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Identification of zygomycetes 9. . 01 ed Professor Retno Wahyuningsih 2 v Professor of Medical Mycology er Department of Parasitology ly s Faculty of Medicine u e Universitas Indonesia and Universitas Kristen IndonesiaJ r Jakarta, Indonesia s 21 t 0- gh 2 ri N ll T . A M er t M ak a pe ed f s nt o se ht rIDENTIFICATIONe ig OF MUCOROMYCOTINA P (ZYGOMYCETES)r py o Retno Wahyuningsih C Department of Parasitology, Faculty of Medicine, Universitas Indonesia, Department of Parasitology, Faculty of Medicine, Universitas Kristen Indonesia , © Jakarta, Indonesia

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Mucormycoses

• Emerging fungal infection caused by a group fungi called mucormycetes (zygomycetes) . 19 d. • A life threatening infection 0 e 2 rv • An aggressive & highly destructive invasive fungal infectiony in e immunocompromised patients l s Ju re 1 ts -2 gh 0 Ibrahim et al., CID 2012;54(S1):S16–22 2 ri N ll T . A M er t M ak a pe ed f s Incidence:nt France o 1997-2006 se ht e g • 828 hospital, 531 incident cases were r i identified P r • 283 males and 248 females (ratio 1:1); y • mean age: 57.1 years (median 60 p years, range: <1 month–96 years). o • The annual incidence rate (AIR) increased from 0.7 cases/million C persons in 1997 to 1.2/million persons in 2006 © • yearly increase was +7.4% (p<0.001).

Bitar et al. Emerg Infect Dis. 2009; 5: 1395-1401

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Five patients with sinusitis (only) & dissemination to adjacent tissue were diagnosed as mucormycoses.

Diabetes (adults), one patient with leukemia (pediatric)9. . was infected with rhizopus 01 ed 2 rv ly se Jakarta, January – November , 2018 u e Sent from one hospital only J r 1 ts -2 h 0 Datag Dept. Parasitology, FKUI, 2018 2 ri N ll T . A M er t M ak a pe Epidemiology:d India s te of • a steadyen increase in tthe number of patients: • 129s cases over 10 yearsh (13 cases/year during 1990–1999), •e178 cases overi nextg 5 years (36 cases/year during 2000– 2004), r• 75 cases overr 18 months (50 cases / year during July 2006 - December 2007) P• Diabetes mellitusy as major risk factors (65.1 million), ca. 70% are uncontrolled DM. p • Environmentalo factors: the tropical & sub-tropical humid climate & high environmentalC temperature accommodates the survival of the fungi, ©

Chakrabarti & Dhaliwal. Curr Fungal Infect Rep (2013) 7:287–92 Chakrabarti & Singh. Mycoses, 2014, 57 (Suppl. 3), 1–6

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Taxonomy

9. . 01 ed 2 rv ly se Ju re 1 ts -2 h 0 Kyung J. Kwon-Chungg CID 2012;54(S1):S8–15 2 ri N ll T . A M er t M ak a pe The causesd s te of • Consistingen of two importantt genera: • Entomophthoromycotina,s h a natural insect pathogens i.e. e• Conidiobolus &g Basidiobolus, r • are found in tropicali and subtropical regions P • cause chronicr subcutaneous infections in immunocompetent host py • The Mucoromycotina: found worldwide as common saprobe in soil, recycling of organico materials, e.g. leaves, compost, rotten wood C• invasive infection in immunocompromised host ©

Hoffmann et al., Persoonia 2013; 30: 57–76

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9. . 01 ed 2 rv ly se Ju re 1 ts -2 h 0 Binder et al. Cling Microbiol Infect 2014; 20 (Suppl. 6): 60–66 2 ri N ll T . A M er M k at ea sp Mucormycoses:ed portalf of entry nt o • Inhalationse of hsporest to the respiratory tract, re ig P• injured yskinr or percutaneous route: inoculation of sporesop by contaminated needles or catheters C ©• ingestion of contaminated food.

