Method for predicting hepatitis d relapse after completion of Anti-viral therapy

Abstract

The invention relates to medicine, and more particularly to hepatology, infectious diseases and oncology, and can be used for predicting hepatitis D relapse after completion of anti-viral therapy. In a liver biopsy sample from the patient, hepatitis D virus RNA is detected using a polymerase chain reaction (PCR) method. In the event of a positive PCR result for hepatitis D virus RNA in the liver biopsy sample, irrespective of a negative PCR result for hepatitis D virus RNA in the blood serum, a relapse of hepatitis D is predicted within 4-24 weeks after completion of treatment. The method provides an increase in the accuracy of predicting hepatitis D relapse after completion of anti-viral therapy by early detection of hepatitis D virus RNA in a liver biopsy sample.

Description

[0001] Method for predicting hepatitis D recurrence after completion of antiviral therapy

[0002] FIELD OF INVENTION

[0003] The invention relates to medicine, namely to hepatology, infectious diseases and oncology, and is intended to predict the recurrence of hepatitis D after completion of antiviral therapy.

[0004] LEVEL OF TECHNOLOGY

[0005] Hepatitis D virus (HDV) is a defective satellite virus of the hepatitis B virus (HBV), discovered in 1977, which does not have its own enzyme systems and envelope, using cellular polymerases for replication and HBsAg to build the envelope. The prevalence of chronic HDV infection is on average 4.5% of HBsAg-positive individuals, or at least 12 million infected with HDV worldwide. The addition of HDV to an ongoing HBV infection (HDV superinfection) is highly frequent.

[0006] (up to 90%) leads to the development of chronic hepatitis D (CHD), the most severe and rapidly progressing form of chronic viral hepatitis, characterized by accelerated, within 5-10 years, formation of liver cirrhosis in 70-80% of patients, in 15% within 2 years, and a significantly higher risk of liver failure, hepatocellular cancer and death compared to chronic hepatitis B.

[0007] Evaluation of response to antiviral therapy in patients with

[0008] HDV is based on the detection and quantification of viral RNA in serum. However, the development of reliable methods for analyzing the response to antiviral therapy is difficult due to the lack of correlation between aviremia and the achievement of a sustained virological response, i.e., the persistent absence of hepatitis D virus RNA in serum after completion of treatment. The absence of a valid test system for the quantitative assessment of viremia is also due to the heterogeneity of the virus (at least 8 genotypes), the rapid evolution of the pathogen, and the features of the secondary structure of RNA. Different methods of HDV RNA extraction, the design of test primers and probes, and general deficiencies in standardization contribute to significant variability in the performance characteristics of research and commercial tests.

[0009] Many laboratories use the now more available

[0010] 10-year WHO standard for HDV RNA, which facilitates comparison of RNA levels across different research centers.

[0011] The main goal of treatment of chronic viral hepatitis is persistent suppression of viral replication, ideally maintained after completion of antiviral therapy. In the treatment of chronic hepatitis C, this is called achieving a sustained virological response; achieving a sustained virological response in the treatment of chronic hepatitis B is extremely unlikely and requires continuous antiviral therapy.

[0012] The possibility of achieving a sustained virological response in the treatment of hepatitis D is possible, but the duration of therapy has not yet been determined. A disadvantage of the existing method for assessing the effectiveness of antiviral treatment is the lack of the ability to predict hepatitis recurrence

[0013] D after completion of antiviral therapy despite a negative PCR result for hepatitis D virus RNA in the blood serum. Despite a long-term, from 36 to 96 weeks, negative PCR result for hepatitis D virus RNA in the blood serum against the background of antiviral therapy, Most patients develop a relapse of hepatitis D.

[0014] The probability of hepatitis D recurrence does not correlate with the rate of onset and duration of a negative PCR result for hepatitis D virus RNA in the blood serum during antiviral therapy.

