Hepatitis B Virus DNA Quantification Research Articles

CRISPR/Cas12b assisted loop-mediated isothermal amplification for easy, rapid and sensitive quantification of chronic HBV DNA in one-pot

BackgroundCurrently, millions of people suffer from undiagnosed chronic hepatitis B (CHB) infection each year, which leads to high mortality rates attributed to cirrhosis and hepatocellular carcinoma. Previously reported assays, such as PCR-based assays, have limitations in terms of convenient for CHB screening in high-burden regions and resource-limited settings. Recently, diagnosis based on CRISPR/Cas, which has been considered as a potential method of point-of-care test (POCT) in resource-limited settings, offers a significant advantage in terms of high sensitivity and specificity. Therefore, there is an urgent need for the hepatitis B virus (HBV) detection utilizing CRISPR/Cas system. ResultsWe have proposed a one-pot of one-step method for CRISPR/Cas12b assisted loop-mediated isothermal amplification (LAMP) to facilitate the quick, sensitive, and precise quantification of HBV DNA. This method is designed for point-of-care testing following genomic extraction or sample heat treatment. We have optimized several critical factors, such as the reaction buffer, AapCas12b-gRNA concentration, reporter and its concentration, reaction temperature, and chemical additives, to significantly enhance the performance of the one-pot assay for HBV. Importantly, it exhibited no cross-reactivity between HBV and blood-borne pathogens. Moreover, the assay is capable of quantifying HBV DNA within 1 h with a limit of detection (LOD) of 25 copies per milliliter. Additionally, when tested on 236 clinical samples, the assay demonstrated a sensitivity of 99.00 % (198/200) and a specificity of 100.00 % (36/36) at the 99 % confidence level compared to real-time quantitative PCR. SignificanceThe utilization of convenient and reliable point-of-care diagnostic methods is crucial for reducing the burden of CHB globally. The assay we developed was helpful to improve the ability of HBV diagnosis for practical clinical translation, especially in high-burden regions and resource-limited settings. It has great advantages for rapid screening of CHB as well as evaluation of therapeutic efficacy as a companion diagnostic method.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patient on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA.

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

HBeAg testing is better than quantitative HBsAg assay as an alternative to HBV DNA assay among HBV-infected pregnant women.

Using tenofovir disoproxil fumarate (TDF) is recommended in the 3rd trimester for pregnant women with HBV DNA ≥ 200,000 IU/mL to prevent mother-to-child transmission (MTCT) of hepatitis B virus (HBV). However, HBV DNA quantification is unavailable in many resource-limited areas worldwide, hence prophylaxis is often missed. The aim of this study was to determine whether HBeAg or qHBsAg is a better alternative to HBV DNA testing in HBV-infected pregnant women. In this prospective cohort study, pregnant women with HBV infection were recruited in 3 hospitals from October 2019 to November 2020. Socio-demographic and clinical data were collected. Blood samples were taken for qHBsAg and HBV DNA testing. HBeAg results were collected from the medical records of the participants who visited a doctor during the study. 465 pregnant women met the study criteria. 41.9% were HBeAg positive, 33.3% had high qHBsAg levels (> 104 IU/mL), 38.3% had high HBV DNA levels (≥ 200,000 IU/mL). Pregnant women with high qHBsAg levels were 27 times more likely to have high HBV DNA levels (aOR = 27.0, 95% CI: 11.1-65.5, p < 0.001). Participants who were HBeAg positive were 57.5 times more likely to have high HBV DNA levels (aOR = 57.5, 95% CI: 23.0-140.0, p < 0.001). The sensitivity of qHBsAg and HBeAg was 80% and 94%, respectively; and specificity was 95% and 90%, respectively. HBeAg testing should be considered over qHBsAg assay as an alternative to HBV DNA assay because of its technical simplicity, lower cost, and fewer missed treatments.

