Differentiation of varieties by culture on CGB agar and COPT medium. III. Detection of antibodies by CFT ,IF or tube agglutination IV. Detection of cryptococcal antigen by LA or ELIZA

29. Treatment of human cryptococcosis a) In immunocompotent patient CNS form: combination of amphotericin and flucytosine followed by fluconazole . Pulmonary and other non CNS forms: fluconazole b) In immunocompromised patient Amphotericin B with fluconazole followed by fluconazole

30. Genus Malassezia The genus Malassezia is lipophilic basidiomycetous yeast composed of 13 species. It is present in human skin and animals as cutaneous microflora. Under the influence of some predisposing factors , it cause pityriasis versicolor, folliculitis, sebonrraeic dermatitis, atopic dermatis or even systemic mycosis in human and otitis or malassezia dermatitis in animals.

31. Malassezia cultivate on Sabouraud dextrose agar with sterile olive oil or on special media as Dixon's medium Malassezia grow within 10-15 days at 31ºC as creamy to tan color, flat or raised, smooth or rough colonies Microscopically, it may be oval, spherical or bottle shaped with broad base or narrow base buds

32. Medically important Malassezia species 1- Malassezia furfur 2- Malassezia globosa. 3- Malassezia sympodialis. 4- Malassezia pachydermatis 5 -Malassezia restricta 6- Malassezia obtusa. 7- Malassezia slooffiae.

33. Methods used for identification of Malassezia species a) Phenotypic 1) Growth on different temperature: Subculturing on Dixon's medium and incubated at 31ºC, 37ºC and 41ºC for 7-10 day then tested for growth. 2) Micromorphology: Film stained with Gram then examined microscopically for the size, shape, base of bud if narrow or broad and occurrence of sympodial buds or filaments.

34. Difference between Malassezia spp.

35. 3) Catalase reaction 4) Lipid assimilation: Assimilation of tweens 20, 40, 60, 80 and caster oil 5) Splitting of esculin b) Genotypic by pcr

36. Human malasseziasis A) Superficial Malassezia infections 1 - Pityriasis versicolor Hypo or hyperpigmented patches covered with fine scales on the upper trunk, neck, upper portion of the arms or other sites usually caused by M.furfur,M.globosa and M.sympodialis 2- Seborrheic dermatitis Seborrheic dermatitis scaly reddish patches or whitish dry flakes localized in sebum-reach area as the scalp, face, trunk, ear and eye lid margin caused by different species.

37. 3- Malassezia folliculate Follicular papules and pustules which are often diffusely scattered on the shoulders and back. 4- Neonatal cephalic pustulosis A non follicular pustular eruption characterized by cephalic location in neonates. B) Deep-seated Malassezia infections Deep-seated Malassezia infections occur mostly in neonates especially those receiving lipid infusions and immunocompromised patients in adults.

38. Animal malasseziasis 1-Malassezia dermatitis in dogs characterized by pruritis,alopacia,erythema and lichenfication 2-Interdigital dermataitis as inflammed skin between feet pads 3-Acne and perioral dermatitis in cats 4-Generalized alopacia with seborrhea oleosa 5-Mycotic otitis externa in dogs Animal malasseziasis usually caused by M.pachydermatis

39. Laboratory diagnosis of malasseziasis 1- Direct microscopy a) Superficial Malassezia infections Pityriasis versicolor characterized microscopically after KOH 10 % by bented short hyphae and collections of spores (spaghetti and meat balls). In seborrheic dermatitis and folliculitis only yeast cells without hyphae are predominant. b) Deep-seated (disseminated) Malassezia infections In disseminated, Malassezia infections with fungimia buffy coat of centrifuged blood used. Film Stained by Gram’s, Giemsa or calcuflour are suitable to demonstrate yeast cells.

40. 2- Culture a) Superficial Malassezia infection Culture of Malassezia is not necessary for routine diagnosis of tinea versicolor or other superficial infections. It may be done to identify the causative species by cultur on Dixon's medium. b) Deep-seated Malassezia infections Culture for Malassezia in disseminated cases is often useful

41. Treatment of human malasseziasis 1) Superficial infection Topical - Ketoconazole 2% shampoo - Chlotrimazole, micoconazole cream Oral - Itraconazole 200 mg/day for one week. - Fluconazole 300 mg/week for two weeks. 2- Deep seated Malassezia Amphotericin B 0.7 mg/kg/day (intravenous). Fluconazole 400 mg/day (oral).

42. Genus Trichosporon Trichosporon is basidiomycetous yeast widely distributed in nature It may be a part of normal flora of human skin, nail, oral and respiratory tract. Trichosporon grows rapidly on almost all standard fungal media, appeared firstly as flat smooth or wrinkled white to creamy colonies then turned waxy with central folds . Microscopically, hyaline mycelium which is septated and fragmented into rectangular arthrospores .Blastoconidia of various size may be formed sympodially on the hyphae.

43. Medically importantTrichosporon species 1- Trichosporonasahii 2-Trichosporonasteroides. 3- Trichosporoncutaneum. 4- Trichosporoninkin. 5- Trichosporonmucoides. 6- Trichosporonovoides

44. Methods used for Trichosporon species identification 1. Macromorphology of colonies characterized by heaped centers and wrinkled surface 2. Micromorphology depending on the presence of arthroconidia or frequency blastoconidia, true hyphae or appressoria formation. 3- Physiological and biochemical characters: Standard items used for identification are: a- Growth at 37 ºc. b- Carbohydrate assimilation used commercial methods as API 20C Aux. c - Hydrolysis of urea. d- Growth in the presence of 0.1% cyclohexamide.

45. Difference between important Trichosporn species

46. Human trichosporonosis 1- White piedra hair of beard, mostache, axillary and scalp affected by white soft nodules 2- Skin and nail infections 3- Ottomycosis mycoticotitisexterna 4- Invasive (deep –seated) characterized by 3 forms - cutaneous - Renal - Pulmonary

47. Animal trichosporonosis 1-White piedra in horses and monkeys 2-Mycotic mastitis in cattle 3-Nasal granuloma, cystitis and dissiminatedtrichosporonosis in cats

48. Laboratory diagnosis of trichosporonosis 1- Direct microscopic examination a) in case of superficial infection hair ,skin or nail treatedbykoh or calcufuor with koh to demonstrate fungal elements b) in case of invasive trichosporonosis tissue biobsy subjected for histopatholoy 2- Culture on SDA then colonies identified by physiological characters or molecular methods 3- In case of invasive trichosporonosis detection of antibodies by serological tests

49. Treatment of trichosporonosis In white piedra shaving or cutting the hair topical azoles Deep- seated trichosporonosis Amphotericin B with combination of flucytosine or voriconazole

50. Geotrichum Genus Geotrichum is ascomycetous yeast comprises from 14 species the only important one is G.candidum.The yeast cause pulmonary geotrichosis superfical infections on the skin, oral mucosa and gastrointestinal tract. G. candidum grow rapidly as white to cream, flat and smooth, yeast-like, which turned mold- like Microscopically showed septated branched hyphae break up into arthroconidia It can identify by morphological characters and API C 20