[0015] Thus, in the prior art there is a known method for predicting relapse or stable remission of chronic hepatitis C after interferon therapy (patent of the Republic of Belarus BY 11036 Cl, published on 30.08.2008), which consists in the fact that before and after interferon therapy, the histological activity index is determined in a liver biopsy, and after interferon therapy, the number of aggressive lymphocytes is additionally determined per 100 liver sinusoids, and if the histological activity index decreases to 4 or less and there are no or few aggressive lymphocytes in 100 liver sinusoids, stable remission is predicted, and if the histological activity index decreases to 4 or less and there are 30 or more aggressive lymphocytes in

[0016] 100 liver sinusoids predict relapse. The disadvantages of this method are, firstly, that the method is not intended to predict relapse of hepatitis D, and, secondly, that the prognostic markers of this method are indirect.

[0017] Also known in the prior art is a method for predicting the recurrence of hepatitis D after completion of antiviral therapy.

[0018] (Galimova S. F., Chronic hepatitis D, Evidence-based gastroenterology, 3-4, 2015, pp. 32-42), consisting of assessing the prognostic index of the expected event for patients with chronic HDV infection (baseline-event-anticipation — BEA) and assessing the level of anti-HDV IgM. Factors associated with an unfavorable long-term prognosis were age (over 40 years), male gender, thrombocytopenia, high levels of bilirubin and prothrombin time, and the origin of patients (residents of the Mediterranean).

[0019] The level of anti-HDV IgM can also be used to assess disease activity, prognosis, and when deciding on the timing of antiviral therapy. However, the disadvantages of this method are its complexity, extremely low accuracy, and the indirectness of prognostic criteria.

[0020] Also known from the prior art is a method for predicting the recurrence of hepatitis D after completion of antiviral therapy.

[0021] (Chronic viral hepatitis D (CHVD) In ​​adults,

[0022] Clinical guidelines, 2021, p. 14), including PCR to determine the RNA of the hepatitis D virus in the patient's biomaterial (blood serum) obtained at the end of treatment, which we have chosen as a prototype. Qualitative determination of RNA of the hepatitis D virus (Hepatitis D virus) in the blood by PCR is recommended for patients with XBTD who have completed etiotropic (antiviral) treatment, 24 weeks after completion of treatment, to predict the relapse of hepatitis D. The disadvantage of the method is its low accuracy and lack of correlation with persistent disappearance

[0023] HDV RNA in patient blood after completion of antiviral therapy.

[0024] Thus, there is a need for a method for predicting hepatitis D recurrence after completion of antiviral therapy that is free from the above-mentioned drawbacks.

[0025] The technical result of the proposed method is to increase the accuracy of predicting the recurrence of hepatitis D after completion of antiviral therapy due to early detection.

[0026] Hepatitis D virus RNA in a liver biopsy prepared in a specific way, which in turn will improve the results of hepatitis D treatment.

[0027] DISCLOSURE OF INVENTION

[0028] The following is described below.

[0029] In order to achieve the specified technical result in the method for predicting hepatitis D relapse after completion of antiviral therapy, which includes performing a polymerase chain reaction (PCR) to determine hepatitis D virus RNA in the patient's blood serum obtained upon completion of treatment, it is proposed to also use as a biomaterial the patient's hepatobiopsy obtained as a result of a liver puncture biopsy, to which 0.5 ml of a lysis solution is added and mixed by pipetting 3-4 times, the resulting sample is placed in a thermostat at 65 ° C for 10 minutes, during the thermostatting process the sample is mixed by pipetting 2 times, after which it is centrifuged for 1 minute at 13,000 rpm, the supernatant obtained from the sample and a set of reagents for the qualitative determination of hepatitis D virus RNA in blood serum are used to perform PCR;and if a positive PCR result for hepatitis D virus RNA is obtained in a liver biopsy, despite a negative PCR result for hepatitis D virus RNA in the blood serum, a relapse of hepatitis D is predicted within 4-24 weeks after the end of treatment.

