Mutacije virusa hepatitisa C u hipervarijabilnoj regiji 1 i razvoj kvazispecijesa u bolesnika s kroničnim hepatitisom C [Determination of hepatitis C virus quasispecies in hypervariabile region 1 in patients with chronic hepatitis C]

Hrstić, Irena (2008) Mutacije virusa hepatitisa C u hipervarijabilnoj regiji 1 i razvoj kvazispecijesa u bolesnika s kroničnim hepatitisom C [Determination of hepatitis C virus quasispecies in hypervariabile region 1 in patients with chronic hepatitis C]. PhD thesis, Sveučilište u Zagrebu.

Preview

PDF Download (2MB) | Preview

Abstract

INTRODUCTION: Clinical implication of HCV quasispecies is not well-known. Available methods for their detection are time consuming and expensive. ----- AIMS & METHODS: Aim of this study was detection of HCV quasispecies. Serums from 109 subjects were first genotyped by standard INNO-LIPA HCV II method. After that, same samples were analyzed by high-resolution DNA melting analysis as follows: ribonucleic acid was isolated with commercial kits and afterwards transcript by polymerase chain reaction. Amplification of hypervariable region 1 (HVR1) was done in two phases. Obtained mix for each sample was cycled in Light-cycler and fluorescence was detected. Collected Tm's for each sample was showed as melting curve with 4 variables: Tm, high (H), wide (W) and area (A). Each variable was statisticaly correlate with non achieved treatment response. ETVR means undetectable HCV RNA at the end of treatment and SVR means maintenance of undetectability of HCV RNA at 6 months after the completion of treatment. Non achieved treatment response means still detectable HCV RNA at those treatment points. Second sample was collected in 40 untreated pts, and in 24 treated pts (45/69 achieved ETVR). ----- RESULTS: total of 109 subjects, 18 to 71 years old, 64 men, more with 1 (59.6%), majority with medium fibrosis according to Ishak score (53.8%). Obtained results were: mean Tm 86.8±2.10; mean H 1.64±0.74; mean W 3.38±0.14 and median A 4.42. Correlation of no achieved ETVR and SVR was positive with Tm, H and A and negative with W. Statistical significance was found for A as positive predictor of SVR: OR=1.44, 95%CI (OR) =1.03-2.03, p 0.033. Statistical significance was not found between pared samples in any of parameters but difference in mean has been noticed: Tm2, W2 and H2 increased but A2 decreased in untreated group. In treated group: Tm2 increased while H2 and A2 decreased. Statistical significance was noticed for W2 (Wilcoxon Signed Ranks -1,871; p = 0,061). ----- CONCLUSION: Determination of HCV quasispecies in single sample can predict the treatment response. Changes of quasispecies in untreated and treated group of patients have been observed.

Abstract in Croatian

UVOD: Klinička značajnost HCV kvazispecijesa nije razjašnjena. Dostupne metode određivanja kvazispecijesa sekvencioniranjem tehnički su zahtjevne i skupe. ----- CILJ I METODE: osnovni cilj bio je detekcija HCV kvazispecijesa. Uzorci 109 ispitanika primarno su genotipizirani standardnom INNO-LIPA HCV II metodom. Nakon toga isti uzorci su analizirani metodom taljenja DNK: RNK je izolirana komercijalnim kitovima a potom prepisana lančanom reakcijom polimeraze. Amplifikacija hipervarijabilne regije 1 rađena je u 2 faze a dobivena mješavina analizirana je u Light-cycleru. U njemu je visokom rezolucijom detektirana fluorescencija uzorka. Rezultat analize predstavlja krivulja taljenja označena s 4 parametra: temperatura taljenja 50% uzorka DNK (Tm), širina (W), visina (H) i površina (A). Svaki parametar statistički je obrađen na sposobnost predikcije terapijskog učinka. Dinamičke promjene analizirane su u parnom uzorku koji je dobiven u 40 neliječenih ispitanika te u 24 liječena ispitanika (45/69 imalo je ETVR). ----- REZULTATI: uključeno je 109 ispitanika, dobi 18-71 godinu, više muškaraca (64), najveći broj genotipa 1 (59,6%), većina srednjeg stadija po Ishaku (fibroza 3 i 4 u 53,8%). Parametri kvazispecijesa jesu: srednja Tm1 86.8±2.10; srednja H1 1.64±0.74; srednja W1 3.38±0.14 a medijan A1 iznosi 4.42. Nije nađena statistički značajna korelacija parametara s genotipom, viremijom ili fibrozom. Tm1, H1 i A1 u pozitivnom su odnosu s ETVR i SVR dok je odnos s W1 obrnuto proporcionalan. Statistička značajnost za SVR utvrđena je za A1 (OR=1.44, 95%CI=1.03-2.03, p 0.033). U odnosu bazalnog i parnog uzorka postoji pomak raspona u svim parametrima: u neliječenoj grupi porasla je Tm2, W2 i H2, dok se A2 smanjila. U liječenoj grupi Tm2 je porasla, dok su se H2 i A2 smanjile. Statistička značajnost dobivena je za W2 (Wilcoxon Signed Ranks -1,871; p = 0,061). ----- ZAKLJUČAK: prediktivnu vrijednost za terapijski odgovor ima površina kvazispecijesa. Tijekom vremena zamijećene su promjene parametara kvazispecijesa u neliječenoj i liječenoj grupi.

Actions (login required)

View Item

Downloads