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Identificazione molecolare di Candida lusitaniae
No full textCandida lusitaniae è un patogeno emergente, causa di gravi infezioni nell’ospite immunocompromesso, e presenta una ridotta sensibilità all’amfotericina B. Una rapida e precoce identificazione di tale specie nei campioni clinici riveste, pertanto, una notevole importanza. L’utilizzazione di metodiche molecolari può costituire una valida alternativa ai convenzionali metodi colturali.
Allo scopo sono stati utilizzati primers selezionati dalla regione costante del gene ERG11 di Candida albicans per amplificare un segmento di 350 bp dal DNA genomico di C. lusitaniae. L’amplicone ottenuto è stato clonato, sequenziato e comparato con le sequenze geniche di C. albicans, C. dubliniensis, C. glabrata, C. guillermondii, C. kefyr, C. krusei, C. parapsilosis e C. tropicalis, riscontrando un’omologia del 62% - 97%. L’amplicone è stato successivamente sottoposto a digestione (Restriction Enzyme Analysis) con le seguenti endonucleasi Sau3A, HincII e NotI, che in un precedente lavoro avevano consentito di differenziare le 7 principali specie patogene di Candida.
I pattern elettroforetici ottenuti consentivano di differenziare C. lusitaniae dalle altre specie di Candida ad eccezione di C. glabrata, i cui pattern risultavano pressocchè identici. L’aggiunta di un quarto enzima di restrizione RsaI differenziava C. lusitaniae da C. glabrata.
Sono attualmente in corso saggi di PCR su campioni simulati (urine e sangue) per valutarne l’eventuale utilizzazione diretta sui campioni clinici
Helicobacter pylori : ureA, cagA and vacA expression during conversion to the coccoid form
No full textAs viability of coccoid forms of Helicobacter pylori can only be verified by demonstrating the integrity of the DNA and active protein synthesis, we analysed the expression of ureA, cagA, vacA genes after prolonged incubation in a liquid medium. Exponentially growing and ageing phase cultures were used. Our results showed that, although the coccoid forms had decreased DNA and RNA levels after 31 days, they were not degraded and still expressed the urease, cytotoxic island and vacuolating toxin genes. Coccoid forms are therefore viable and may act as a transmissible agent that plays a crucial role in disease relapses after antibiotic therap
Molecular identification of Candida lusitaniae using a PCR-REA method
No full textA pair of primers selected from the costant region of the ERG11 gene of C. albicans, was used to amplified a 350 bp segment from the genomic DNA of C. lusitaniae strain. The PCR product was cloned in plasmid pCR-TOPO (Invitrogen) and sequenced (GenBank submitted). The sequence of the fragment was compared with the published gene sequences of C. albicans, C. tropicalis, C. krusei, C. glabrata, C. guillermondi, C. dubliniensis, C. parapsilosis and C. kefyr. The homology range observed was 62% to 97% among all species.
The amplicon obtained from C. lusitaniae was therefore subjected to REA using Sau3A, HincII and Not I according to a precedent study in which restriction enzyme analysis (REA) of PCR product was used to identify Candida strains at the species level.
The REA pattern obtained allowed to differenziate C. lusitaniae from the other species of Candida except from C. glabrata whose patterns were very similar. Thus we tested another restriction enzyme, RsaI, with all species of Candida previously described. The results showed that only C. lusitaniae and C. krusei had a recognition site for this enzyme but the patterns were different from each other . The use of Rsa I for PCR-REA, in addition to the other enzyme, makes the identification of C. lusitaniae possible.
An early and rapid identification of this species in clinical specimens is of great importance because C. lusitaniae is an emerging human pathogen that can cause severe infections in immunocompromised hosts
Molecular typing of Aspergillus fumigatus isolates collected from cystic fibrosis patients
No full textTwenty-seven strains of Aspergillus fumigatus was isolated from the respiratory tract secretions of patients with cystic fibrosis (CF) together with P. aeruginosa and in some cases S. aureus.
The potential pathogenic role of A. fumigatus in this patients category is controversial and it is supposed to be a colonizer.
The aim of this work was to verify if C.F patients are colonized by a unique Aspergillus fumigatus genotype because literature data report that some genotypes are dominant and persistent in immunocompromised hosts.