[0030] The advantage of the proposed method for predicting hepatitis D relapse after completion of antiviral therapy is the use of a standard PCR test to detect hepatitis D virus RNA with 100% specificity in liver tissue, which, along with a many times higher concentration of hepatitis D virus RNA in liver tissue, allows for the verification of active replication of the hepatitis D virus even with repeated negative PCR results in blood serum.

[0031] THE BEST OPTION FOR IMPLEMENTATION

[0032] INVENTIONS

[0033] The method is carried out as follows.

[0034] Patients with chronic hepatitis D are prescribed antiviral therapy: daily subcutaneous injections of bulevirtide at a dose of 2 mg / day with or without pegylated interferon alpha-2b at a dose of 1.5 mcg / kg subcutaneously once a week for at least 48 weeks. Treatment continues for 36 to 144 weeks based on the achievement and maintenance of a negative polymerase chain reaction (PCR) result for hepatitis D virus RNA (HDV RNA) in the blood serum. PCR was performed using the RealBest HBV RNA kit (cat. No. D-0998) with a specificity of detecting hepatitis D virus RNA of 100% and a sensitivity of 100 copies / ml.

[0035] In order to assess the dynamics of histological activity and liver fibrosis of hepatitis D against the background of antiviral therapy, as well as to study HDV RNA in liver tissue, patients undergo a Menghini liver biopsy. The resulting liver tissue biopsies are separated for histological and virological examination. The hepatobiopsy for morphological examination is fixed in 10% neutral formalin buffered according to Lilly. Liver tissue measuring 3-

[0036] 5 mm for virological testing are placed in a saline solution and delivered to the PCR laboratory.

[0037] Delivery of hepatobiopsy at a temperature of 18-25 ° C is carried out within 2 hours, at a temperature of 2-8 ° C no more than 24 hours, at a temperature of minus 18-60 ° C up to 2 weeks.

[0038] DNA / RNA extraction from biopsy is performed using a reagent kit for the qualitative determination of hepatitis virus RNA

[0039] D in blood serum (we used RealBest extraction

[0040] 100", manufactured by JSC Vector-Best). The biomaterial was placed in an Eppendorf with 0.5 ml of lysing solution from the reagent kit.

[0041] "RealBest extraction 100".

[0042] If the hepatobiopsy was previously frozen, it is defrosted. The hepatobiopsy is mixed in the lysing solution 3-4 times by careful pipetting. The resulting sample is placed in a thermostat at 65 ° C for 10 minutes. During thermostatting, the sample is mixed by pipetting 2 times, then centrifuged for 1 minute at 13,000 rpm. To perform PCR, 100 μl of the supernatant obtained from the sample and a reagent kit for the qualitative determination of hepatitis D virus RNA in blood serum are used. Nucleic acids are isolated according to the instructions for the reagent kit.

[0043] Amplification is carried out using a reagent kit for detecting hepatitis D virus RNA by the RT-PCR method in real time (we used RealBest RNA VGO manufactured by Vector-Best JSC) and a reagent kit for detecting and quantifying hepatitis virus DNA by the PCR method in real time (we used

[0044] "RealBest VGV PCR" (option 2), manufactured by JSC "Vector-

[0045] Best» ). In each test tube with lyophilized ready-made reaction mixture for PCR, 50 µl of the corresponding solution of isolated RNA (from test tubes installed in a magnetic stand) are added using a pipette with a separate tip with a filter. Attention is paid to the inadmissibility of capturing sorbent particles. The test tubes are closed with lids or optical film and placed in the amplifier.

