Clinical and prognostic significance of hepatitis b rna in chronic viral hepatitis B

Clinical and prognostic significance of hepatitis B RNA in chronic viral hepatitis B

Authors
Rakhmanova. A.M., Kan.N.G

The Scientific-Research Institute of Virology of the Republic specialized scientific practical medical center of epidemiological, microbiological, infections and parasitic diseases, Tashkent, Uzbekistan.


Background: Chronic hepatitis B viral infection is a global health problem, especially in the Central Asian region. Approximately 3.8 % population of the world are chronically infected with hepatitis B virus (HBV) and about 650,000 people die annually from HBV infection related liver failure, liver cirrhosis and liver cancer. Although the first line antiviral agents such as nucleos(t) ide analogues (NAs) can efficiently inhibit HBV replication and control disease progression in almost all patients, these drugs rarely lead to elimination of chronic HBV infection completely due to their little effect on covalently closed circular DNA(cccDNA),which is believed to be the main cause of viral persistence . HBV RNA is derived only from intrahepatic cccDNA, and its quantification is not affected by viral antigens or antibody complexes. Hence, the aim of this cross-sectional study was to evaluate the levels of HBV RNA in the consecutive phases of HBV infection in patients not receiving antiviral therapy and to evaluate the significance of HBV RNA quantification in monitoring the natural history of chronic HBV infection.


Material and methods: Between October 2022 and September 2018, employees of the Reference Laboratory of the Research Institute of Virology conducted a clinical and laboratory test of the ROSSAmed HBV-RNA reagent kit for detecting hepatitis B virus (HBV) RNA using the RT-PCR method with real-time detection of results, produced by ROSSA Limited Liability Company, Uzbekistan. The ROSSAmed HBV-RNA reagent kit is designed for the qualitative detection of hepatitis B virus RNA by RT-PCR with real-time detection of results.Group 1. The material for PCR is RNA samples isolated from blood plasma. polymerase chain reaction, with real-time detection of results. HBV RNA was examined in 55 patients among them. Group 2. 13 samples of plasma of patients with clinical manifestations of hepatitis, unclear etiology. In the blood of patients there are no markers of viral hepatitis B, C and D. 3 group. 55 samples positive for PCR contain DNA of the hepatitis B virus. According to ELISA, among 55 samples:- 3 samples contain HBsAg;


- 1 sample contains antibodies of viral hepatitis D;
- 16 samples of antibodies to viral hepatitis D are missing;
- 4 samples contain RNA and DNA of viral hepatitis B and C.


Results: Comparative clinical trials were carried out on blood plasma samples from patients of the Research Institute of Virology, with clinical manifestations of viral hepatitis, with markers of viral hepatitis B, C and D according to the results of testing by enzyme immunoassay and PCR. In view of the absence of similar kits on the market of Uzbekistan, a set of reagents was used for testing: "Ample Sense HVV-FL", FGBU "TsSP" FMBA, Russia. REF: R-V5-MOD (RG, iQ, Mx, Dt), LOT: 06/01/22, expiration date 2023-06-01, which was used to determine the presence or absence of HBV DNA in the samples. Extraction of RNA from blood plasma was carried out with a kit for RNA/DNA isolation "Ribo-sorb", Federal State Budgetary Institution "TsSP" FMBA, Russia. REF: K2-1-Et-100, LOT. Data on the content or absence of markers of viral hepatitis by ELISA or PCR are presented in the table along with the data of the test kit (Table 1). In group No. 2: samples of patients with clinical manifestations of hepatitis, unclear etiology. Patients do not have markers of viral hepatitis. As a result of testing, one sample containing HBV RNA was identified;( Table 2) When testing 55 samples containing viral hepatitis B DNA, the test kit detected viral hepatitis B RNA in 55 samples.(Table 3)


Conclusion: Samples with confirmed occult hepatitis cccDNA, samples of the dissertation work were collected in 2018. As a result of testing, one positive sample was found. We believe that the result obtained can be directly related to the instability of RNA to long-term storage.
According to the literature, chronic hepatitis B is one of the leading etiological causes of liver failure, cirrhosis, and hepatocellular carcinoma (HCC) worldwide. This condition cannot be completely cured by currently available drugs due to the persistent presence of covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV), which is a reliable template for the transcription of HBV RNA, in infected hepatocytes. Quantification of cccDNA requires an invasive procedure, so serum biomarkers that reflect the intrahepatic activity of cccDNA are needed. Recently, a growing body of research suggests that circulating HBV RNA can serve as a serum biomarker of HBV infection, treatment, and prognosis. Table 1. Sample test results with cccDNA