Development of a Reagent Kit for the Quantitative Determination of Hepatitis B Virus (HBV) DNA in Clinical Material by PCR with Hybridization-Fluorescence Detection

Relevance.Hepatitis B virus (HBV) is one of the most common viral infections affecting people worldwide and can lead to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Currently 3% of the world's population are infected with hepatitis B virus and are at risk of developing life-threatening liver disease. Immunological and molecular biological methods of detection of HBV are currently used in laboratory diagnostics. The polymerase chain reaction (PCR) is currently the most sensitive method for the detection and quantification of HBV. HBV DNA quantification is widely used to monitor the antiviral treatment of HBV infection.Aim.To develop a real-time PCR kit for the quantification of HBV DNA.Materials and methods.A total of 200 plasma and serum samples positive and negative for HBV were used in the development. The performance of the developed kit was compared with the use of other commercially registered HBV diagnostic kits in Russia. Additionally, the nucleotide sequences of all existing virus genotypes analysed for the selection of primers using GeneBank system.Results and discussion.Comparison analysis of the results of quantitative determination by real-time PCR in 200 clinical serum and blood plasma samples showed that the diagnostic sensitivity of the developed kit was 100% and specificity 100%. The primers developed specific to the POL gene region. The kit is capable of detecting all types of virus genotypes.Conclusions.The developed reagent kit allows detection of hepatitis B virus and determination of its quantity within 70 minutes. In addition to a large number of genotypes and subgenotypes, the virus is characterized by mutational changes in the genome, which complicates its diagnosis and, as a consequence, the ongoing therapy with drugs. Conservative regions for primer and probe selection taken into account in the development, and the sequencing results obtained are applicable to all HBV genotypes. The reagent kit is designed to monitor HBV infected patients and will allow the analysis of different HBV viral loads.

Hepatitis B core-related antigen: a novel and promising surrogate biomarker to guide anti-HBV therapy.

The current requirement for biomarkers to detect hepatitis B virus (HBV) infection is polarized. One is a fully-automated and highly sensitive measurement system; the other is a simple system for point-of-care testing (POCT) in resource-limited areas. Hepatitis B core-related antigen (HBcrAg) reflects intrahepatic covalently closed circular DNA and serum HBV DNA. Even in patients with undetectable serum HBV DNA or HBsAg loss, HBcrAg may remain detectable. Decreased HBcrAg levels are associated with reduction of the occurrence of hepatocellular carcinoma (HCC) in chronic hepatitis B (CHB). Recently, a fully-automated, novel high-sensitivity HBcrAg assay (iTACT-HBcrAg, cut-off value: 2.1 log U/mL) has been developed. This attractive assay has been released in Japan very recently. iTACT-HBcrAg can be useful for monitoring HBV reactivation and prediction of HCC occurrence, as an alternative to HBV DNA. Moreover, monitoring HBcrAg may be suitable for determining the therapeutic effectiveness of approved drugs and novel drugs under development. Presently, international guidelines recommend anti-HBV prophylaxis for pregnant women with high viral loads to prevent mother-to-child transmission of HBV. However, >95% of HBV-infected individuals live in countries where HBV DNA quantification is not available. Worldwide elimination of HBV needs the scaling-up of examination and medication services in resource-limited areas. Based on this situation, a rapid and easy HBcrAg assay as a POCT is valuable. This review provides the latest information regarding the clinical use of a new surrogate marker, HBcrAg, in HBV management, based on iTACT-HBcrAg or POCT, and introduces novel agents targeting HBV RNA/protein.

Hepatitis B virus genotypes and drug resistance mutations circulating in blood donors in Beira, Mozambique.

Hepatitis B virus (HBV) infects nearly 300 million people and is the leading cause of hepatitis and hepatocellular carcinoma worldwide. Despite the high burden of HBV in sub-Saharan Africa, countries such as Mozambique have limited data available on circulating HBV genotypes and the presence of drug resistance mutations. Blood donors from Beira, Mozambique were tested for HBV surface antigen (HBsAg) and HBV DNA at the Instituto Nacional de Saúde in Maputo, Mozambique. Regardless of HBsAg status, donors with detectable HBV DNA were evaluated for HBV genotype. PCR was performed with primers amplifying a 2.1-2.2 kilobase fragment of the HBV genome. PCR products were submitted for next generation sequencing (NGS), and consensus sequences were evaluated for HBV genotype, recombination, and the presence or absence of drug resistance mutations. Of the 1281 blood donors tested, 74 had quantifiable HBV DNA. The polymerase gene could be amplified from 45 of 58 (77.6%) individuals with chronic HBV infection and 12 of 16 (75%) with occult HBV infection. Among these 57, 51 (89.5%) sequences belonged to HBV genotype A1, while 6 (10.5%) were HBV genotype E. All genotype E sequences were E/A recombinants, and clustered separately from other genotype E references. Genotype A samples had a median viral load of 637 IU/mL, while genotype E samples had a median viral load of 476,084 IU/mL. No drug resistance mutations were observed in the consensus sequences. The current study demonstrates the genotypic diversity of HBV in blood donors in Mozambique, but the absence of dominant (consensus) drug resistance mutations. Studies in other at-risk populations are essential for understanding the epidemiology, risk of liver disease, and likelihood of treatment resistance in resource-limited settings.

