FTA Technology for Fungal and Bacterial DNA Storage

“Old pathogens, new species:Library the role of the new taxonomy”

Lecture author Dr Andy Borman Deputy Director Onlineby UK National Mycology Reference Laboratory,© Bristol, UK

ECCMID12 August 2011,ESCMID 2003 Milan Library Potential impact of new developments

in the molecular identification Lecture of pathogenic fungi on clinical decision making: author Are the vast numberOnline of taxonomicby changes that are resulting © from molecular approaches clinically relevant?

ESCMID Conventional identification and taxonomy (of moulds) Library Historically relies on the characterisation of the structures produced by fungi during their sexual cycle (3 Phyla):

(now 7 phyla with additional 4 sub-phyla in polyphyletic Zygomycota - Mucoromycotina) Ascomycetes ZygomycetesLecture Basidiomycetes author Onlineby ©

ESCMID Female mould with nice fruiting bodies seeks male with huge conidia for raunchy time on Sabouraud’s agar………. Library Unfortunately, many fungi can not be induced to enter a sexual cycle in the lab:

Lack of a compatible matingLecture partner (usually a clinical isolate is a single mating type) author Incorrect growthOnline conditionsby (natural substrates/host) – Deuteromycetes© or “imperfect fungi”

Instead, identification usually relies upon characterisation of the mechanismESCMID of production of asexual spores (conidia) and the type of spore produced Some asexual spores (conidia) Library

Lecture author Onlineby ©

ESCMID Why we need molecular techniques for Fungi

• Fungal kingdom is vast – we can’t beLibrary expected to recognise all members, or ID them on basis of conidia > 100 000 species of Fungi described (estimated >106). > 700 species of yeast. >1000 new species/year > 600 species isolated from infections (>200 regularly isolated) 1 new/emerging pathogen per month Lecture • Virtually any fungus capable authorof growth at 37oC is a potential pathogen in patients with impaired immunity Onlineby © • Many fungi from real (esp. deep) infections fail to produce structures by which they can be identified ESCMID • Taxonomic affiliations for imperfect fungi (no known sexual cycle) Taxonomic impact of molecular approaches to fungal ID

Re-named fungi : Library Neoscytalidium dimidiatum; Lichtheimia corymbifera (ex Absidia corymbifera via Mycocladus corymbiferus [briefly]); Paecilomyces lilacinus (Purpureocillium lilacinum??); Fusarium lichenicola (ex Cyclindrocarpon lichenicola); Main (only) clinical impact resultsLecture from potential confusion surrounding new names author Description of a large number of “cryptic” species (sister species within well-known morphospeciesOnline that areby only distinguishable by molecular approaches). ©

Aspergillus fumigatus species complex Fusarium spp. Scedosporium apiospermum species complex ESCMID Clinical impact/relevance? DNA extraction (MRL)

• Apply homogenous aqueous suspension of yeastLibrary cells or mould spores and hyphal fragments directly to Whatman FTA matrix cards and microwave treat (2 x 30 sec) cards to dry them and kill organism

• Filter punches removedLecture from the area of the dried spot using a Micro Punch. author

0 h • TheOnline discs areby transferred into a PCR tube, washed, ©and then directly used for molecular detection. Aspergillus fumigatus LSU μ hp PCR -ve 0 h hp μ (Borman et al., 2006 Med. Mycol. 44, 389-398) 38 species of yeast;ESCMID 75 species of mould. Now tested in excess of 2000 isolates; new improved protocol (Borman et al. 2010 Mycopathologia 169, 445-449 Amplification Targets we use for Fungi Library •Should be highly conserved across species – “pan-fungal” PCR primers

•But be sufficiently variable to allow accurate identification to species level after sequencing of the PCR products Lecture Nuclear Ribosomal Repeat – Multi-copy genes ITS1 author LSU

Nuclear Small rDNA Nuclear Large rDNA Onlineby 5.8S 18S (LSU) Internal© Internal Transcribed Transcribed Spacer 1 Spacer 2 (ITS1) (ITS2) Moulds to species level Yeasts to species level ESCMID(for some genera) Moulds to genus level 2 most “populated” loci in public databases Fusarium spp.

