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Amplificazione Delle Regioni ITS1 E ITS2 Per L’Identificazione Di Muffe Di Interesse Clinico

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Amplificazione Delle Regioni ITS1 E ITS2 Per L’Identificazione Di Muffe Di Interesse Clinico Blanca M. Fernandez Rodriguez Lavoro di diploma Formazione Tecnico in Analisi Biomediche SSS Scuola Superiore Medico Tecnica, Locarno Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico Lavoro svolto presso Istituto Cantonale di Microbiologia, Bellinzona 2007 Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico 1 Indice 1. Riassunto, Abstract 3 2. Introduzione 4 3. I funghi 5 4. La classificazione 6 5. Gli aspetti clinici 7 6. Le regioni ITS1 e ITS2 8 7. Materiali e metodi 10 7.1. Campioni 10 7.2. Coltura 10 7.3. Estrazione 10 7.4. PCR 11 7.5. Seminested PCR 12 7.6. Controlli 12 7.7. Elettroforesi 13 7.8. Purificazione dei prodotti PCR 13 7.9. Reazione di sequenza 13 7.10. Purificazione dei prodotti della reazione di sequenza 13 7.11. Determinazione automatica della sequenza nucleotidica 14 7.12. Analisi delle sequenze 14 7.13. Costruzione dell’albero filogenetico 14 8. Risultati 15 8.1. Confronto fra Metodica 1 e Metodica 2 per l’estrazione del DNA 15 8.2. Coltivazione su piastra 17 8.3. Estrazione e amplificazione 17 8.4. Sequenze 18 8.5. Confronto fra identificazione morfologica e identificazione mediante analisi 18 delle sequenze 8.6. Albero filogenetico 19 9. Discussione 27 10. Bibliografia 28 Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico 2 11. Allegati 30 11.1. Tavole illustrate delle caratteristiche morfologiche degli Ascomiceti, 31 Zigomiceti, e basidiomiceti 11.2. Lista dei campioni 34 11.3. Lista delle sequenze di riferimento 37 11.4. Anamorfismo e Telomorfismo delle diverse specie usate in questo lavoro 39 11.5. Albero filogenetico 42 11.6. Classificazione secondo .S. de Hoog, J. Guarro, J. Gené, M. J. Figueras, 44 delle specie usate in questo lavoro Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico 3 1. Riassunto Abstract Il numero delle micosi é in continuo aumento Invasive fungal diseases are increasing in soprattutto nei pazienti ospedalizzati ed immunocompromised and hospitalized immunocompremessi. Una rapida ed accurata patients. Early detection and accurate diagnosi permette d’iniziare una terapia identification of the fungi permits a rapid and antimicotica adeguata. Le tecniche optimal initiation of antifungal therapy. diagnostiche comunemente usate in The diagnosis of mould is generally based on laboratorio per l’identificazione dei miceti biochemical, serological and phenotype sono tecniche biochimiche , sierologiche e identification. However these methods take fenotipiche. Purtroppo queste tecniche time and do not always lead to a precise prevedono tempi d’esecuzione lunghi e non identification of the microorganism. For this sempre forniscono risultati accurati reason, the Cantonal Institute of Microbiology nell’identificazione della specie. decided to test a molecular biological Per questo motivo l’Istituto Cantonale di technique, the polymerase chain reaction Microbiologia ha deciso di testare come (PCR), as identification procedure. In fact tecnica d’analisi per l’identificazione delle PCR technology promises more rapid and muffe patogene l’utilizzo della polymerase specific identification. chain reaction (PCR). Una tecnica che si In this work we have evaluated the feasibility presenta con un’alta sensibilità e specificità. of the sequence analysis of the internal In questo lavoro abbiamo sfruttato la PCR per transcribed spacer regions (ITS) of the ottenere le sequenze di una regione altamente ribosomal DNA for the identification of variabile presente nel DNA ribosomale, e pathological moulds. regioni ITS1 e ITS2, mediante l’utilizzo di The ITS sequences were obtained by primers universali (ITS1, ITS4).Le sequenze amplification of the ITS region (ITS1-5,8S- ottenute, appartenenti a 56 ceppi di 36 specie ITS2) with universal fungal primers (ITS1, diverse, sono state confrontate con le ITS4). The sequence analyses of 56 strains for sequenze presenti nella GenBank a total of 36 different species were compared (NCBI).L’identificazione genotipica che to reference data available at the GenBank questa ha fornito é stata confrontata con database, and the genotypic identification was l’identificazione morfologica. compared with phenotype identification. Le due tecniche d’analisi mostrano For 98,2% of the strains, both methods gave un’identificazione identica a livello di genere concordant results at the genus level. Of per il 98,2% e a livello di specie per il 44,6% . these, 44,6% were assigned to the identical L’identificazione fenotipica é risultata più species. Phenotypic criteria were more specifica a livello di specie per il 21,4%. specific in 21,4% and genotypic criteria were Mentre l’identificazione genotipica é risultata more discriminative for 7,14%. 