A nomogram based on HBeAg, AST, and age to predict non-minimal liver inflammation in CHB patients with ALT

Precise assessment of liver inflammation in untreated hepatitis B e antigen (HBeAg)-positive patients with chronic hepatitis B virus (HBV) infection can determine when to initiate antiviral therapy. The aim of this study was to develop and validate a nomogram model for the prediction of non-minimal liver inflammation based on liver pathological injuries combined with age and alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatitis B surface antigen (HBsAg), HBeAg, and HBV DNA quantification. We retrospectively included 735 HBeAg-positive chronic hepatitis B (CHB) patients with ALT<80U/L as the primary cohort and prospectively enrolled 196 patients as the validation cohort. Multivariate logistic regression analysis identified independent impact factors. A nomogram to predict significant liver inflammation was developed and validated. Multivariate logistic regression analysis showed that HBeAg, AST, and age were independent risk factors for predicting non-minimal liver inflammation in untreated CHB patients. The final formula for predicting non-minimal liver inflammation was Logit(P) = -1.99 - 0.68 × Log10HBeAg + 0.04 × Age + 0.06 × AST. A nomogram for the prediction of non-minimal liver inflammation was established based on the results from the multivariate analysis. The predicted probability of the model being consistent with the actual probability was validated by the calibration curves, showing the best agreement in both the primary and validation cohorts. The C-index was 0.767 (95%CI=0.734-0.802) in the primary cohort and 0.749 (95%CI=0.681-0.817) in the prospective validation cohort. The nomogram based on HBeAg, AST, and age might help predict non-minimal liver inflammation in HBeAg-positive CHB patients with ALT<80U/L, which is practical and easy to use for clinicians.

Control of occult hepatitis B virus infection.

The diagnosis of hepatitis B virus (HBV) infection requires HBV DNA testing and serologic testing for detection of the surface antigen (HBsAg) and the hepatitis B core antibody (anti-HBc). There is a population of patients with occult HBV infection (OBI), which is not detected by HBsAg or HBV DNA quantification in blood, despite the presence of active replication in the liver. This document provides a definition of OBI and describes the diagnostic techniques currently used. It also addresses the detection of patients with risk factors and the need for screening for OBI in these patients. Correct diagnosis of OBI prevents HBV reactivation and transmission. Diagnosis of OBI is based on the detection of HBV DNA in patients with undetectable HBsAg in blood. A high number of patients with OBI may remain undiagnosed; therefore, screening for OBI in patients with factor risks is essential. For a correct diagnosis of OBI, it is necessary that new markers such as ultrasensitive HBsAg are incorporated, and a more comprehensive marker study is performed by including markers such as cccDNA.

Evaluation of Hepatitis B viral Serological Patterns and HBV DNA levels in Hepatitis B subjects at Nauth Nnewi, Southeastern Nigeria

