Review of the 2005 Mycology QAP Results

Transcription

1 Review of the 2005 Mycology QAP Results Royal North Shore Hospital, Sydney Wednesday 28 th June 2006 Dispatch 2005:2:7A,B,C Maria Carino,, SDS Pathology Dispatch 2005:4:7A,B,C Kerry Weeks, PaLMS Dispatch 2005:6:7A,B,C Okcha Lee & Catherine Wu, ICPMR Dispatch 2005:8:7A,B,C Mirka Rej, Symbion Health

2 RCPA QUALITY ASSURANCE PROGRAM DISPATCH 2: 2005 MYCOLOGY Maria Carino

3 Clinical: Case 1 Culture A was isolated from a CAPD bag from a 30 year old man with peritonitis.

4 Any ideas?

5 Rhodotorula mucilaginosa

6 Rhodotorula mucilaginosa On Sabourauds dextrose agar cultures are: fast growing smooth, glistering or dull sometimes roughened or soft moist to mucoid yeast-like in appearance cream to pink, coral red, orange in colour. Microscopic morphology: spherical to elongate budding yeast-like cells or blastoconidia, x um in size. India ink preparation: small capsules present.

7 Culture on Cornmeal and Tween 80 agar: budding blastoconidia only. No pseudohyphae are formed. Physiological tests: germ tube test is negative hydrolysis of urea is positive growth on cycloheximide medium is negative growth at 37ºC is variable. Fermentation reactions: negative for Glucose; Sucrose; Lactose; Galactose; Maltose;Trehalose.

8 Rhodotorula mucilaginosa is the current name for the species formerly known as Rhodotorula rubra.

9 Final Results % of laboratories got it correct to the genus level 60% of laboratories got it to correct to the species level Morphologically the isolate is very distinctive and a presumptive identification can be made. Identification systems. (ID32C) Keeping up to date with name changes.

10 Case 2: Culture B grew from a scalp scraping taken from a 12 year old girl who recently arrived from Sudan.

11 Any ideas?

12 Trichophyton soudanense

13 Trichophyton soudanense On Sabourauds dextrose agar cultures are: slow growing flat to folded, suede-like surface often there is a broad fringe of submerged growth surface and reverse pigment are characteristically a deep apricot-orange in colour. Microscopy morphology: hyphae often show reflexive or right-angle branching pyriform microconidia may occasionally be present numerous chlamydoconidia are often found in older cultures

14 Clinical significance: T. soudanense is an anthropophilic fungus which is a frequent cause of tinea capitis in Africa. Invaded hairs show an endothrix infection but do not fluoresce under Wood s ultra-violet light. Distribution is mainly in Africa with occasional isolates from Europe, Brazil and U.S.A.

15 Final results % of laboratories got it correct to the genus level 57% of laboratories got it correct to the species level Follow a schematic key. Process of elimination Selective media. If available The absence or the occasional presence of microconidia Reflexing hyphae. Bamboo affect Geographical information

16 Case 3: Culture C was isolated from a subcutaneous abscess on the forearm in a 29 year old HIV positive patient.

17 Any ideas?

18 Cladophialophora carrionii

19 Cladophialophora carrionii On Sabourauds dextrose agar cultures are: slow growing (reaching 3-4cm in dia. after 1 month compact suede-like to downy surface colonies are olivaceous-black in colour and have well defined margins Microscopy morphology: elongate conidiophores producing branched acropetal chains of smoothwalled conidia, x um in size maximum growth temperature 35-37ºC

20 Clinical significance: Cladophialophora carrionii is a recognised agent of chromoblastomycosis and it has been isolated from soils and fence posts made from Eucalyptus sp. Cases of chromoblastomycosis caused by C.carrionii are commonly found in Australia, Venezuela, Madagascar and South America.

21 Final Results % of laboratories got it correct to the genus level 59% of laboratories got it correct to the species level Cladosporium carrionii has been transferred to the Cladophialophora genus Cladophialophora can be distinguished from Cladosporium by the absence or poorly differentiated conidiophores, by unpigmented conidial scars and by being unable to liquify gelatin.