Library Plant pathogen; can cause nail infections; most common mould associated with keratitis (contact lens users; traumatic inoculation); emerging opportunistic pathogen in immuno-compromised patientsLecture – disseminated infections with extremely poor prognoses. author Onlineby ©

ESCMID Fusarium cryptic species

Morpho-species Species complexes (MLSTLibrary including tEF-1α) 12 assoc. with human >69 assoc. with human infections Infections

Fusarium solani - now FSSC >45 spp. (~20 from infections) Lecture Fusarium dimerum - now FDSC - at least 12 species

Fusarium oxysporum - now FOSCauthor >30 distinct sequence types Online(speciesby limits remain unresolved within FOSC) Fusarium moniliforme © Fusarium verticilloides - Giberella fujikuroi species complex ~40-50 species; all species from human infections fully described ESCMID Fusarium incarnatum-equiseti SC ~30 spp. incl ~20 from infections Fusarium chlamydosporum species complex (>4 species) Clinical implications?

Library FSSC, FOSC, GFSC account for ~80% human infections; to date no obvious clinically significant differences in antifungal susceptibility profiles, or “virulence / pathogenicity” Lecture However, some species show restricted disease patterns (FOSC and v. low occurrence in ocular infections)author Onlineby Main utility is therefore probably© epidemiological surveillance and outbreak source identification

Data principally taken from K. O’Donnell 1998 (Mycologia 90; 465-93); 2000 (Mycologia 92; 919-38); 2007 (J. Clin. Micro. 45: 2235-48); 2008 (J. Clin. Micro. 46; 2477-90); and MRL unpublishedESCMID data We used to think this was an easy fungus to ID !!

A. brevipes Library A. duricaulis MIC90 AMB ITR VOR CAS A. fumigatus A. fumigatus 1 0.25 0.25 0.5 A. lentulus 1-2 11-2 2-16 A. fumigatiaffinis A.thermomutans 1-2 1 2-4 0.03 A. fumisynnematous A.udagawae 2-4 0.5 1 0.125 A. lentulus A. novofumigatus S.A. BalajeeLecture (Eukaryotic Cell, 2005; Eukaryotic Cell 2006) A. thermomutatus (N. pseudofischeri) NumberSeveral cryptic1 cause species of invasive within mould A. fumigatus infections which in can A. turcosus only be reliablyneutropenicauthor identified patients by β-tubulin gene sequencing : A. viridinutans Most common mould to colonise CF patient lungs - ABPA A. unilateralis OnlineAllergiesbyA. lentulus N. assulata Otitis externaA. thermomutatus N. aureola Keratitis© A. udagawae N. denticulata N. fennelliae A. novofumigatus N. fischeri A.fumigatiaffinis N. galapagensis N. hiratsukae Found in an analysis of “A. fumigatus” isolates that N. quadracintaESCMID sporulate poorly and/or slowly N. udagawae MRL – 50 isolates selected as poorly-sporing and/or aberrant– sequencing ITS1 and β-Tubulin Library

In UK: Cryptic species are currentlyLecture very rare (<1% of isolates of A.mut. fumigatushelvola ) var alba All 100% identical to A.author fumigatus To date, none have been found to exhibit resistance to Onlinecurrentlyby employed antifungals in vitro© (caveat ~10 isolates, mainly single examples of various species)

NeosartoryaESCMID Aspergillus Aspergillus Neosartorya quadracinta fumigatiaffinis thermomutatus hiratsukae Scedosporium apiospermum complex Library Specific features: Clinically indistinguishable from invasive aspergillosis Infection often follows near-drowning accidents Pneumonia can progress to cerebral infectionLecture Neutropenia is a risk factor – emerging pathogen Refractory to treatment ~50% isolates author exhibit elevated in vitro MICsOnline to Amphotericinby B ©

ESCMID Cryptic species within the S. apiospermum complex Library Historical (morphospecies): Pseudallescheria boydii (teleomorph) of Scedosporium apiospermum

Based on β-tubulin sequences: (Gilgado et al., 2005 [J.Clin. Micro. 43; 4930-45]; 2008 [J.Clin. Micro. 46; 766-71]).

P. boydii – anamorph S.boydii P. angusta Lecture P. minutispora P. ellipsoidea P. fusoidea Scedosporium apiospermum author S. aurantiacum Most virulent in mouse models of infection (Gilgado et al. 2009; Med. Mycol. 47: 371-4; S. dehoogii Harun et al. 2010;Online Med. Mycol 48S:by 37-44) © Relative prevalence of each species varies: S. aurantiacum very prevalent in Australia (Heath et al. 2009 Clin. Microbiol. Infect. 15; 689-93); all species are more commonly isolated from urban environments; S. apiospermum predominates in industrialised Europe where it is the most common member of the complex isolated from human infections (Kaltseis et al. 2009 Med. Mycol. 47; 398-405). ESCMID Antifungal susceptibilities

Presence of cryptic species could explain ~50% of isolates R toLibrary Amb??