Binder et al. Clin Microbiol Infect 2014; 20 (Suppl. 6): 60–66

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Classification of mucormyocses

Anatomic location disease . Sinus & adjacent tissue Rhino – orbito- cerebral 19 d. Lung Pulmonary 0 e Skin Cutneous/subcutaneous 2 rv Gastrointestinal Ingestion of contaminatedly food se Diseminated form Dissemination from uprimary site re others Bones, kidney,J etc 1 ts -2 h 0Spellberg et al Clin Microbiolg Rev 2005; 18: 556–69. 2 Marpaung et al.; J Penyakri it Dalam Indonesia; 2018; 5. N ll T . A M er t M ak a pe Major riskd factors s te of • uncontrolleden diabetest mellitus (ketoacidosis) • otheres forms of metabolich acidosis, •rCorticosteroidsi treatmentg P• organ & boneyr marrow transplantation • neutropeniap • traumao & burns, • malignant C hematologic disorders, ©• deferoxamine therapy in patients receiving hemodialysis

Chakrabarti & Dhaliwal Curr Fungal Infect Rep (2013) 7:287–92 Binder et al. Clin Microbiol Infect 2014; 20 (Suppl. 6): 60–66

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9. . 01 ed 2 rv ly se Ju re 1 ts -2 h 0 Spellberg et al Cling Microbiol Rev 2005; 18: 556–69 2 ri N ll T . A M er t M ak a pe Clinical presentationd s te of • Based non vascular invasiont that causes thrombosis & tissue infarction/necrosisse h •rblacke eschar ig P• occurs in patients:r • With defectsy in immune defense &/or with increased available serum iron, • p Changeso in their metabolism (DM- ketoacidosis) • Very rare in normal hosts • most C cases, are progressive infection & lethal, unless identified early & ©treated promptly

Spellberg et al Clin Microbiol Rev 2005; 18: 556–69

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. 19 d. 20 ve DIAGNOSIS OF MUCORMYCOSISy er ul es J s r 21 t 0- gh 2 ri N ll T . A M er t M ak a pe ed f s nt o se ht re ig P yr op C ©

Cornely et al., Clin Microbiol Infect. 2014; 20 (suppl.3: 5-26

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rhino cerebral mucormycosis in diabetic patient

9. . 01 ed 2 rv ly se Ju re 1 ts -2 h Day - 1 Day - 2 0 Singhg et al., BMJ Case Rep 2013. 2 ri N ll T . A M er t M ak a pe Rhinocerebrald mucormycosis s in diabetic patient te of en t es gh Intraoral photograph of r ri rhinocerebral mucormycosis. P y Started by left maxilary op region discoloration C ©

Sahota et al. Ethiop J Health Sci. 2017; 27

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Rhino-cerebro-orbital mucormycosis in DM

. 19 d. 20 ve y er ul es J s r 21 ht Oladeji0 et- al. J West Africang Coll Surge 2013; 3: 93-100 2 ri N ll T . A M er M k at ea sp Cutaneoused mucormycosisf nt o se ht re ig P yr op C ©

Gardiner et al., Med Mycol Case Reports 7(2015) 8–11

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Mucormycoses in human tissue

• The amount of fungi that cause mucormycosis is very large but in human tissues they grow as coenocytic hyphae (septum is quite rare) that are similar to one another and rarely spores production . 9 d. • The mucormycetes hyphae generally do not have septa, excessive1 e manipulation of clinical material will cause leakage of the cells0 which results vin fungal death & does not grow on culture 2 r ly se • Direct examination (KOH) is quite important in the identificatu ion of thee disease J s r 21 t 0- gh 2 ri N ll T . A M er M k at ea sp Direct examination:ed f KOH wet slide nt o • Braine tissue, of a girlt with tubulares acidosisg h P•ra coenocyticr ihyphae, no septump y • thick owalled, refractile • 400C ×magnification ©

Pic. Wahyuningsih, Dept. Parasitology FKUI; Case of Dr. Satari, Dept of Pediatric FKUI

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KOH wet slide

• Orbital tissue from a patient with rhino-orbito-cerebral mucormycoses 9. . • Branched coenocytic (aseptate) 1 d hyphae among eye tissue 0 ve • 400 ×magnification 2 r ly se Ju re 1 ts -2 h Pic. Wahyuningsih,0 Dept. Parasitology FKUI;g Case of Dr. Satari, Dept of Pediatric FKUI 2 ri N ll T . A M er M k at ea sp Histopathologyed f nt o • Rhinocerebralse mucormycosesht •rHEe staining ig P• Coenocyticyr hyphae (aseptate) • Inflammatoryop cells C ©

Pic. Wahyuningsih Dept. of Parasitology FKUI

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Histopathology

9. . 01 ed 2 rv ly se Ju re 1 ts HE staining (left) and PAS-2 staining (right)h 0Gardiner et al., Medg Mycol Case Reports7(2015)8–11 2 ri N ll T . A M er M k at ea sp Calcofluored white f nt o se ht re ig P yr op C Direct microscopy using calcofluor white, © a clear large hyphae