In order to verify genetic relatedness, molecular typing methods including random amplified polymorhic DNA (RAPD) with two different primers (NS3 and R108) and Afut I restriction fragment lenght polymorphism (RFLP), were performed. Use of combination of methods resulted in a greater degree of discriminatory power than that can be achieved by a single method.
Our results suggest that different types of A. fumigatus exist in CF patients as colonizers.
For comparative purposes, we also tested the in vitro susceptibility to various antifungals (voriconazole, itraconazole and amphotericin B). No significant differences were observed except for one strain with high MIC value for itraconazole (16 mg/ml). Furthermore there was no correlation between genotype and susceptibility patterns.
The strain with high MIC for itraconazole (ITZ) was furtherly investigated for the full coding region sequences of 14a-sterol demethylase genes (cyp51A and cyp51B). Sequence analysis revealed no significant point mutation related to the different behaviour regarding ITZ.
The expression of efflux pumps are now under evaluation
Antimicrobial Susceptibility Testing of Helicobacter pylori Determined by Microdilution Method Using a New Medium
No full textAntibiotic susceptibility testing of Helicobacter
pylori isolates was performed by broth microdilution
method with MegaCellTM RPMI-1640 Medium (SIGMA).
Fifty five clinical isolates of H. pylori were tested against
metronidazole, tinidazole, amoxicillin, and clarithromycin.
The results were compared to those obtained by standard
agar dilution method. The microdilution method performed
with new medium, showed excellent correlation with agar
dilution results, with 100% agreement for metronidazole,
96.3% for amoxicillin, 90.7% for clarithromycin, and
92.8% for tinidazole. MICs determined by proposed
method were highly reproducible: replicate results were
variable within one-two-fold dilution by using different
inocula and different batches of medium
Epidemiologia molecolare di ceppi di Clostridium difficile isolati in reparti geriatrici
No full textClostridium difficile (Cd) è un microrganismo anaerobio, sporigeno, gram positivo. Si può trovare come commensale nella flora intestinale dell’uomo. C.d e’ il principale agente eziologico di diarrea associata a terapia antibiotica acquisita in ambiente ospedaliero. I ceppi patogeni producono tossine (A e B) con effetti citotossici a carico dell’epitelio intestinale. Alcuni ceppi produttori di tossina binaria, sono causa di infezioni più severe. A tutt’oggi non è noto il ruolo specifico di questa tossina.
Nei pazienti istituzionalizzati con età superiore ai 60 anni aumenta il rischio di acquisizione dell’infezione in relazione alle patologie concomitanti, alle modifiche della flora intestinale, alla pressione selettiva degli antibiotici, all’esposizione all’ambiente ospedaliero. Scopo del nostro lavoro è stato quello di effettuare una tipizzazione molecolare dei ceppi di Cd isolati da pazienti ospedalizzati presso ASP Pio Albergo Trivulzio. Nell’analisi di campioni sequenziali si è voluto verificare se le infezioni ricorrenti fossero dovute a recidive o reinfezioni
In vitro testing of Aspergillus fumigatus clinical isolates for susceptibility to voriconazole, amphotericin B and itraconazole : comparison of sensititre versus NCCLS M38-A using two different inocula
No full textVoriconazole, amphotericin B and itraconazole were tested in vitro against 18 strains of Aspergillus fumigatus isolated from cystic fibrosis patients. Susceptibility was tested with the broth microdilution method (M38-A protocol-NCCLS). Results of this reference method were compared with those of an experimental commercial microdilution broth method (Sensititre). Two different inocula, prepared from 2- and 7-day cultures, were used. Minimum inhibitory concentrations (MICs) of the reference method ranged from 0.25 to 2 microg/ml for voriconazole, 0.06 to 1 microg/ml for amphotericin B, 0.016 to >16 microg/ml for itraconazole. There were no significant differences in the MIC ranges or MIC90 values obtained with the two testing methods or with the two types of inocula. These findings confirm the good in vitro activity of voriconazole, itraconazole and amphotericin B against A. fumigatus. They also indicate that reliable susceptibility data can be generated more rapidly by commercial systems and use of 2-day cultures for inoculum preparation
Tipizzazione molecolare di ceppi di Aspergillus fumigatus isolati da pazienti con fibrosi cistica
No full textI pazienti affetti da Fibrosi Cistica (CF) sono esposti al rischio di infezioni delle basse vie respiratorie, in genere gli agenti causali sono Staphylococcus aureus e Pseudomonas aeruginosa con tutte le problematiche connesse alla formazione di biofilm e, soprattutto, alla farmaco-resistenza dei ceppi isolati. La colonizzazione e/o una superinfezione con funghi possono rappresentare un ulteriore complicanza infettiva, in particolare se si tratta di funghi filamentosi. Scopo del nostro lavoro è stato quello di verificare se i pazienti con CF fossero colonizzati da un genotipo predominante di Aspergillus fumigatus, il fungo filamentoso di più frequente isolamento dalle secrezioni delle basse vie respiratorie in tale categoria di pazienti.