Nigeria is one of the countries with the highest incidence of Hepatitis B Virus (HBV) infection worldwide. The inaccessibility and unaffordability of HBV DNA quantification (viral load) assay which is the key laboratory test for therapy initiation, and monitoring is a challenge to HBV management. This study aimed at evaluating the HBV DNA viral load differences across the serological markers of HBV in order to develop a more cost-effective diagnostic algorithm for Hepatitis B management. Cross sectional study design was used with a total of 264 subjects comprising of 88 HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects on antiviral therapy as case subjects and 88 age-matched apparently healthy HBsAg seronegative individuals were recruited as control subjects. Hepatitis B Virus DNA assay was performed using real time PCR technique, Hepatitis B core Antibody Immunoglobulin M and Hepatitis D Virus Immunoglobulin G assay. Immunochromatography was used for HBV Panel, Hepatitis C Virus assay and Human Immunodeficiency Virus testing. The non-treatment group has higher viral load (M=805.50 IU/ml) compared with treatment group (M = 65.50 IU/ml) (p&lt;0.001). There was a significant difference in the HBV DNA levels of the four serological patterns observed in the study (P&lt; 0.001). Among the four serological patterns observed, the second pattern with positive surface antibody, positive envelope antigen and negative envelope antibody showed highest viral load (M= 46850189.50 IU/ml) followed by the third pattern with negative surface antibody, negative envelope antigen, and negative envelope antibody (M=46555 IU/ml). The first pattern which has negative surface antibody, negative envelope antigen and positive envelope antibody has the lowest viral load (M=21.00 IU/ml) followed by the fourth pattern with positive surface antibody, negative envelope antigen and positive envelope antibody (M=493.00 IU/ml). This study showed that HBV serological markers can predict viral load and should be used as its alternative in resource poor settings.

Rapid Point-of-Care Test for Hepatitis B Core-Related Antigen to Diagnose High Viral Load in Resource-Limited Settings

Global elimination of hepatitis B virus (HBV) requires scale-up of testing and treatment services in low-income and middle-income countries (LMICs). Following the screening for hepatitis B surface antigen (HBsAg), quantification of serum HBV DNA is invariably required to identify a subset of HBV-infected individuals who should immediately start antiviral therapy. Anti-HBV treatment is recommended for patients with chronic HBV infection (CHB) who have high HBV DNA levels (≥2000 or ≥20,000 IU/mL) in the presence of liver fibrosis or inflamation.

Immunoglobulin-free strategy to prevent HBV mother-to-child transmission in Cambodia (TA-PROHM): a single-arm, multicentre, phase 4 trial

Prevention of mother-to-child transmission (MTCT) of hepatitis B virus (HBV) is based on administration of vaccine and immunoglobulins (HBIg) to newborns at birth and maternal antiviral prophylaxis for those with an HBV-DNA viral load of at 5·3 log10 IU/mL or more. Many low-income and middle-income countries face difficulty in accessing HBIg and HBV-DNA quantification. The aim of this study was to evaluate the effectiveness of an HBIg-free strategy to prevent MTCT of HBV. TA-PROHM was a single-arm, multicentre, phase 4 trial done in five maternity units in Cambodia. Pregnant women who were positive for hepatitis B surface antigen (HBsAg), aged 18 years or older were included. Women who were HCV or HIV positive, had creatinine clearance of less than 30 mL/min, severe gravid disease, and planned to give birth outside the study sites were excluded. From Oct 4, 2017, to Jan 9, 2019, HBsAg positive pregnant women who tested positive for hepatitis B e antigen (HBeAg) with a rapid diagnostic test were eligible to receive tenofovir disoproxil fumarate. From Jan 9, 2019, women who were HBeAg negative with an alanine aminotransferase concentration of ≥40 IU/L were also eligible to receive tenofovir disoproxil fumarate. Women in the tenofovir disoproxil fumarate eligible group received 300 mg of tenofovir disoproxil fumarate orally once a day from the 24th week of gestation until 6 weeks postpartum. The primary outcome was the overall proportion of infants who were HBsAg positive at 6 months of life, confirmed by positive HBV DNA quantification. For the primary outcome, the proportion (95% CI) of infants with HBsAg at 6 months was stratified according to infant's HBIg status, duration of maternal tenofovir disoproxil fumarate treatment (>4 weeks and ≤4 weeks), and study period (before and after the change in therapeutic algorithm) and was measured in a modified intention-to-treat analysis, which excluded infants lost to follow-up or who were withdrawn before 6 months. The study is registered with ClinicalTrials.gov, NCT02937779. From Oct 4, 2017, to Nov 27, 2020, 21 251 pregnant women were screened for HBsAg, of whom 1194 (6%) were enrolled in the study: 338 (28%) were eligible to receive tenofovir disoproxil fumarate. For the tenofovir disoproxil fumarate eligible group, four (1% [95% CI 0·34-3·20]) of 317 infants had HBV infection at 6 months; in the subgroup of 271 children who did not receive HBIg, four (1% [0·40-3·74]) had HBV infection at 6 months. In absence of HBIg, MTCT HBV transmission occurred in none (0% [0-1·61]) of 227 women who received tenofovir disoproxil fumarate for more than 4 weeks before giving birth and three (8% [1·75-22·47]) of 36 women who received tenofovir disoproxil fumarate for less than 4 weeks. In the tenofovir disoproxil fumarate ineligible group, seven (1% [0·40-2·02]) of 712 infants had HBV infection at 6 months; in the subgroup of 567 children who did not receive HBIg, six (1% [0·39-2·30]) had HBV infection at 6 months. An immunoglobulin-free strategy using an HBeAg rapid diagnosis test and alanine aminotransferase-based algorithm to assess eligibility for tenofovir, is effective at preventing MTCT of HBV when tenofovir was initiated at least 4 weeks before birth. French Agency for Research on AIDS and Viral Hepatitis and Emerging Infectious diseases. For the French translation of the abstract see Supplementary Materials section.