Lass-Florl. CMI. 2009; 15 (Suppl. 5), 60–65

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9. . 01 ed 2 rv CULTURE & PHENOTYPICl y se Phenotypic identifications u e J s r 21 t 0- gh 2 ri N ll T . A M er t M ak a pe Schematic diagram labeling thes morphologic structures seen in the sporangium-producing ed f Mucorales (not drawn to scale). nt o se ht re ig P yr op C ©

Julie A. Ribes et al. Clin. Microbiol. Rev. 2000; doi:10.1128/CMR.13.2.236

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Rhizopus oryzae (most common cause)

Culture & microscopy Schematic diagram

9. . 01 ed 2 rv ly se Ju re 1 ts -2 h Bonifazg et al Clin Dermatol (2012; 30: 413-9 0 Thomas PA. CMR; 2003; 16: 730-97. 2 ri N ll T . A M er t M ak a e d sp Apophysomyceste o variabilisf H. Sporangiphoren t I. Sporangosporese h re ig P yr op C ©

Chander et al. Rev Iberoam Micol. 2015;32(2):93–98 Diagram modified from Alvarez et al. Rev Iberoam Micol. 2010;27(2):80–89 dela Cruz et al. JCM. 2012; 50: 2814 –2817

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Cunninghamell a bertholletiae Microscopy LPCB mounts: . . • branching 19 d sporangiospores, 0 e vesicles, 2 rv • Sporangiolum & y e sporangiospores l es & denticles Ju r • hyalinous broad s hyphae & septae 1 t -2 h Tadepalli et al., Case Repg Infect Dis. 2015, Article ID 703240 0Mycology online 2 ri N ll T . A M er t M ak a pe Lichtheimia d s corymbifera = eMucor f corymbiferat = Absidia o corymbiferaen t A commons human h pathogen,re ig pulmonary, rhino-r Pcerebral, y disseminated,p & cutaneous mucormycosis.o world-wide C distribution, can be ©found in soil & decaying plant debris.

Mycology online; Thomas PA. CMR; 2003; 16: 730-97 Vyas & Shah. Indian J Otol. 2011; 17: 33-6 |

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Lichtheimia ramosa syn. Mycocladus ramosus, Absidia ramosa . In nature: soil, decaying plant debris & 9 . foodstuffs. 1 d immunocompromised hosts, c 0 e becoming increasingly common in individuals 2 v without predisposing factors (e.g. in traumatic r injuries). ly se Associated with cutaneous, pulmonary, rhinocerebral, CNS & disseminated form u e a. CultureJ of 3 days (30 ˚)r b. Sporangiophore, intacts sporangium & 1ruptured sporangiumt with columella -2c. sporangiosporeh gLIFE 2018; 0 Bibashi et al., Med Mycol Case Rep 2013; 2: 7 2 ri N ll T . A M er M k at ea sp Mucor circinelloidesed f nt o se ht a e r ig b P yr op C ©GMS of skin tissue: nonseptate hyphae & intercalary (A) Branched circinate sporangiophores, sporangia, & oval to subglobose chlamydospores (arrow, a). Culture collumellae (B) chlamydospores formed successively in on PDA, 6 days, 30°C, sporangium (white arrow), sporangiospores (black arrowhead), & chlamydospores chains. 400× produced singly & short (b) Iwen et al., JCM. 2007; 45: 636–40 Khan et al., JCM. 2009, 47:1244–8

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Dimorphic stage of M. M.circinelloides

. 19 d. 20 ve y er ul es (A and B) BHIJ agar, 37°C, hyphaer & arthroconidium Bactec blood culture: gram stain showed branching hyphae& yeast (a) forms with single, bipolar, and multipolar buds. & yeast phase resembling P. brasieliensis 600×1 ts -2 h 0 Khan etg al., JCM. 2009, 47:1244–8 2 Arroryio et al., Case Rep Infect Dis 2016; ID 3720549 N ll T . A M er M k at ea sp M.circinelloidesed culturef RT nt o se ht re ig P r Microscopic from PDA “tape prep”, LPCB y (a)Sympodially branched sporangiophores (100x); p (b)circinate sporangiophores (200 ×); o (c) deliquescent sporangia (100×); C (d)columella with collarette (200×). ©