22 Cladosporium species have occasionally been found as opportunists in humans, having a wide clinical spectrum. In contrast, each Cladophialophora species provokes a mycoses which is characteristic for that species. Cladophialophora is related to the black yeast Keeping up to date with name changes Following a schematic key Clinical information

23 Acknowledgements Voula Henry - Biomerieux Dr Pitman - SDS Pathologist Dr fungus website - Images Quality Assurance Program

24 Review of Mycology QAP Results for 2005:4: 7A, B, C By Kerry Weeks Mycology Laboratory PaLMS, NSCCAHS

25 Item 4: 7A Clinical history A 60 y.o. farmer presented with swelling & a dark, blood-tinged discharge from the nose. A biopsy taken from the paranasal sinus grew culture 4: 7A

26 4: 7A Conidiobolus coronatus This fungus didn t survive freeze drying by QAP = most labs failed to isolate this fungus Conidiobolus spp. Phylum: Zygomycota, Order: Entomophthorales 27 species Most common sp: C. coronatus, C. incongruus & C. lamprauges

27 Conidiobolus spp. Rapid growth of waxy colonies (routine agar), wide hyphae, coenocytic or irregular septate Simple conidiophores single forcibly discharged terminal conidium papilla scar remains. Ballistospores- growth on petri dish lid Zygospores intercalary, thick walled, hyaline, w/o beaks Photo from Mycology Online

28 Differentiating between Conidiobolus species Species Colony (PDA 3 days) Forcibly discharged conidia w or w/o secondary conidia Primary conidia size/shape Villose (hair-like) conidia Conidiobolus coronatus > 40 mm Yes 40 um/ prominent papillate base Yes (in older cultures) C. lamprauges < 30 mm No um/ spherical, thin walled, 1-1 many papilla after liberation No C. incongruus < 30 mm Yes um, single tapering basal papilla No

29 Conidiobolus coronatus Multiplicative conidia Papilla RNSH photo (high magnification) Photo from Mycology Online Primary conidia of C. coronatus

30 C. coronatus Papillae Villose conidia C. coronatus -mature, spherical villose conidia (hair-like protrusions), & papillae (site of former attachment to conidiophore) Photo from Mycology Online

31 Differentiating between Basidiobolus & Conidiobolus Microscopic features Basidiobolus spp. Conidiobolus spp. Ejected sporangioles with papilla Sporangiophores with swollen apices Zygospores Zygospores with conjugation beaks No Yes Yes Yes Yes No No- C. coronatus Yes- C. incongruus C. lamprauges No (Yes 2 beaks in C. lamprauges)

32 Basidiobolus ranarum B. ranarum- beaked zygospores Discharged globose conidia & conidiophores Photos from Mycology Online

33 Rhino-facial infections caused by Conidiobolus Photos from Mycology Online Infections mainly in tropics- eg. Africa, Asia, Central America, India. Infection not evident until swelling/ deformity occurs.

34 Epidemiology Found in soil & decaying debris (found on bananas, rotten wood & a fruit warehouse etc). Ubiquitous- wide range of areas- Alsaksa,, temperate to tropical regions (eg( eg.. PNG soil) Insect pathogen (mosquitoes, termites, aphids) Infects man (80% cases are males) (typically) nasal mucosa & para-nasal sinus, (sometimes) subcutaneous, (rarely) pulmonary & pericardial infections firm, subcutaneous nodules/polyps Immune-competent & immune-suppressed suppressed- emerging opportunist. Occur sporadically but rarely. Route: inhalation Infects animals- horses, sheep, deer, dogs & chimpanzees. Hard to treat- high MIC s?itraconazole, Amp B + terbinafine, flucon, keto, iodides, plastic surgery

35 Item 4: 7B Clinical history This culture was isolated from sputum collected from a lung transplant patient who presented with a chronic cough

36 4:7B Geotrichum candidum Teleomorph: Galactomyces candidum Description: Colonies, fast growing (routine agar), white, rough texture, hairy, creeping, mostly submerged, dry. Fruity odour. Hyaline hyphae, aerial hyphae fragment forming arthroconidia (cylindrical, barrel shaped or ellipsoidal). No blastospores produced. No capsules. [Trichosporon spp. produce blastospores along pseudohyphae] Differential features of G. candidum: Hyphae 12 um wide with dichotomous branching at colony margin. Assimilation: xylose POS, cellobiose NEG NG at 40C.