Species Study : number of isolates (% resistant to Amphotericin B) Alastruey-Izquierdo 2007 Gilgado 2006 Heath 2009 MRL 2008-2010 (Spain) (Spain) (Australia) (United Kingdom) N=64

P. boydii ND 30 (<100%)LectureND 25 (47%) S. apiospermum 42 (<100%) ND author26 (<100%) 26 (50%) S. aurantiacum 4 (100%) 7 (100%) 27 (<100%) 5 (100%) Onlineby S. dehoogii ND ND© ND 1 P. angusta ND 4 (100%) ND -

P. minutispora ND 4 (<100%) ND - P. ellipsoidea ESCMIDND 6 (<100%) ND 1 P. fusoidea ND 2 (100%) ND - Molecular ID of unusual moulds: the agents of dark grain mycetoma

Library Fungal mycetoma: Most infections occur in tropical areas but imported cases of fungal mycetoma do occur in the UK. Infections follow the traumatic implantationLecture of fungal spores or hyphal fragments present in soil or on plant material. Destructive infection of the skin and subcutaneousauthor tissues, which if untreated, progresses to involve muscle and bone. Chemotherapy with antifungalOnline drugsby is often not effective - surgical methods, often involving amputation© of the affected limb.

ESCMID Library Several moulds known to cause dark grain mycetoma (Madurella grisea, Madurella mycetomatis, and Pyrenochaeta romeroi).

Major problems for diagnosis. LectureOrganisms when cultured in vitro from biopsies of mycetomal lesions frequently fail to produce spores or other structuresauthor by which they can be formally identified.Online by Additionally, virtually no© data exist concerning the genetic relationships between these organisms. Or possibility that other mould species could provoke mycetomasESCMID Mycetoma continued………

Library •Organisms isolated from dark grain mycetomas, and stored in National S. Africa Collection of Pathogenic Fungi (NCPF). CulturedAMB, inGroup vitro IVITR, ……… VOR, CAS

M.mycetomatis •DNA extracted using FTA Lecture mycetomatisTYPE AMB, ITR, VOR, •PCR amplification of LSU and ITS regions,Group II sequencing, BLASTCAS searchesIndia 100% Only reliable matches obtained with MadurellaL.author senegalensis mycetomatis isolates (which produce distinctive sporulation patterns eventually in vitro anyway). • “Madurella grisea” is poly-specificS. Americaand OnlineGroup IIIby AMB, ITR, VOR, CAS 40% contains species100% from several different genera For all other organisms,* very poor© matches - (80% similarity) with “leaf litter * ascomycetes” • Many organisms identified as Madurella grisea are * * in fact P. romeroi isolates that “refuse” to sporulate * * P.romeroi (Group I) * •P. romeroi is a more common mycetomal agent than •Phylogenetic studies* AMB, ITR, VOR, CASrecords suggest. ESCMID33% 50% 100%

(Borman et al., in preparation) Role of molecular techniques for clinical yeast isolate ID Library > 700 species of yeast described

> 200 species (25 genera) isolatedLecture from human infections Conventional (fermentation/assimilation)author ID kits cover 30 - 40 most commonlyOnline encounteredby species Kits don’t work if the organism© is misbehaving

Many species-specific trends in antifungal susceptibility profilesESCMID Yeast species identified 2004-2006 as a function of frequency of isolation

Organism Number Number requiring % requiring received molecular ID Librarymolecular ID C. albicans 1192 7 0.6 C. glabrata 659 6 0.9 C.parapsilosis 430 3 0.7 C.tropicalis 156 0 0 C.lusitaniae 99 19 19.2 C.krusei 74 4 5.4 Cr.neoformans 64 3 4.7 S.cerevisiae 60 4 6.7 C.guilliermondii 53 19 35.8 M.pachydermatis 36 2 5.5 C.dubliniensis 33 3 9.1 Trichosporon sp. 23 4 17.4 Rhodotorula sp. 19 Lecture0 0 C.inconspicua 17 5 29.4 C.kefyr 15 4 26.7 C.famata 12 3 25 >9/10 OK C.nivariensis 11 11 100 Geotrichum sp. 10 3 30 C.pelliculosa 822author 5>2/3 OK C.fabianii 5 5 100 C.lipolytica 5360 C.blankii 4 4 100 <2/3 OK C.utilis 4125 Cr. albidus 400 C.rugosa 3 Onlineby 1 33.3 C.norvegensis 3 3 100 C.pararugosa 3 3 100 C.catenulata 3 © 3 100 Kloeckera sp. 3 1 33.3 S.elongasporus 2 2 100 C.zeylanoides 2 2 100 C.eremophila 2 2 100 C.lambica 2 2 100 C.ciferii 1 1 100 C.boidinii 1 1 100 C.palmioleophila 1 1 100 C.freyschussii 1 1 100 C.magnoliae 1 1 100 C.viswanathii 1 1 100 C.haemulonii 1 1 100 C.pseudointermedia ESCMID1 1 100 C.pseudoglaebosa 1 1 100 Linton et al., J. Clin Microbiol. 2007. 45; 1152-8 A common question (or criticism) Library Q: Why bother identifying yeast isolates to species level ?

