Vpeljava ultracentrifugiranja v postopek genotipizacije virusa hepatitisa B

Vidic, Blaž (author), Jeras, Matjaž (mentor), Klemenc, Polona (co-mentor)

Okužbe z virusom hepatitisa B (HBV) so kljub številnim preventivnim ukrepom še vedno velik svetovni zdravstveni problem. Danes poznamo 10 genotipov HBV in več študij je pokazalo povezanost med genotipom virusa hepatitisa B ter potekom in uspešnostjo zdravljenja okužb. Virus hepatitisa B se lahko prenese tudi s transfuzijo okužene krvi. Z uvedbo presejalnega testiranja vseh krvodajalcev z metodo pomnoževanja nukleinskih kislin (NAT) na prisotnost DNA virusa hepatitisa B, se je možnost za njegov prenos s transfuzijo znatno zmanjšala. V transfuzijski medicini je zelo pomembna diagnostika okultne okužbe s HBV (OBI), ki še vedno predstavlja izziv z vidika laboratorijske diagnostike. V okviru magistrskega dela smo v postopek genotipizacije HBV vpeljali ultracentrifugiranje. Ultracentrifugirali smo plazemske vzorce 24 slovenskih krvodajalcev, pri katerih je bila diagnosticirana okultna okužba s HBV, nastale usedline resuspendirali, iz njih izolirali DNA HBV in določili virusne genotipe. Genotipe HBV smo uspešno določili v vseh 24 (100 %) plazemskih vzorcih preiskovanih krvodajalcev, ki smo jih ultracentrifugirali pred izolacijo virusne DNA, ter v le 5 od omenjenih 24 (21 %), ki jih nismo ultracentrifugirali. Genotipe HBV smo določili v 21 od 24 vzorcev (88 %), pri katerih smo usedlino po ultracentrifugiranju resuspendirali v vodi in v 15 od 24 (63 %), pri katerih smo jo resuspendirali v lizirajočem pufru. Ugotovili smo, da pri slovenskih krvodajalcih z OBI prevladuje genotip D, ki smo ga določili pri 21 od 24 preiskovancev (88 %), 2 (8 %) sta imela genotip A, pri 1 (4 %) pa smo odkrili mešano okužbo z genotipoma A in D. Z vpeljavo ultracentrifugiranja smo izboljšali občutljivost metode verižne reakcije s polimerazo DNA v realnem času (qPCR) za genotipizacijo HBV. S tem smo še korak bližje k varnejši uporabi krvi ter krvnih komponent in pripravkov iz krvi. Izziv za prihodnost pa še vedno ostaja laboratorijska diagnostika HBV v vzorcih z ekstremno nizkim virusnim bremenom (0,15 IU/ml).

• Language: Slovenian
• Keywords: virus hepatitisa B, genotipi virusa hepatitisa B, okultna okužba z virusom hepatitisa B, ultracentrifugiranje, genotipizacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-106825
• Publication date in RUL: 18.03.2019

Secondary language

• Language: English
• Title: Implementation of ultracentrifugation into genotyping of hepatitis B virus

Abstract

Hepatitis B virus (HBV) infection remains a major global health problem. Ten HBV genotypes have been identified and several studies have shown correlations between the actual HBV genotype and the course and effectiveness of infection treatment. HBV can be transmitted through transfusion of infected blood. With implementation of nucleic acid amplification technology testing (NAT) of all blood donors for the presence of HBV DNA, the possibility of viral transmission by blood components and products has significantly decreased. However, diagnostics of occult HBV infection (OBI) is still an important challenge in transfusion medicine. In our study, ultracentrifugation was implemented in the HBV genotyping procedure. We performed ultracentrifugation on 24 plasma samples of 24 Slovenian blood donors which were diagnosed with OBI. Following ultracentrifugation, the resulting pellets were resuspended, HBV DNA was isolated and viral genotypes were successfully determined in all 24 samples. As stated, HBV genotype was identified in all 24 (100 %) blood donors whose plasma samples were ultracentrifuged prior to DNA isolation and in only 5 (21 %) out of the same 24 plasma samples which were not ultracentrifuged before DNA isolation. HBV genotypes were identified in 21 out of 24 samples (88 %), where, after ultracentrifugation, the resulting pellets were resuspended in water, and in 15 out of 24 (63 %) where they were resuspended in lysis buffer. Our results revealed that the D genotype is predominant in Slovenian blood donors with OBI, as this genotype was identified in 21 out of 24 blood donors (88 %). The A genotype was detected in 2 blood donors (8 %), while in the remaining 1 (4 %), a mixed infection with the A and D genotypes was identified. With the implementation of ultracentrifugation we improved the sensitivity of real-time polymerase chain reaction (qPCR) used for HBV genotyping. This is a step closer to even safer use of blood and blood components. However, laboratory diagnostics of HBV in samples with extremely low viral load (0,15 IU/ml) still remains a challenge for future.

• Keywords: hepatitis B virus, hepatitis B virus genotype, occult hepatitis B virus infection, ultracentrifugation, genotyping