[0046] The analytical sensitivity (detection limit) of the RealBest HDV RNA kit is guaranteed (in 100% of samples) to be 100 copies / ml of HDV RNA when isolating RNA from 100 μl of sample; the specificity of detection is 100%. The RT-PCR reaction protocol: Stage 1: 45 ° C for 30 min; Stage 2: 94 ° C for 1 min; Stage 3: 50 cycles (94 ° C for 10 sec, 60 ° C for 20 sec). Fluorescence is measured at

[0047] 60 °C. Analysis and recording of results is carried out automatically using the RealBest Diagnostics software.

[0048] We used the amplifier "CFX96", manufactured by

[0049] BIO-RAD, USA.

[0050] If a positive PCR result for hepatitis D virus RNA is obtained in a liver biopsy, despite the presence of a negative PCR result for hepatitis D virus RNA in the blood serum, a relapse of hepatitis is predicted.

[0051] D within 4-24 weeks after the end of treatment.

[0052] What is new is that PCR for hepatitis D virus RNA in patients with hepatitis D receiving antiviral therapy is proposed to be performed in an optimally prepared sample from a liver biopsy, rather than in blood serum. Given the many times higher concentration of HDV RNA in hepatocytes than in blood serum, a positive PCR result for hepatitis D virus RNA in a liver biopsy against the background of antiviral therapy allows one to reliably predict an early relapse of HDV infection with a negative PCR result for hepatitis D virus RNA in blood serum. The result consists in increasing the effectiveness of antiviral treatment in patients with hepatitis D by preventing unjustified termination of antiviral therapy despite a negative PCR result for hepatitis D virus RNA in blood serum.

[0053] INDUSTRIAL APPLICABILITY

[0054] The high accuracy and value of the proposed method are confirmed by the clinical examples below.

[0055] Example 1. Patient K., female, 48 years old. Has been observed by a hepatologist since 2015 for compensated liver cirrhosis resulting from hepatitis D (HBV DNA "-", HDV RNA "+"). History includes 48 weeks of antiviral therapy with PEG-interferon without achieving a virological and biochemical response.

[0056] At the initial assessment of the data (before antiviral therapy), mild leukopenia was detected 3.46 thousand, ACN

[0057] 1, 69, mild thrombocytopenia - 129 thousand, moderate increase in ALT 180 U / l with normal liver function tests: total bilirubin - 19.4 μmol / l, albumin 39.2 g / l, MNO 1.22, PTI 73%, prothrombin time 15.3 sec., cholinesterase 9.89 μmol / l. According to the results of virological tests, HBV DNA is "negative", HDV RNA was detected, the amount of 10 * 8 cop / ml. Ultrasound of the abdominal organs revealed signs of minor diffuse changes in the liver and pancreas, splenomegaly, the portal and splenic veins are not dilated. The liver density according to liver elastography data was 12.3 kPa (F4 according to

[0058] METAVIR) .

[0059] Based on the examination results, liver cirrhosis was diagnosed as a result of hepatitis D without signs of liver dysfunction.

[0060] (clinical - laboratory compensation, absence of ascites, signs of portal hypertension, slight splenomegaly according to ultrasound data). Diagnosis: Liver cirrhosis as a result of hepatitis D (HDV RNA "+" amount 10* 8 cop / ml), compensated. Child A. Sequestration neutro-, mild thrombocytopenia.

[0061] Since 05 / 27 / 2020, the patient has been prescribed combination antiviral therapy: Pegylated interferon alpha-2a

[0062] (hereinafter Peg-IFN) 180 mcg per week, subcutaneously + Bulevirtide 2 mg per day, subcutaneously for 48 weeks, then monotherapy

[0063] Bulevirtide 2 mg daily subcutaneously for another 96 weeks.

[0064] During antiviral therapy, the patient's virological (HDV RNA, HBV DNA), biochemical (blood test) and histological (including non-invasive diagnostics of liver fibrosis) responses were assessed, as well as the safety of the treatment.