A: Epidemiology, surveillance and also antifungalLecture choice (many species-specific trends in antifungal susceptibility profiles) plus proposed changes to CLSI guidelines = species-specific antifungalauthor interpretive breakpoints

Q: Yes but you can performOnline antifungalby susceptibility testing faster than you can arrive at© a molecular identification

A: fair point / still needed for epidemiological studies / oh go away / NOT ANY MOREESCMID Pyrosequencing ® works …….. Organisms tested so far which give unique pyrosequencing profiles:

ORGANISM ITS2 PYROSEQUENCING PROFILE SIMILARITY NUMBER TESTED C. albicans GTCAAA----GTTTGAAGATATAC G-TGG-TAGACGTTACC 35 21 L. elongisporus GTCgAA----GTTTGAA-ATATAga-TtG-gAGcttTTAtt 24 Library2 C. dubliniensis GTCAAA----GTTTGAAGAataAa a-TGG-gcGACGccaga 23 14 C. tropicalis GTCAAA----GTTatgAaATAaAt tgTGG-TgGcCacTAgC2334 N = 40 sp. S. cerevisiae* GTCAAA----cTTTaAgaAcATtg ttcGccTAGACGcTctC2216 468 isolates C. metapsilosis GTCgAA-----TTTGgAagaATgt t-TGG--AGtttgTACC 21 4 C. pelliculosa GTCAAA----cTTTtAgttTATt- GtTGt-TAagCcgagCC 21 10 C. parapsilosis GTCgAA-----TTTGgAagaAgtt t-TGG--AGtttgTACC 20 69 C.palmioleophila GTCAAActt-GTTTGttGtTtTtt aagGcaaAGcCtaacAC 20 2 C. orthopsilosis GTCgAA-----TTTGgAagaA-tt t-TGG--AGtttgTACC 20 10 Filobasid.unigut. GTCAgA----tgTcaAAG-TATAC acaGG-aAGcaacacCa 20 1 P.membranifaciens GTCgA-----GcTcaAtGATATAtttTcGcTcGgCGgcAaa 20 3 C.glaebosa* GTCAAActt-GTTTGt-GtTtTAt aagGcaaAGcCtaatga 19 1 Zygosacc. bailii GTCAAA----cTTTGAgagTATtg ttTGc-ccaLectureAgGcgcgC1 9 1 C. kefyr GTCAAA----cTTTGAgagTtTtg GtTaa-agcggtaTgCC 19 16 C. bracarensis GTCAAA----cTTaaAgGtTtctg t-TtG-ccGtgGTcACa 18 1 C. eremophila* GTCgAgcttaGTTaaAAGtTcggC G--GccaAagCGTgcta 18 1 C. blankii GTCAAA----tTTTGgAGcggTtg ttacGcctGtCtcgAaC18 1 Kazachstania sp* GTCAAA----cTTaaAAGAacact GtTcG--ccACGgctgtauthor18 3 C. nivariensis GTCAAA----cTTaaAgGtTcctg t-TtGccAGcaGacttC1726 C. utilis GTCAAg----cTTaGAAaggtgtt a-aGccgAG-CtcTgCC 17 1 Cr. neoformans* GTCAAA-----caaaAAGAgATgg t-TGttatcAgcaagCC 17 17 Sporopachydermia sp. GTCAAA---gaTTTAgAtcTtTgt caaGbyc-aAGAC aaacta 17 2 Sporob. salmonicolor aTCtAA----tcTaaOnlineAAGgTAgAC ttTaGgg AttaGaagCt 17 2 C. fabianii* GTCAAA----cTTatgAagaAatt GtTaGgccGAgcccAaa 16 9 C. glabrata GTCAAA----cTTaaAGacgtctg tcTGccTcagCGacgCa 16 47 C. inconspicua GTCgAg----cTT-GAttAaAag©T t-c GGcggGgaGaacgC1611 C. lusitaniae GgCgAA----aTgTcgtGcTgTAa caaGctTAactGTTtta 16 59 C. zeylanoides GTCAAA----cTTTGtttgTtgtt GtaaGgccGAgccTgtg 16 1 C. famata* GTCAAA----cTTgtttGtTATAt tgTaaggccgaGcctag 15 4 C. guilliermondii* GTCAAA----cTTgtttGgTtgtt G-TaaggccggGccAaC1527 C. norvegensis GTCgAg----cTTaGAtttaAaAa aaaaGtTcGgCGggcca 15 4 Pseudoxyma aphidis* GcCgAt---gaaTTGAAatTAaAt cccttcctctCcTTcCg 15 1 R. mucilaginosa aTCtAA----tcTTaAA-ATgTAg acattcTgattagaAgC15 7 Cr.saitoi complex GcCAgA----tgTTatgaATATta a-TccgaAGAtcaatgg 14 1 Cryp. terreus* GcCAgA----taaTaAA-AaAgtc a-TGtccAcgaGgTgga 14 1 (Borman et al 2010. C. pseudointermedia*ESCMIDGgCgAA--aaGaa TaAAGtTgaAg taacG-TAttgcaacaa 14 2 J. Clin. Microbiol. M. pachydermatis acCAgA----aaTgaAAaAggaAt G-TactTttcaGTTcgt 14 2 C. krusei GTCgAg----cTTTttgttgtctCGcaacactcgCtcTcgg 11 28 48, 3648-53.) C. rugosa aatAAc----GTcaaAgGgTccgt aacaagcttAacTgttt 9 6 47, 2307-2310.