37 Colonial morphology G. candidum Sabs agar (28C 4 days)

38 Microscopy Photo from Mycology Online Geotrichum candidum- Course true hyphae (no pseudohyphae) that segment into arthoconidia. main & lateral branches with arthroconidia. Dichotomously branching.

39 Similar species to Geotrichum Trichosporon sp- blastoconidia & pseudohyphae (not seen in Geotrichum has true hyphae) Scytalidium sp- dematiacous arthroconidia (hyaline arthos in Geotrichum) Arthrographis & Oidiodendron sp- have condiophores (which Geotrichum lacks) Malbranchea sp- arthroconida release by disjunctor cells ( (Geotrichum produces arthros by fission) Coccidioides sp- arthros alternate with empty cells ( (Geotrichum- consecutive)

40 Biomerieux ID 32c result Profile number: Xylose assimilation POS Cellobiose NEG Result: Geotrichum candidum

41 Differentiating between Geotrichum species G. candidum G. capitaum G. clavatum Cellobiose Neg Neg Pos D-xylose Pos Neg Neg Salicin Neg Neg Pos Vitamin free Pos Neg Neg Arbutin Neg Neg Pos Growth at 37C V Pos Pos [Ref: dehoog, Guarro et al. Atlas of Clinical Fungi p. 227]

42 4:7:B QAP results Geotrichum candidum Geotrichum sp. G. pennicillatum G. captatum G. klebahni Candida kefyr Unable to ID 82 labs (73.2%) 16 (14.3%) 7 (6.3%) 1 (0.9%) 1 (0.9%) 1 (0.9%) 4 (3.6%) 96% labs correct to genus level 73% correct to species level Results obtained from Microbiology QAP

43 Epidemiology G. candidum found worldwide (tundra, temperate, tropical climates) in soil, marine habitats, water, air, sewage, plants,, freq dairy, cereals (eg.frozen( fruit cake, bread), tomatoes, animals skin & in droppings etc. Found in gas oil in Germany, paper pulp factory in France, isolated from irradiated soil. Normal human flora- isolated from faeces & sputum Opportunistic infections in immune compromised ( Geotrichosis ) Endogenous- oral, bronchial, systemic & exogenous- skin, allergic & trauma. Disseminated infection- poor prognosis. Route: Ingestion, inhalation or trauma?also may cause environmental damage- eg. destroy data- storing polycarbonate resin found in CDs! (disc becomes transparent). Also sig cause of citrus fruit rot.

44 Item 4: 7C Clinical history This culture was isolated from a corneal scarping collected from a 12 y.o. with an eye injury

45 4:7:C Candida kefyr Synonym: Candida pseudotropicalis Teleomorph: Kluyveromyces marxianus Colony- white, cream- coloured, smooth texture Microscopy (CMA tween 80) shows elongated blastospores, parallel logs in a stream, curved along pseudohyphae Differential features of C. kefyr: Raffinose POS, 2-keto 2 gluconate (2KG) NEG, ethylamine POS, growth on 0.1% cyclohex G at 37C, G w/o niacin.

