Metodi biochimici e biomolecolari applicati alla medicina clinica: rendimento della real-time TaqMan PCR per la valutazione della resistenza alla claritromicina in H.pylori e tests fecali nella diagnorstica del carcinoma colo-rettale: M2PK, calprotectina e FOBT

1) Background: The most common methods to evaluate clarithromycin resistance is the E-Test, but is time consuming. Resistance of Hp to clarithromycin is due to point mutations in the 23S rRNA. Eight different point mutations have been related to CH resistance, but the large majority of the clarithromycin resistance depends on three point mutations (A2142C, A2142G and A2143G). A novel PCR-based clarithromycin resistance assays, even on paraffin-embedded biopsy specimens, have been proposed. Aims: to assess clarithromycin resistance detecting these point mutation (E-Test as a reference method);secondly, to investigate relation with MIC values. Methods: Paraffin-embedded biopsies of patients Hp-positive were retrieved. The A2142C, A2142G and A2143G point mutations were detected by molecular analysis after DNA extraction by using a TaqMan real-time PCR. Results: The study enrolled 86 patients: 46 resistant and 40 sensible to CH. The Hp status was evaluated at endoscopy, by rapid urease test (RUT), histology and hp culture. According to real-time PCR, 37 specimens were susceptible to clarithromycin (wild type dna) whilst the remaining 49 specimens (57%) were resistant. A2143G is the most frequent mutation. A2142C always express a resistant phenotype and A2142G leads to a resitant phenotype only if homozigous. 2) Background: Colonoscopy work-load for endoscopy services is increasing due to colorectal cancer prevention. We tested a combination of faecal tests to improve accuracy and prioritize the access to colonoscopy. Methods: we tested a combination of fecal tests (FOBT, M2-PK and calprotectin) in a group of 280 patients requiring colonoscopy. Results: 47 patients had CRC and 85 had advanced adenoma/s at colonoscopy/histology. In case of single test, for CRC detection FOBT was the test with the highest specificity and PPV, M2-PK had the highest sensitivity and higher NPV. Combination was more interesting in term of PPV. And the best combination of tests was i-FOBT + M2-PK.

Risposte di resistenza a batteri fitopatogeni di importanti specie coltivate indotte da molecole segnale di diversa natura

A modern management of crop protection should be based on integrated control programmes, including the use of environmentally safe products. Antagonistic/beneficial bacteria and resistance inducers may have a great potential in the prophylaxis of diseases caused by common and quarantine pathogens. This work was carried out to confirm the ability of the known strain IPV-BO G19 (Pseudomonas fluorescens) against fire blight (Erwinia amylovora), as well as to evaluate their efficacy against southern bacterial wilt of tomato (Ralstonia solanacearum) and grapevine crown gall (Agrobacterium vitis). A virulent strain of R. solanacearum race 3 was inhibited by the antagonist on plate. When the pathogen was inoculated 48 h after their application to the root apparatus of tomato plants grown in a climatic chamber, bacterial wilt progression rate was clearly reduced. Moreover the defence response evoked by IPV-BO G19 was studied in tomato plants by monitoring the transcription of genes codifying for three PRs as PR-1a, PR-4, PR-5 and for an intracellular chitinase using multiplex RT-PCR and Real Time RT-PCR. In two field trials during 2005 and 2006, the strain IPV-BO G19 was compared with biofungicides and some abiotic elicitors to protect actively growing shoots of pear scions against fire blight. In both trials, IPV-BO G19 plus Na-alginate gave a high level of protection, three weeks after wound inoculation with E. amylovora. In pear leaf tissues treated with the antagonistic strain IPV-BO G19, catalase, superoxyde dismutase and peroxidise activity was evaluated as markers of induced resistance. The IPV-BO G19 strain was compared with other bioagents and resistance inducers to prevent grapevine crown gall under glasshouse and vineyard conditions.

Infezione da virus dell'epatite E nel suino e nell'uomo: aggiornamento sulle metodiche diagnostiche di laboratorio

The first part of the thesis is a brief review on the most important aspects of HEV infection in human and swine, followed by an update on the laboratory techniques currently in use for the diagnosis of HEV infections in humans and animals. The second part refers on the results of two investigations carried out on the presence of HEV infection in swine farms in Toscana and Piemonte and on the presence of HEV infection in pigs and humans in some rural communities in Bolivia. HEV strains isolated from swine herds in Toscana and Piemonte were all included in the genotype 3, showing particular homology with Dutch porcine isolates, Spanish porcine and human isolates and British human isolates. The investigation carried out, with a random sampling, in the province of Cuneo, detected HEV infection with a prevalence of 46% on farms with a number of pigs greater than 500. HEV was detected in pigs and humans in rural communities in Bolivia and all the viral isolate were included in the genotype 3. Aminoacidic homology of human and swine isolates was estimated to be 92%. Results on the development of a Real Time RT-PCR to detect HEV are also reported. The used Real Time RT-PCR protocols, one step and two steps, exhibited good sensitivity to detect several Italian swine HEV strains with high rate of genetic variability.