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C. parapsilosis C. metapsilosis C. orthopsilosis C. parapsilosis (contains 3 distinct species [2 new ones] on from 11 genes; of MLST using fragments basis Tavanti

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16 isolates in 2 years (17 16 isolates in ESCMID ESCMID ., 2008. al most common C. inconspicua / norvegensis C. nivariensis (Borman et yeast pathogen?? C. nivariensis Potential clinical impact of C.nivariensis Library More than half of isolates received were cultured from deep, usually sterile body sites – indicative of systemic infection (blood, biopsies, ascitic fluid, abscess) Lecture AMB ITR VOR FLU FLY POS CAS

C. nivariensis MIC90 0.5 >16 4author >64 0.5 2 1 MFC 2 >16 >16 >64 32 >16 2 90 Onlineby © C. glabrata MIC90 110.532 <0.1 4 2

MFC90 2 >16 >16 >64 <0.1 >16 >64

ESCMID Summary/conclusions

Library Moulds : Cryptic species likely to be described in most key genera of pathogenic moulds; for some species in certain geographic areas - apparently little impact on antifungal choice, for other species ID to species level mayLecture be important.

Yeasts : molecular methods are becomingauthor an essential adjunct to conventional (biochemical/phenotypic) ID methods. Onlineby © Overall clinical relevance remains to be determined, but is likely to depend on: - particular species involved - countryESCMID of isolation (different antifungal susceptibility profiles?) - local epidemiological considerations (prevalence) ISHAM Working group recommendations for molecular ID (of moulds) Library

Lecture author Onlineby

© If molecular ID is deemed important: 1) ITS1 sequencing and comparison against validated databases (~ 20% of fungal sequences in public databases are erroneous) – ID to species complex level 2) Sequencing of secondaryt targets to discriminate cryptic species (β-tubulin for S. apiospermum SC, ESCMID A. fumigatus SC; tEF-1α for Fusarium spp). Acknowledgments

HPA Mycology Reference Library Laboratory, Bristol. Haroun Shah, NCTC, HPA, Colindale, UK.

Mark Fraser Paul Bridge, British Antarctic Survey, Debbie Oliver Cambridge,Lecture UK. Adrien Szekely Eric Dannaoui, Marie Desnos-Ollivier, Institut Pasteur, Chris Linton Paris, France.author Mike Palmer Frank Odds, Aberdeen Fungus Group, Institute of Colin Campbell OnlineMedicalby Sciences, Aberdeen, UK.

(Sarah-Jane Miles) Biotage (Rebecca Petch) (Ann Holmes)ESCMID Whatman International Promoting professional mystique………….. Library Metschnikowia pulcherrima Pseudochaetosphaeronema larense

Staphylotrichum coccosporum LectureMycoleptodiscus indicus Coniothyrium fuckeliiauthor Onlineby ©Dichotomophthoropsis nymphaearum

Colletotrichum gloeosporioides ESCMID Sporobolomyces salmonicolor Thanks for listening Library

Trichoderma harzianumESCMID