46 Colonial morphology Candida kefyr colony white, cream coloured, butyrous (buttery) ie. typical yeast morphology. (Sabs 28C 3 days)

47 Microscopy Pseudohyphae Candida kefyr elongated blastoconidia Elongated blastoconidia Photo from Boekhout et al. CD-ROM Yeasts of the World

48 Kluyveromyces marxianis asci (Teleomorph of C. kefyr) asci with ascospores ascospore Photo from Boekhout et al. CD-ROM Yeasts of the World

49 Biomerieux ID 32c result Profile number: Raffinose assimilation POS 2 KG Neg Result: C. kefyr 99.9% T 0.88

50 4:7:C QAP results Candida kefyr Candida sp. Candida (not albicans) Candida tropicalis Geotrichum candidum Unable to ID 102 labs (90.3%) 6 (5.3%) 1 (0.9%) 1 (0.9%) 1 (0.9%) 2 (1.8%) 97% labs correct to genus level 90% correct to species level Results obtained from Microbiology QAP

51 Epidemiology Found worldwide in humans, other mammals & dairy foods Emerging fungal pathogen. Infections- occasionally superficial candidiasis. Reported cases of pulmonary infections, disseminated infection, oesophagitis in immune compromised.

52 A Review of 2005 Mycology QAP (despatchdespatch 6) Okcha Lee Catherine Wu Mycology Unit, CIDMLS, ICPMR Westmead hospital

53 Item 2005:6:7A Clinical notes: Culture was isolated from fingernail scrapings taken from a 23 year-old African man with suspected onychomycosis. Direct microscopy showed the presence of septate hyphae.

54 Colony Initially whitish, finally becoming dark gray Spreading, fast

55 Microscopy Hyphae: melanized or hyaline, falling into athroconidia Chlamydospore: conidia single or in chains, dark brown, thick walled, swollen up to 7µm 7 m wide

56 Pathogenicity Identification Scytalidium spp Onychomycosis Dermatophytosis like infection Ecology Soil fungus or plant pathogen in tropical and subtropical areas * 60% correct to genus level

57 Item 2005:6:7B Clinical notes: Culture was isolated from a wound swab taken from a 30 year-old man recovering from severe burns.

58 Colony Bright yellow with dark yellow reverse.

59 Microscopy

60 Microscopy Hyphae: sparsely septated Sporangiophores: Sporangia: <100µm Columella: spherical Sporangiospores: variable in size and shape Chlamydospores: present Rhizoids: present Growth temperature: growth at 37ºC no growth at 42 ºC

61

62 Comparison between Rhizomucor variabilis and Mucor hiemalis colony Sporangio- phores sporangia columella Rhizomucor variabilis Greyish-ochraceous Up to 2mm long, 9-23um 9 wide, branched Subspherical,, up to 100um diameter Spherical, ellipsoidal to cylindrical in various shape, up to 40µm wide Mucor hiemalis Whitish to ochraceous Up to 15mm long, 14um wide, branched Up to 80um diameter Ellipsoidal, spherical, up to um

63 Comparison continued sporangios pores chlamydos pores rhizoids Maximum growth Tm Rhizomucor variabilis Hyaline, smoothwalled,, very variable um abundant abundant 38ºC Mucor hiemalis ellipsoidal, smooth-walled um ºC

64 Molecular ITS1 ITS3 LR1 D1 D2 ITS2 ITS4 LR16 18S rdna (SSU) ITS 1 5.8S rdna ITS 2 28S rdna (LSS) Schematic diagram of ribosomal gene cluster of fungi

65 Comparison of sequencing results between Rhizomucor variabilis and Mucor hiemalis ITS 1 and ITS 2 region Rhizomucor variabilis not in database Mucor hiemalis 92% (452/487 bp) SSU Base pairs ( bp) 99% (487/488 bp) 98% (484/488 bp) Base pairs ( bp) 100% 557/557 bp) 98% (547/557 bp) LSU D1/D2 region ( bp) 99% (638/644 bp) 97% 642/658 bp)

66 Relationship between Rhizomucor variabilis and Mucor hiemalis Rhizomucor variabilis may concern degenerate cultures of Mucor hiemalis Phylogenetically it proved to be very close to Mucor hiemalis Identification Rhizomucor variabilis * 63% answered to Mucor spp and 3.6% to Rhizomucor variabilis

67 Item 2005:6:7C Clinical notes: Culture was isolated from a lung biopsy taken from a 15 year-old girl with acute lymphocytic leukaemia.