Epatite E: ruolo del suino nella trasmissione dell'infezione all'uomo, studio di prevalenza in aree rurali della Bolivia

L’infezione da virus dell’ epatite E (HEV) nei suini e nell’uomo è stata segnalata in diversi Paesi. Nei suini, il virus causa infezioni asintomatiche, mentre nell’uomo è responsabile di epidemie di epatite ad andamento acuto nei Paesi a clima tropicale o subtropicale con condizioni igieniche scadenti, di casi sporadici in quelli sviluppati. HEV è stato isolato anche in diversi animali e l’analisi nucleotidica degli isolati virali di origine animale ha mostrato un elevato grado di omologia con i ceppi di HEV umani isolati nelle stesse aree geografiche, avvalorando l’ipotesi che l'infezione da HEV sia una zoonosi. In America del Sud HEV suino è stato isolato per la prima volta in suini argentini nel 2006, mentre solo dal 1998 esistono dati sull’ infezione da HEV nell’uomo in Bolivia. In questa indagine è stato eseguito uno studio di sieroprevalenza in due comunità rurali boliviane e i risultati sono stati confrontati con quelli dello studio di sieroprevalenza sopra menzionato condotto in altre zone rurali della Bolivia. Inoltre, mediante Nested RT-PCR, è stata verificata la presenza di HEV nella popolazione umana e suina. La sieroprevalenza per anticorpi IgG anti-HEV è risultata pari al 6,2%, molto simile a quella evidenziata nello studio precedente. La prevalenza maggiore (24%) si è osservata nei soggetti di età compresa tra 41 e 50 anni, confermando che l’ infezione da HEV è maggiore fra i giovani-adulti. La ricerca di anticorpi anti HEV di classe IgM eseguita su 52 sieri ha fornito 4 risultati positivi. Il genoma virale è stato identificato in uno dei 22 pool di feci umane e l'esame virologico di 30 campioni individuali fecali e 7 individuali di siero ha fornito rispettivamente risultati positivi in 4/30 e 1/7. La Nested RT-PCR eseguita sui 22 pool di feci suine ha dato esito positivo in 7 pool. L’analisi delle sequenze genomiche di tutti gli amplificati ha consentito di stabilire che gli isolati umani appartenevano allo stesso genotipo III di quelli suini e presentavano con questi una elevata omologia aminoacidica (92%).

Analisi del profilo tossicologico e cancerogeno di contaminanti ambientali e identificazione dei rischi per la salute umana correlati all'esposizione

L’attenta analisi della letteratura scientifica su argomenti riguardanti i contaminanti ambientali oggetto di studio (i policlorodifenili) ha permesso di raccogliere dati utili riguardanti le proprietà di queste molecole, la loro diffusione e la loro pericolosità. Oggetto della ricerca è stato lo studio in vitro del potenziale citotossico e trasformante dei PCB, utilizzando come riferimento una miscela commerciale di PCB, l’Aroclor 1260, e di un MIX di 18 congeneri ricostituito in laboratorio. Il lavoro è proseguito con la valutazione degli effetti di queste miscele e di due congeneri singoli (PCB 118 e PCB 153) su linee cellulari diverse in test di vitalità a breve termine. L’utilizzo di test specifici ha poi permesso la valutazione di un possibile potenziale estrogenico. Una volta ottenuto un quadro generale sui possibili effetti delle miscele grazie ai risultati dei test funzionali, è stata valutata la modulazione, da parte delle molecole e/o di miscele delle stesse, dell’espressione di geni coinvolti nella risposta ad estrogeni o a composti diossino simili, andando ad effettuare un’analisi di tipo molecolare con Real-Time PCR (RT-PCR) e analizzando nello specifico marcatori di pathway dell’Aryl Hydrocarbon Receptor (AhR) o dell’Estrogen Receptor (ER). In ultima analisi al fine di verificare l’applicabilità di biomarkers di espressione a situazioni di contaminazioni reali, ci si è focalizzati su campioni estratti da matrici ambientali, ed in particolare linee cellulari di interesse sono state esposte a estratti di sedimenti provenienti da siti inquinati. L’approccio scelto è stato di tipo molecolare, con lo scopo di individuare pathway da valutare in un secondo momento in test funzionali specifici. L’attività di ricerca si è avvalsa della tecnica del DNA-microarray per valutare la modulazione dell’espressione genica in risposta all’esposizione a contaminanti ambientali. In questo modo è possibile definire i profili di espressione genica che sottendono a risposte biologiche complesse nell’intento di individuare biomarcatori in grado di predire il rischio per l’uomo, e di consentire la stima di una relazione diretta tra esposizione ed effetti possibili.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

Development of new molecular methods for the diagnosis and the study of viral diseases of fish

The increase in aquaculture operations worldwide has provided new opportunities for the transmission of aquatic viruses. The occurrence of viral diseases remains a significant limiting factor in aquaculture production and for the sustainability. The ability to identify quickly the presence/absence of a pathogenic organism in fish would have significant advantages for the aquaculture systems. Several molecular methods have found successful application in fish pathology both for confirmatory diagnosis of overt diseases and for detection of asymptomatic infections. However, a lot of different variants occur among fish host species and virus strains and consequently specific methods need to be developed and optimized for each pathogen and often also for each host species. The first chapter of this PhD thesis presents a complete description of the major viruses that infect fish and provides a relevant information regarding the most common methods and emerging technologies for the molecular diagnosis of viral diseases of fish. The development and application of a real time PCR assay for the detection and quantification of lymphocystivirus was described in the second chapter. It showed to be highly sensitive, specific, reproducible and versatile for the detection and quantitation of lymphocystivirus. The use of this technique can find multiple application such as asymptomatic carrier detection or pathogenesis studies of different LCDV strains. The third chapter, a multiplex RT-PCR (mRT-PCR) assay was developed for the simultaneous detection of viral haemorrhagic septicaemia (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and sleeping disease (SD) in a single assay. This method was able to efficiently detect the viral RNA in tissue samples, showing the presence of single infections and co-infections in rainbow trout samples. The mRT-PCR method was revealed to be an accurate and fast method to support traditional diagnostic techniques in the diagnosis of major viral diseases of rainbow trout.