12,5% of the più discriminante solamente per il 7,14%. Il strains showed discrepant results. We can 12,5% dei ceppi analizzati ha mostrato dei therefore conclude that identification of risultati discrepanti fra le due tecniche medically important moulds at the level of d’identificazione. In conclusione si può dire species by ITS sequencing is not a viable che l’analisi delle diverse specie di funghi alternative to conventional identification mediante l’utilizzo delle sequenze ITS non methods. può essere un valido sostitutivo dell’identificazione morfologica . Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico 4 2. Introduzione Il numero delle infezioni fungine é in continuo aumento soprattutto nei pazienti immunocompromess [1]. L’identificazione precisa delle diverse specie nella diagnostica delle micosi gioca dunque un ruolo molto importante per l’orientamento del trattamento medico. Prendiamo per esempio il genere Scedosporium dove il Scedosporium apiospermun é sensibile ad una determinata gamma di antimicotici mentre ill Scedosporium prolificans si mostra resistente alla maggior parte di essi [2]. Oppure consideriamo il genere degli Aspergillus che comprendente più di 180 specie diverse, ma la principale causa di problemi broncopolmonari, delle cheratiti, delle sinusiti, sopratutto nei pazienti immunocompromessi, sono unicamente l’Aspergillus fumigatus, l’A. flavus e A. terreus [3, 4]. L’identificazione delle diverse specie fungine può essere effettuata con l’analisi diretta al microscopio ottico del campione, mediante la coltura su piastra e la differenziazione morfologica delle diverse strutture, con test sierologici per la ricerca di antigeni o anticorpi e test biochimici. Oggi queste tecniche non sono più soddisfacenti soprattutto a livello diagnostico poiché sono tecniche laboriose, poco standardizzabili e con tempi di esecuzione molto lunghi in particolar modo per quanto riguarda la coltura su piastra i qui tempi variano da specie a specie, ma richiedono un minimo di 48h d’incubazione [2]. In oltre nella maggior parte dei casi queste tecniche non forniscono risultati accurati poiché presentano una bassa specificità nell’identificazione della specie [2]. L’utilizzo di tecniche di biologiamolecolare Figura 1. Tempi di crescita su piastra per permette invece un’identificazione rapida e specifica lieviti, muffe e funghi dimorfi [20] con una sensibilità maggiore. Molte pubblicazioni propongono infatti varie tecniche fra le quali troviamo la PCR, la Real-time PCR, la PCR-Elisa, la RFLP (Restriction Fragment Polymorfism) ecc. [5, 6, 7, 8, 21]. In questo lavoro si é deciso di testate come tecnica d’analisi per l’identificazione delle diverse specie di muffe l’amplificazione mediante la PCR della regione altamente variabile ITS1 e ITS2 presente nel DNA ribosomiale. Studi precedenti propongono l’utilizzo dei geni ribosomali poiché si presentano in più copie all’interno del genoma e mostrano regioni conservate, come per esempio i geni 18S, 28S e 5.8S comuni a tutti i funghi, e delle regioni altamente variabili fra le differenti specie, come le regioni ITS. Queste regioni variabili sono quindi sfruttabili per la classificazione filogenetica e dunque anche per un’identificazione a livello di specie necessaria per la diagnostica.[1, 3, 9, 10, 11]. Amplificazione delle regioni ITS1 e ITS2 per l’identificazione di muffe di interesse clinico 5 3. I Funghi Gli organismi che costituiscono il regno dei funghi sono organismi eucarioti, non fotosintetici, aerobi o anaerobi, pluricellulari o unicellulari. Possono presentarsi come lieviti, muffe o funghi dimorfi, essere saprofiti, parassiti oppure vivere in simbiosi con l’organismo ospite. Sono provvisti di una parete cellulare rigida, che può essere pluristratificata, composta da cellulosa, emicellulosa o chitina e presentano un citoplasma spesso multinucleato. I lieviti sono organismi unicellulari ovali di 3-5 µm di diametro; si riproducono principalmente per gemmazione e possono formare delle pseudoife. Le muffe sono funghi pluricellulari caratterizzati da un elemento di crescita tubulare ramificato detto ifa. L’ifa può essere suddivisa da setti parietali o no: ifa settata o cenocitica. Dal suo sviluppo per estensione apicale e laterale viene a formarsi il micelio (o tallo). Il micelio viene detto vegetativo se aderisce e penetra nel terreno di coltura o nel tessuto ospite, riproduttivo o aereo se le sue ife si proiettano sulla superficie del terreno e portano le cellule riproduttive o spore. I fungi dimorfi sono definiti come miceti in grado di presentarsi sotto forma di lieviti o muffe al variare delle condizioni ambientali [4, 12]. I funghi si riproducono in maniera asessuata o in maniera sessuata
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