68 Colony Usually green, cream-buff or honey-yellow yellow where cleistothecia form Reverse may be olive to purple-brown

69 Microscopy

70 Microscopy Conidiophores: short (< 250um) Conidial heads: short columnar (< 80um) Conidiogenous cells: biseriate, metulae and phialide Conidia: globose ( um in diam) Teleomorph: Emericella nidulans cleistothecia with reddish ascospores are often surrounded by HülleH cells (up to 25um in diam)

71 Identification Aspergillus nidulans Pathogenicity Opportunistic infection Ecology Soil fungus Prominent colonizer of decomposing plant debris * 94% correct to genus level and 74% to species level

72 RCPA Quality Assurance Programs A Review of the 2005 Mycology QAP Mycology Items 2005:8:7 A,B,C Mirka Rej Microbiology Department Symbion and Laverty Pathology North Ryde

73 Item 2005:8:7A Clinical Notes: Culture was isolated from a knee aspirate taken from a ten year old leukemic boy with osteomyelitis of the right knee.

74 Identification: Macromorphology Young colony is cottony or moist (yeasty) and light grey to black. Mature colony becomes dark grey to black and may develop white mycelial tufts with age. Reverse is grey to black.

75 Identification: Micromorphology Septate hyphae Unbrached conidiogenous cells (annellides). Swelling of the basal portion of the annellides. Swan necks

76 Conidia are one celled, smooth and ovoid with a slightly narrowed, truncated base. Conidigenous cells locally aggregated into small brushes. Sympotial conidia

77 Clinical importance of Scedosporium prolificans Documented as a cause of: osteomyelitis septic arthritis onychomycosis otomycosis endophthalmitis sinusitis gangrenous skin ulcers pulmonary infections peritonitis systemic infections (may be isolated from blood cultures)

78 Clinical importance of Scedosporium prolificans Scedosporium prolificans in nature occurs in soil and is traumatically introduced into humans by thorns or splinters. In the body it has a predilection for cartilage and joint areas. In immunocompromised patients or transplantation patients fatal dissemination may occur. A fatal case of endocarditis was reported. Isolates are often resistant to antifungal agents.

79 RCPA Quality Assurance Programs Unable to identify Yeast species Candida guillermondii Pseudallescheria boydii Sporothrix schenckii Sporotrichum schenckii Wangiella dermatidis Exophiala jeanselmei Fonsecaea species Phialophora richardsiae Phialophora species Phialophora verrucosa Scedosporium inflatum Scedosporuim species Scedosporium apiospermum Scedosporium prolificans 84% correct to genus level. 76% correct to species level

80 growth on media contraining cycloheximide Differences between S. apiospermum and S. prolificans S. apiospermum S. prolificans + - annellides cylindrical flask shaped, swollen basal portion annellidic rings difficult to see long thin appendages (swan neck) formation of + (P.boydi) - clastothecia synnemata + - cultures white cottony mycelium later turns grey to brown first moist grey to black later dark grey, white tufts in the centre. growth at 45 degrees - +

81 Scedosporium prolificans Bottle shaped annellides Annellides with parrallel sides may also be seen.

82 Item 2005:8:7B Clinical Notes: Culture was isolated from the fingernail scraping taken from a 50 year old woman with chronic paronychia.

83 Identification: Macromorphology White to cream coloured, smooth, glabrous yeast-like colonies.

84 Identification: Micromorphology Budding yeast like cells. cells spherical to broadly ellipsoidal x µm.

85 Identification For the identification of germ tube negative yeasts, morphological (Dalmau plate culture) physiological and biochemical tests are essential. Reliable commercially available yeast identification kits are the API 20C AUX, ATB32C, Vitek systems.

