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

AGENDA Clinical overview of ocular fungal 14:15 -14:35 Peter McCluskey, AUS infections #935

14:35 - 14:50 Neglected fungal infections of eye #722 Ariya Chindamporn, TH

Fungal virulence factors in ocular 14:50 - 15:10 Philip A Thomas, IN infections #936

The challenges in Mycotic keratitis in 15:10 - 15:25 Andrew Turner, AUS horses and dogs #938

15:25 - 15:45 Host factors in mycotic keratitis Lalitha Prajna, IN

19th 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

Pythium insidiosum Histopathology: GMS Kingdom Straminipila/ Stramenopila Pythiosis in Thailand : 1985-2003 Natural resource 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 1st d 2nd d 2wk 1st d 2nd d 2 wk 1st d 2nd d 2 wk Cornea Scraping/ + + + - - + - + biopsy Aqueous ------Vitreous - - - Lens + - - Vitreous and Iris

+ -

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 • Broad, rare septate hyphae Broad hyphae by confocal microscopy • by KOH preparation 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, , , ……

Referred to tertiary hospital

- Result from ocular confocal micro. & frequency of pythiosis - oral , & 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 Clinical Enviromental spp. P. insidiosum 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 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 Lagenidium albertoi

Parsimony Analysis ITS 1000 Bootstrap

Parsimony analysis, ITS 1000 Bootstrap 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