ISHAM Symposium: S33: Ocular aspects of Fungal Infections Friday, 8 May 2015, 14.15 15.45; MR101/102 Level 1 Chairs: Ariya Chindamporn, TH and Phillip A Thomas, IN 14:15-14:35 AGENDA Clinical overview of ocular fungal infections #935 Peter McCluskey, AUS 14:35-14:50 Neglected fungal infections of eye #722 Ariya Chindamporn, TH 14:50-15:10 15:10-15:25 Fungal virulence factors in ocular infections #936 The challenges in Mycotic keratitis in horses and dogs #938 Philip A Thomas, IN Andrew Turner, AUS 15:25-15:45 Host factors in mycotic keratitis Lalitha Prajna, IN
19 th Melbourne Ocular aspects of Fungal Infections Neglected fungal infections of eye Ariya Chindamporn Department of Microbiology Faculty of Medicine, Chulalongkorn University May 8, 2015
Outline Laboratory investigation Risk factors Epidemiology Cases
Flow chart : laboratory investigation Cornea scrapings, biopsy Direct from specimens Fungal Isolation KOH preparation: 75-90% Macroscopic Exam. Calcofluor: 80-90% Microscopic Exam. GMS: 89-98% Zoospore production PCR & Sequencing PCR & Sequencing %sensitivity (Thomas PA., 2003 Eye:17 (852-62)
Corneal scraping, staining, inoculation
Specimen collection for PCR Cornea, Vitreous, Aqueous For PCR FFPE
Risk factors HISTORY is important. o Trauma : Dust, Leaf, Stone, Water, Contact len o Swimming o Occupation : working outdoor/indoor o Host factors: diabetes mellitus o Fungal virulence factors o > 5-10 days
Frequence isolates of Fungal MOLD Hyaline hyphae : Fusarium spp.: F. solani, F. oxysporum Aspergillus spp. : A. fumigatus, A. flavus Scedosporium spp. : S. apiospermum Penicillium spp. : P. spinulosum, P. citrinum Acremoniium spp. ( A. potronii, A. kiliense) Dematiaceous fungi Curvularia spp. (C. lunata, C. geniculate, C. senegalensis ) Bipolaris spp. ( B. spicifera, B. hawaiiensis) Exserohilum spp. (E. rostratum, E. longirostrata) Coelomycetes (Lasiodiplodia, Colletotrichum) Alternaria Yeast Candida spp. (C. albicans) Rhodotorula Others Pythium insidiosum Lagenidium albertoi Modified from Thomas PA., 2003 Eye :17:852-62
Epidemiology 1352 proven cases, a tertiary care hosp. South India -10 yr. (1991-2000) (Gopinathan et al., 2002 Cornea:21:555-9) > 99% caused by mold; Male > Female: 2.5:1; Age: 16-49 Risk: systemic factors : DM 654 eyes in Shandong Eye Institute, North China, Jan1999 - Dec 2004 (Zhong XL., et al 2006 Opthalmol 113(11):1943-8) 62% of total cases : fungal keratitis Fusarium spp. : F. solani 73%, 437 eyes) Aspergillus spp. : A. flavus 12%, 72 eyes Prevalence : the season, more : Oct to Dec than Jul to Sep. KOH preparation 88% positive, Culture 91% Histopath. staining: most effective, resulting 98% but need to reveal genus and species. 997 cases in eastern India, Jul 2006 and Dec 2009 26.4% as fungal keratitis. The geographical place and climate affected the prevalence (2012) : 36.7% in Southern, 36.3%, 25.6%, 7.3% in Western, North-eastern, and Northern India, in order. Katara SR., et al., 2013 51(6); Acta Medica Iranica
Mimiking fungal infection
Conventional approach KOH-preparation: rare septate hyphae Histopathology: GMS Pythium insidiosum Kingdom Straminipila/ Stramenopila
Natural resource Pythiosis in Thailand : 1985-2003 Clinical case HIGHEST INCIDENCE in Human Supanpandu et al., Med Mycol 2008 C : Central NE: Northeastern S : Southern N : Northern E : Eastern 12 Krajejun et al., CID 2006
Specimens KOH Culture PCR Cornea Scraping/ biopsy 1 st d 2 nd d 2wk 1 st d 2 nd d 2 wk 1 st d 2 nd d 2 wk + + + - - + - + Aqueous - - - - - - Vitreous - - - Vitreous and Iris Lens + - - + - Lens IFN ITS Cornea Rt.Lens KOH: Positive
Rt.Cornea KOH: Positive Culture: Positive PCR: Positive
IMPORTED CASE
Lagenidium sp. Ocular Infection Mimicking Ocular Pythiosis U Riengprayoon et al., JCM 2013:51(8):2778 43 yr. housewife admitted in a tertiary care hospital: lid swelling, pain, redness, & itching for 3 weeks, visit physician,.. visual acuity: finger counting at 1 foot slit lamp ex.: ciliary injection & a yellowish, midstromal, reticular pattern corneal infiltration measuring 5x5.6 mm. with feathery edge corneal epitherial defect 4x3 mm in dia. over the infiltrated area posterior eye segment by ultrasound: no sign of endophthalmitis unsuccessfully treated with systemic and topical drugs
Lagenidium sp.: Mimicking Ocular Infection Fungal elements in corneal stroma by confocal microscopy Broad, rare septate hyphae by KOH preparation Broad hyphae by GMS U Riengprayoon et al., JCM 2013:51(8):2778
Treatment - First : systemic & topical antibiotics: worse - Presumed to be fungal infection: - topical : cefazolin, amikacin, natamycin, voriconazole, Referred to tertiary hospital - Result from ocular confocal micro. & frequency of pythiosis - oral terbinafine, itraconazole & topical natamycin with Pythium Insidiosum Antigen (PIA) vaccine - worse - Twice of Penetrating keratoplasty (PK) - 10 mo. later : no recurrent of infection
Susceptibility result *The value of Minimal Inhibitory Concentration (MIC) Agents Lagenidium spp. Clinical P. insidiosum Enviromental P. insidiosum Amphotericin B 16 4-16 2-16 Terbinafine 2 2-4 2-4 Voriconazole 1 2-16 0.5-2 Itraconazole 1 1-8 0.5-2 Fluconazole 2 2-8 0.5-2 Anidulafungin 1 2-8 0.5-1 Caspofungin 1 2-8 0.5-1 TB/VO Indifference Indifference Indifference TV/IT Indifference Indifference Indifference
Parsimony Analysis ITS 1000 Bootstrap Parsimony analysis, ITS 1000 Bootstrap Lagenidium albertoi Vol. 21, No. 2, February 2015
Sharma et al., 2014 Calcofluor white staining 35C, 3 d. PDA LCB wet mount Sporangia with zoospore
Take home message Rapid and accurate diagnosis, leading the right treatment is required: Communication with physicians Direct exam. & Culture still gold standard Molecular identification Very rapid growth, no sporulation No response to antifungal agents: Think about parafungal infection