86 Microscopy Morphology on Cornmeal-Tween 80 agar Fairly short, fine pseudohyphae. Clusters of blastoconidia at septa.

87 Identification: Physiological tests: Germ Tube test: Hydrolysis of Urea: Growth on Cycloheximide: Growth at 37C: Negative Negative Positive Positive

88 Fermentation reactions: Identification: Glucose: Lactose: Sucrose: Galactose: Trehalose: Maltose: Variable - -

89 Differential Diagnosis The species can be recognised by the following growth characters: D-glucosamine D-arabinose cellobiose inulin erythritol L-arabinitol D-glucuronate

90 Clinical Importance of Candida guilliermondii is found from normal skin, in sea water, faeces of animals, fig wasps, buttermilk, leather, fish and beer. has been isolated from numerous human infections.

91

92 Clinical Importance Yeasts are considered opportunistic pathogens causing disease in patients: with a breakdown in the body s immune system on prolonged treatment with antibiotics on corticosteroids or cytotoxic drugs with intravascular catheters with diabetes mellitus known to be intravenous drug abusers

93 RCPA Quality Assurance Programs No growth Unable to identify Candida glabrata Candida famata Candida species Candida guilliermondii % correct to genus level. 89% correct to species level.

94 Item 2005:8:7C Clinical Notes: A 40 year old market gardener presented with a tender subcutaneous nodule on the leg. Fluid aspirated from the nodule grew Culture C.

95 Identification: Macromorphology Initially smooth, black, mucoid and yeast like colonies. Revers is black Grows slower or not at all at 37C

96 Identification: Macromorphology Colonies become raised and develop tufts of aerial mycelium with age. Often dome shaped and suede like in texture.

97 Identification: Micromorphology Yeast like cells Torulose hyphae

98 Identification: Micromorphology The conidiogenous cells are slender annellides. Tips of annellides are narrow, elongated and rocket shaped. Conidia gather in clusters at the end, sides of conidiophore and at points along the hyphae. Inflated germinating cells. Budding yeast like cells. Conidia 1-3 x 2-5 µm in size, hyaline, smooth, thin walled and broadly ellipsoidal.

99 Clinical Importance of Exophiala jeanselmei Mainly associated with phaeohyphomycotic cysts. The fungus has also been documented as a cause of black-grained mycetoma. Has been reported from a small number of cases of chromoblastomycosis.

100

101 RCPA Quality Assurance Programs Unable to identify Yeast species Cladosporium carrionii Cladosporium species Sporothrix schenckii Homonema deratioides Hortaea werneckii Phialophora gougerotii Phialophora species Philophora verrucoas Fonsecaes pedrosoi Fonsecaea dermatidis Fonsecaea species Wangiella dermatitidis Exophila castellanii Exophila spinifera Exophila dermatitidis Exophiala species Exophiala jeanselmei 76% correct to genus level 52% correct to species level

102 Differences betweem Exophiala jeanselmei and Wangiella dermatitidis (Exophiala dermatitidis) growth at 42 degrees conidiogenous cell Exophiala Wangiella jeanselmei dermatitidis - + annellide tapering extensions to the tip. phialide. do not show exidence of extension.

103 Wangiella dermititidis Exophiala jeanselmei

104 Books: References Ellis, D., Davis, S., Alexiou,, H., Pfeiffer, T., Manatakis,, Z. (1992). " "Descriptions of Medicat QAP fungi". Mycology Unit, Adelaide Children's Hospital. Hocking, A., Woodgyer,, A. "Clinically Significant Non-Dermatophyte Fungi a Practical Guide to Identification". AIMS National Scientific Meeting 2001, Melbourne Hoog G., Guarro,, J. (1995). " "Atlas of Clinical Fungi" Centraalbureau voor Schimmelcultures,, The Netherlands, and Universitat Rovira i Virgili,, Spain] Larone,, D. (1995). "Medically Important Fungi" 3rd Edition.. American Society for Microbiology, Washington. Murry,, P., Baron, E., Pfaller,, M., Tenover,, F., Yolken,, R. (1995). " "Manual of Clinical Microbiology" 6th Edition.. American Society for Microbiology, Washington. Websites: microbiology.mtsinai.on.ca/mig/defungi/index2.shtml ematiaceous)/exo phiala/jeanselmei.html dii/ onychomycosis.com/images/table%201.htm