Ventisette ceppi di A. fumigatus sono stati sottoposti a RAPD (Random Amplified Polymorphic DNA) con due differenti primers (NS3 and R108) ed a RFLP (Restriction Fragment Lenght Polymorphism) con AfutI. La combinazione di più sistemi di tipizzazione molecolare ha consentito di poter discriminare tra i ceppi e di dimostrare che diversi genotipi erano presenti nei nostri pazienti.
Il contemporaneo riscontro di un ceppo con una ridotta sensibilità all’itraconazolo ci ha spinti ad indagare il coinvolgimento di meccanismi molecolari di resistenza valutando la presenza di eventuali mutazioni nei geni (cyp51A e cyp51B) che codificano per l’enzima bersaglio degli azoli e l’espressione delle pompe di efflusso. L’analisi di sequenza dei geni cyp51A e cyp51B non ha rilevato mutazioni puntiformi significative correlabili al fenotipo resistente così come l’espressione delle pompe di efflusso. Sono in corso esperimenti di valutazione della diversa espressione delle pompe di efflusso in questo ed in ceppi con fenotipo sensibile dopo esposizione a concentrazioni sub-mic di itraconazolo
In vitro activity of voriconazole and other antifungal agents against clinical isolates of Candida glabrata and Candida krusei
No full textThe antifungal susceptibility of 309 Candida glabrata and 63 Candida krusei clinical isolates was tested via the Sensititre YeastOne-3 system (Trek Diagnostic Systems, East Grinstead, UK) to compare the in vitro activity of voriconazole with that of five other antifungal agents (amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine). Voriconazole was highly active (MIC90, 0.5 microg/ml) against isolates of both species, including those for which the MICs of itraconazole and fluconazole were high (MIC90s of itraconazole, 2 microg/ml for C. glabrata and 0.5 microg/ml for C. krusei; MIC90s of fluconazole, 32 microg/ml for C. glabrata and 64 microg/ml for C. krusei). Ketoconazole MIC90 values for both species were identical to those of voriconazole. The MIC90 of amphotericin B was similar for both species (0.125 microg/ml for C. glabrata and 0.25 microg/ml for C. krusei). As expected, flucytosine was only moderately active against C. krusei isolates (MIC90, 16 microg/ml) but was highly active against C. glabrata isolates (MIC90, 0.03 microg/ml). Potential cross-resistance within the azole class was noted for some strains of C. glabrata (5.5%) that presented high MIC values for all the azoles tested. In order to consider voriconazole a viable alternative to other triazoles for the treatment of infections caused by Candida species, susceptibility testing of all clinically significant isolates of C. glabrata and C. krusei is recommended because of the potential for azole cross-resistance. The Sensititre YeastOne-3 seems to be a suitable commercial tool for this purpose
Utilizzazione della PCR per la diagnosi di zigomicosi
No full textLe zigomicosi sono infezioni fungine invasive gravi ad evoluzione fatale nell’ospite immunocompromesso.
La diagnosi convenzionale è resa spesso difficoltosa dall’impossibilità di ottenere prelievi o campioni bioptici significativi. La diagnosi molecolare potrebbe rappresentare una valida alternativa.
Recentemente, ci è stato richiesto di accertare in preparati istologici (vetrini non colorati) la presenza di DNA appartenente a zigomiceti, il cui parassitismo tessutale era stato riscontrato utilizzando colorazioni istologiche specifiche per i fungh
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