[0065] Virological response:

[0066] The amount of HDV RNA was estimated by PCR after 12, 24, 48,

[0067] 72, 96, 144 weeks of antiviral therapy. Aviremia was achieved 12 weeks after the start and a negative HDV RNA test result was maintained until completion of therapy. Thus, a complete virological response to antiviral therapy was achieved. HBV DNA was not detected during the entire period of antiviral therapy.

[0068] Biochemical response: Beginning with the 4th week of antiviral therapy, a gradual decrease in transaminase levels was noted. Complete biochemical response was achieved after 12 weeks of therapy. Subsequently, ALT and AST levels remained within the upper limit of normal (ULN) throughout the entire treatment period.

[0069] During treatment, the level of bile acids in the blood serum was monitored, which remained elevated throughout the therapy. The maximum content of bile acids was recorded after 96 weeks of treatment and amounted to 112 μmol / l, which may be, in part, an indicator of the effectiveness of the therapy.

[0070] Histological answer:

[0071] The severity of liver fibrosis was assessed using a non-invasive diagnostic method of transient liver elastography on the Fibroscan device. Initially, the fibrosis level F4 (12.3 kPa) was recorded on a scale

[0072] METAVIR. After 48 weeks of therapy, a repeat study showed F3 (10.1 kPa) on the METAVIR scale. The decrease in liver density according to transient elastography continued during antiviral therapy and at the end of treatment after 144 weeks was 9.2 kPa (METAVIR).

[0073] In order to assess the achievement of a virological response in the liver tissue of a patient with long-term aviremia in the blood serum upon completion of treatment, i.e. 144 weeks after the start of antiviral therapy, a liver biopsy was performed with PCR for HDV RNA in a hepatobiopsy sample using the proposed method. 0.5 ml of lysis solution was added to the hepatobiopsy sample and mixed by pipetting 3 times. The resulting sample was placed in a thermostat at 65 ° C for 10 minutes, during thermostatting the sample was mixed by pipetting 2 times. The sample was centrifuged for 1 minute at 13,000 rpm. The supernatant obtained from the sample and a reagent kit for the qualitative determination of hepatitis B virus RNA were used to perform PCR.

[0074] D in blood serum. PCR result in hepatobiopsy is positive.

[0075] After completion of antiviral therapy, the patient was recommended to be monitored with monthly control of general, biochemical and virological blood tests. Two weeks after completion of antiviral therapy, with a negative PCR result for HDV RNA in the patient's blood serum, an increase in ALT to 1.4 vgn was recorded. Four weeks after completion of antiviral therapy, a positive test for HDV RNA was obtained: a relapse of hepatitis was verified.

[0076] D .

[0077] Safety of the therapy.

[0078] During the 144-week observation period of the patient during antiviral therapy, the patient developed progressive leukopenia and, as a result, neutropenia, as well as thrombocytopenia. These changes were assessed as adverse events of interferon therapy, but did not require modification of the dose of Peg-IFN. Adverse events associated with the use of Bulevirtide were not registered during the entire period of treatment of the patient.

[0079] After 2 weeks of treatment, the tests showed a decrease in the leukocyte level to 2.38 x 10*9 / l, while the absolute neutrophil count was 0.95 x 10*9 / l. IFN-induced leukopenia and neutropenia were registered. The platelet level decreased to a minimum value of 94 x 10*9 / l with subsequent stabilization and throughout the entire period of interferon therapy. The platelet level did not decrease further. Monitoring of the patient was continued without changing the doses of the antiviral drugs received.

[0080] Diagnosis after 144 weeks of antiviral therapy and 12 weeks of observation after its completion: Main diagnosis: Liver cirrhosis as a result of hepatitis D (HDV RNA

[0081] "+", quantity 10*8 cop / ml; HBV DNA "-"), compensated.

[0082] Child A. PEG-IFN + Bulevirtide (05.2020-04.2021) with achievement of virological response (08.2020 negative PCR result), achievement of biochemical response. IFN-induced thrombocytopenia of mild st.

[0083] Bulevirtide 2 mg / day (04.2021-02.2023). Relapse (07.2023)

[0084] HDV RNA «+»).

