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.

Chirurgia dermatologica pediatrica

Le caratteristiche istologiche, immunologiche e fisiologiche della cute in età pediatrica sono responsabili di quadri dermatologici differenti nel bambino rispetto all’adulto, per cui la dermatologia pediatrica sta acquisendo sempre maggiore importanza come branca specifica nell’ambito sia della dermatologia generale che della pediatria. Il problema cruciale che si incontra nel management delle dermatosi pediatriche è legato alle difficoltà diagnostiche incontrate, che comportano spesso la necessità di eseguire una biopsia cutanea. Mentre gli studi epidemiologici relativi alla frequenza delle patologie dermatologiche pediatriche siano ampiamente riportati in letteratura, i dati e le revisioni relative alla chirurgia pediatrica dermatologica, nell’ambito dei servizi di Dermatologia Pediatrica, sono ridotti. Nell’arco dei tre anni di dottorato, la mia attività è stata finalizzata a valutare la possibilità di organizzare un servizio ambulatoriale per i prelievi bioptici in età pediatrica, con il solo ausilio di anestetici topici e locali. Durante i tre anni di Dottorato di Ricerca sono stati eseguiti 296 prelievi. Le biopsie eseguite sono state suddivise in 3 gruppi: biopsie diagnostiche su patologie dermatologiche (108 pz, 36%), biopsie su neoformazioni cutanee (174 pz, 59 %) e biopsie su lesioni follicolari ( 14 pz, 5%). Di ciascun gruppo sono state valutate le patologie riscontrate, l’età, il sesso, l’impiego di anestetico topico associato ad anestetico locale. In 180 (61%) pazienti dopo la biopsia si è proceduto all’applicazione di punti di sutura. Si sono valutati inoltre i vantaggi e gli svantaggi di tale attività ambulatoriale rispetto ai prelievi eseguiti avvalendosi di una sedazione profonda.

Impatto della glomerulopatia cronica da trapianto sulla disfunzione tardiva del rene trapiantato

INTRODUCTION. Late chronic allograft disfunction (CAD) is one of the more concerning issues in the management of patients (pts) with renal transplant (tx). Humoral immune response seems to play an important role in CAD pathogenesis. AIM OF THE STUDY. To identify the causes of late chronic allograft disfunction. METHODS. This study (march 2004-august 2011) enrolled pts who underwent renal biopsy (BR) because of CAD (increase of creatininemia (s-Cr) >30% and/or proteinuria >1g/day at least one year after tx). BR were classified according to 1997/2005 Banff classification. Histological evaluation of C4d (positive if >25%), glomerulitis, tubulitis, intimal arteritis, atrophy/fibrosis and arteriolar-hyalinosis were performed. Ab anti-HLA research at BR was an inclusion criteria. Pts were divided into two groups: with or without transplant glomerulopathy (CTG). RESULTS. Evaluated BR: 93/109. BR indication: impaired s-Cr (52/93), proteinuria (23/93), both (18/93). Time Tx-BR: 7.4±6.3 yrs; s-Cr at BR: 2.7±1.4 mg/dl. CTG group(n=49) not-CTG group(n=44) p Time tx-BR (yrs) 9.3±6.7 5.3±5.2 0.002 Follow-up post-BR (yrs) 2.7±1.8 4.1±1.4 0.0001 s-Cr at BR (mg/dl) 2.9±1.3 2.4±1.5 NS Rate (%) of pts: Proteinuria at BR 61% 25% 0.0004 C4d+ 84% 25% <0.0001 Ab anti-HLA+ 71% 30% 0.0001 C4d+ and/or Ab antiHLA 92% 43% 0.0001 Glomerulitis 76% 16% <0.0001 Tubulitis 6% 32% 0.0014 Intimal arteritis 18% 0% 0.002 Arteriolar hyalinosis 65% 50% NS Atrophy/fibrosis 80% 77% NS Graft survival 45% 86% 0.00005 Histological Diagnosis: CTG group (n=49:Chronic rejection 94%;IgA recurrence + humoral activity 4%;IIA acute rejection + humoral activity 2%. Not-CTG group (n=44: GN recurrence 27%;IF/TA 23%; acute rejection 23%;BKV nephritis 9%; mild not specific alterations 18%. CONCLUSIONS: CTG is the morphological lesion mainly related to CAD. In the 92% of the cases it is associated with markers of immunological activity. It causes graft failure within five years after diagnosis in 55% of pts.

Melanocytes: a new potential tool to study and monitoring Duchenne muscular dystrophy

Dystrophin is a subsarcolemmal protein critical for the integrity of muscle fibers by linking the actin cytoskeleton to the extracellular matrix via the dystroglycan complex. It is reported that dystroglycans are also localized in the skin, at dermal-epidermal junction. Here we show that epidermal melanocytes express dystrophin at the interface with the basement membrane. The full-length muscle isoform mDp427 was clearly detectable in epidermis and in melanocyte cultures as assessed by RNA and western blot analysis. Dystrophin was absent in Duchenne Muscular Dystrophy (DMD) patients melanocytes, and the ultrastructural analysis revealed mitochondrial alterations, similar to those occurring in myoblasts from the same patients. Interestingly, mitochondrial dysfunction of DMD melanocytes reflected the alterations identified in dystrophin-deficient muscle cells. In fact, mitochondria of melanocytes from DMD patients accumulated tetramethylrhodamine methyl ester but, on the contrary of control donor, mitochondria of DMD patients readily depolarized upon the addition of oligomycin, suggesting either that they are maintaining the membrane potential at the expense of glycolytic ATP, or that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Melanocyte cultures can be easily obtained by conventional skin biopsies, less invasive procedure than muscular biopsy, so that they may represent an alternative cellular model to myoblast for studying and monitoring dystrophinopathies also in response to pharmacological treatments.

Efficient ultrasonic signal processing techniques for aided medical diagnostics

Ultrasound imaging is widely used in medical diagnostics as it is the fastest, least invasive, and least expensive imaging modality. However, ultrasound images are intrinsically difficult to be interpreted. In this scenario, Computer Aided Detection (CAD) systems can be used to support physicians during diagnosis providing them a second opinion. This thesis discusses efficient ultrasound processing techniques for computer aided medical diagnostics, focusing on two major topics: (i) Ultrasound Tissue Characterization (UTC), aimed at characterizing and differentiating between healthy and diseased tissue; (ii) Ultrasound Image Segmentation (UIS), aimed at detecting the boundaries of anatomical structures to automatically measure organ dimensions and compute clinically relevant functional indices. Research on UTC produced a CAD tool for Prostate Cancer detection to improve the biopsy protocol. In particular, this thesis contributes with: (i) the development of a robust classification system; (ii) the exploitation of parallel computing on GPU for real-time performance; (iii) the introduction of both an innovative Semi-Supervised Learning algorithm and a novel supervised/semi-supervised learning scheme for CAD system training that improve system performance reducing data collection effort and avoiding collected data wasting. The tool provides physicians a risk map highlighting suspect tissue areas, allowing them to perform a lesion-directed biopsy. Clinical validation demonstrated the system validity as a diagnostic support tool and its effectiveness at reducing the number of biopsy cores requested for an accurate diagnosis. For UIS the research developed a heart disease diagnostic tool based on Real-Time 3D Echocardiography. Thesis contributions to this application are: (i) the development of an automated GPU based level-set segmentation framework for 3D images; (ii) the application of this framework to the myocardium segmentation. Experimental results showed the high efficiency and flexibility of the proposed framework. Its effectiveness as a tool for quantitative analysis of 3D cardiac morphology and function was demonstrated through clinical validation.