Arroyo et al., Case Rep Infect Dis 2016; ID 3720549

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Saksenea vasiformis

A special method to stimulate sporulation: . A small block of agar is cut from a 9 . well established culture grown on 1 d PDA & is placed in the center of petri e dish containing 1% agar in distilled 0 v water. 2 r After 21 days at 26˚C sporangium e formation can be seen at the ly s periphery of the petri dish. u e J s r Mycology online 1 t Padhye & Ajello . JCM 1988;2 26: 1861–1863 0- gh 2 ri N ll T . A M er M k at ea sp Rhizomucored pusillusf nt o se ht re ig P yr op C ©

Bard et al., Med Mycol Case Rep. 2014; 5: 20-23 Thomas PA. CMR; 2003; 16: 730-97; Mycology Online

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Rhizomucor microsporus

9. . 01 ed 2 rv ly se Ju re R. microsporus var. rhizopodiformis SDA, 48 hour culture, globose sporangia s 3 weeks-old culture, azygospores was produced 1 t -2 h Cheng et al. JCM 2009; 47: 2834–43; g http://www.pf.chiba-u.ac.jp/gallery/fungi/r/Rhizomucor_microsporus_var_rhizopodiformis.htm0 2 ri N ll T . A M er t M ak a pe ed f s nt o se ht re ig P yr op MOLECULARC BASED ©

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Diagnosis of mucormycosis •mucormycosis remains difficult to diagnose, •Direct methods investigation is the “gold standard”. for . diagnosis, but requires expertise & does not1 allow9 d species identification. 0 e 2 rv •Culture of clinical specimens often faill toy grow (ca.se 50%). Ju re •Require other technique: molecular based s 21 t identification - gh 0 Roden et al., 2005. Clin Infect Dis. 41:634–653. 2 ri N ll T . A M er t M ak a pe Moleculard based method s te of • A retrospectiven study tusing tissue blocks, semi nested PCR continued by sequencingse h •rprimerse developedig from 18S ribosomal DNA, the V4 and V5 variable regions P• The outer primersyr ZM1 (5-ATT ACC ATG AGC AAA TCA GA-3) and ZM2 • (5-TCC GTCp AAT TCC TTT AAG TTT C-3) • Productso of the seminested reaction using primers ZM1 and ZM3 (5-CAA TCC AAGC AAT TTC ACC TCT AG-3) are 175 to 177 bp long • Able to distinguish variability to identify genera but not to species level. ©• 12 positive culture (10 PCR pos & 2 PCR neg); 15 negative culture (12 PCR pos, 3 PCR neg)

Hammond et al., JCM 2011; 49: 2151–3

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Species identification of 9. . culture using universal 01 ed fungal primers (ITS 2 rv regions) ly se Ju re 1 ts -2 h 0 Dannaoui E. gClin Microbiol Infect 2009; 15 (Suppl. 5): 66–70 2 ri N ll T . A M er M k at ea sp Speciese identificationd f nt o •Sequencingse hoft the ribosomal genes: r•eUniversal ifungalg primers - ITS (primers ITS1 & ITS 4) P • D1/D2 yribosomalr DNA (primers NL-1 & NL-4) op •BetaC tubulin © •Calmodulin Romanelli et al., JCM. 2010, 48: 741–52 Atkins & Clark. J Appl Genet. 2004;1:3–15. 2. Balajee et al., JCM. 2009; 47:877–84.

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Algorithm for diagnosis

Treatment: . surgical, anti 9 . Surgical excision of fungal & handling of d the lesion: 1 risk factors e - KOH slide: broad 0 septate hyphae v Clinical 2 r presentations: - Culture: filamentous rhinocerebral, fungi y e sinusitis, skin, l s - histopathol: broad other type septate hyphae u e Patient with - Molecular based r risk factor, J identification e.g. DM, s malignancy 1 t etc. -2 h 0 gPersonal oppinion 2 ri N ll T . A M er M k at ea sp Conclussioned f t o • Suspicionen of mucormycosest should be started when we recognize underlyinges conditiongh (patient at risk) •rClinical presentationri & its relation with underlying condition P• The importancey of direct microscopic investigations: KOH wet slide &p histopathology. Fast result, facilitate fast treatment for the patiento • Species C identification is important which can be done based on ©phenotypic identification (culture) or molecular based method

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COLLABORATION BETWEEN . DOCTORS FROM MYCOLOGY lab., 9 d. BETTER RESULT IN PATIENT 01 e 2 rv ly se Take home message u e J s r 21 t 0- gh 2 ri N ll T . A M er t M ak a pe ed f s nt o se ht re ig P THANKyr YOU Presentedop at MMTN meeting - Penang, Malaysia, July 20-21,2019 C ©

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