[0085] Liver elastography (07.2023): 9.2 kPa - F3 (METAVIR); 221 dB / cm - S0.

[0086] Conclusion: positive PCR test for HDV RNA in liver tissue despite the achievement and long-term (up to 136 weeks) maintenance of aviremia (HDV) against the background of antiviral therapy

[0087] RNA "neg") was a reliable early predictor of relapse

[0088] HDV infections after completion of treatment.

[0089] Example 2. Patient V., male, 51 years old. Has been observed since 2019 for chronic hepatitis D (HBV DNA “-”, HDV RNA

[0090] «+») .

[0091] During examination before antiviral therapy, a moderate increase in ALT of 120 U / L was detected with normal liver function tests: total bilirubin - 17.6 μmol / L, albumin - 40.2 g / L, INR - 0.9, cholinesterase - 8.9 μmol / L.

[0092] According to the results of virological tests, HBV DNA was negative, HDV RNA was detected, the amount was 10*6 cop / ml. Ultrasound of the abdominal organs revealed signs of minor diffuse changes in the liver and pancreas, the portal and splenic veins were not dilated. The density of the liver according to liver elastography was 4.3 kPa (F0 according to METAVIR).

[0093] Based on the examination results, a diagnosis was made: chronic hepatitis D (HDV RNA "+" amount 10*6 cop / ml).

[0094] Since May 2020, the patient has been prescribed combination antiviral therapy: Pegylated interferon alpha-2a

[0095] (hereinafter Peg-IFN) 180 mcg per week, subcutaneously 4- Bulevirtide 2 mg per day, subcutaneously for 48 weeks, then monotherapy

[0096] Bulevirtide 2 mg daily subcutaneously for another 96 weeks.

[0097] During antiviral therapy, the patient's virological (HDV RNA, HBV DNA), biochemical (blood test) and histological (including non-invasive diagnostics of liver fibrosis) responses were assessed, as well as the safety of the treatment.

[0098] Virological response:

[0099] The presence of HDV RNA in the blood serum by PCR was assessed after 12, 24, 48, 72, 96, 144 weeks of antiviral therapy. Aviremia was achieved after 12 weeks from the beginning and the negative result of the HDV RNA test was maintained until the end of therapy. Thus, a complete virological response to antiviral therapy was achieved. HBV DNA was not detected during the entire period of antiviral therapy.

[0100] Biochemical response

[0101] Beginning with the 4th week of antiviral therapy, a gradual decrease in transaminase levels was noted. A complete biochemical response (ALT normalization) was achieved after 8 weeks of therapy. Subsequently, ALT and AST levels remained within the upper limit of normal (ULN) throughout the entire treatment period.

[0102] During treatment, the level of bile acids in the blood serum was monitored, which remained elevated throughout the therapy. The maximum content of bile acids was recorded after 48 weeks of treatment and was 96 μmol / l, which may be an indicator of the effectiveness of the therapy.

[0103] Histological response

[0104] The severity of liver fibrosis was assessed using a non-invasive diagnostic method of transient liver elastography on the Fibroscan device. Initially, the fibrosis level F0 (4.3 kPa) was recorded on a scale

[0105] METAVIR, which was maintained throughout the entire period of antiviral therapy.

[0106] In order to assess the achievement of virological response in the liver tissue of a patient with prolonged aviremia in the blood serum upon completion of treatment, i.e. 144 weeks after the start of antiviral therapy, a liver biopsy with PCR for HDV RNA in a hepatobiopsy was performed. 0.5 ml of lysis solution was added to the hepatobiopsy and mixed by pipetting 4 times. The resulting sample was placed in a thermostat at 65 °C for 10 minutes, during the thermostatting the sample was mixed by pipetting 2 times. The sample was centrifuged for 1 minute at 13,000 rpm. The supernatant obtained from the sample and a reagent kit for the qualitative determination of hepatitis D virus RNA in blood serum were used to perform PCR. The PCR result in the hepatobiopsy was positive.

[0107] After completion of antiviral therapy, the patient was recommended to be monitored with monthly control of general, biochemical and virological blood tests. Sixteen weeks after completion of antiviral therapy, with a negative PCR result for HDV RNA in the blood, the patient's ALT increased to 1.1VGN. Twenty-four weeks after completion of antiviral therapy, a positive test for HDV RNA was obtained: relapse of hepatitis D was verified.

[0108] Safety of the therapy.

[0109] During the 144-week patient observation period during antiviral therapy, no significant adverse events were recorded.

[0110] Diagnosis after 144 weeks of antiviral therapy and 24 weeks of observation:

[0111] Main diagnosis: Chronic hepatitis D (HDV RNA "+" amount 10* 6 cop / ml; HBV DNA "-"). PEG-IFN + Bulevirtide

[0112] (05. 2020-04. 2021) with achievement of a virological response (08. 2020 negative PCR result), achievement of a biochemical response. Bulevirtide 2 mg / day (04. 2021-

[0113] 02. 2023). Relapse (08.2023 HDV RNA “+”).

[0114] Liver elastography (08.2023): 4.2 kPa - F0 (METAVIR); 221 dB / cm - SO.

[0115] Conclusion: A positive PCR test for HDV RNA in liver tissue, despite the achievement and long-term maintenance of aviremia (HDV RNA “neg”) against the background of antiviral therapy, was a predictor of late (after 24 weeks) relapse of HDV infection after completion of treatment.

[0116] In total, we studied 24 patients with chronic hepatitis D who received antiviral therapy.

[0117] Pegylated interferon alpha-2b at a dose of 1.5 mcg / kg subcutaneously once a week for 48 weeks and daily subcutaneous injections of bulevirtide at a dose of 2 mg / day for

[0118] 96 144 weeks. All patients had a negative PCR result for hepatitis D virus RNA in the blood serum 12-24 weeks after the start of antiviral therapy, i.e. a complete virological response was achieved (negative PCR result for hepatitis D virus RNA in the blood serum), which lasted until the end of antiviral treatment (from 84 to 132 weeks). At the end of antiviral therapy, all patients underwent liver biopsy with histological and virological examination of the liver tissue. In 16 patients, PCR revealed hepatitis D virus RNA in the liver biopsy. Within 4-24 weeks after the end of antiviral therapy, all of them had a relapse of hepatitis D: the appearance of hepatitis D virus RNA in the blood serum.

[0119] Thus, the proposed method allows to increase the accuracy of predicting the recurrence of hepatitis D after the completion of antiviral therapy due to early detection.

[0120] Hepatitis D virus RNA in a liver biopsy prepared in a certain way, which in turn will improve the results of hepatitis D treatment.

Claims

Invention formula A method for predicting the recurrence of hepatitis D after completion of antiviral therapy, including conducting a polymerase chain reaction (PCR) to determine the RNA of the hepatitis D virus in the patient's biomaterial obtained at the end of treatment, characterized in that the biomaterial used is the patient's hepatobiopsy obtained as a result of a puncture biopsy of the liver, to which 0.5 ml of a lysing solution is added and mixed by pipetting 3-4 times, the resulting sample is placed in a thermostat at 65 ° C for 10 minutes, during the thermostatting process the sample is mixed by pipetting 2 times, after which it is centrifuged for 1 minute at 13000 rpm, for PCR, the supernatant obtained from the sample and a set of reagents for the qualitative determination of hepatitis virus RNA are used D in the blood serum; and if a positive PCR result is obtained for hepatitis D virus RNA in a liver biopsy, in the presence of a negative PCR result for hepatitis D virus RNA in the blood serum, the onset of a relapse of hepatitis D is predicted within 4-24 weeks after the end of treatment.