Alinity m, a Random-Access System, for Hepatitis B Virus DNA Quantification in Plasma and Whole Blood Collected on Dried Blood Spots.

ABSTRACTThe International Liver Association recommends the use of accurate and sensitive molecular methods for determination of hepatitis B virus (HBV) DNA levels in plasma or serum of chronic HBsAg carriers. The level of HBV replication represents the strongest predictive biomarker associated with disease progression and long-term outcome of chronic HBV infection. The purpose of this study was to evaluate the ability to the new Alinity m System to detect and quantify HBV DNA in plasma and whole blood collected on dried blood spots (DBS). Paired plasma and DBS samples from patients chronically infected with various HBV genotypes were tested in parallel for HBV DNA detection and quantification. There is a linear relationship between HBV DNA levels measured in plasma samples using the Alinity m HBV assay and the Xpert HBV viral load assay, used for comparison. A slight deviation (0.03 ± 0.31 log IU/mL) was observed within the quantitative range. In DBS, HBV DNA levels closely correlated with levels measured in plasma. All patients had detectable and quantifiable HBV DNA by DBS testing, except for one patient with a plasma HBV DNA level above 2,000 IU/mL. In conclusion, the newly developed real-time PCR-based assay Alinity m HBV assay can correctly detect HBV DNA in DBS, especially for patients with blood HBV DNA levels above 2,000 IU/mL, and also accurately quantify HBV DNA in plasma samples.IMPORTANCE Hepatitis B virus is one of the most prevalent blood-borne viruses affecting the liver and causing acute and chronic hepatitis. Only a small proportion of people with HBV infection are diagnosed. HBV DNA measurement is critical in clinical practice for the diagnosis and treatment decisions of patients requiring antiviral therapy. Dried blood spot (DBS) collection provides a simple, practical, and acceptable alternative to venous blood collection, especially in community settings. We have demonstrated high sensitivity and specificity for HBV DNA detection in DBS compared to plasma samples, especially when using clinically relevant cutoffs of 2,000 and 20,000 IU/mL. Results support the use of DBS in community-based settings.

Residual risk of mother-to-child transmission of hepatitis B virus infection despite timely birth-dose vaccination in Cameroon (ANRS 12303): a single-centre, longitudinal observational study

In sub-Saharan Africa, administration of hepatitis B virus (HBV) birth-dose vaccines remains suboptimal. Evidence is scarce on whether African countries should focus on increasing vaccine coverage or developing strategies incorporating additional measures, such as peripartum antiviral prophylaxis to pregnant women at high risk. To better inform decision makers, we estimated the residual risk of mother-to-child transmission despite HBV birth-dose vaccine in Cameroon. We did a single-centre, longitudinal observational study. Pregnant women were systematically screened for HBV surface antigen (HBsAg) at Tokombéré District Hospital (Tokombéré district, Cameroon). Children born to HBsAg-positive mothers in 2009-16 who received the HBV birth-dose vaccine and three subsequent doses of pentavalent vaccine at 6, 10, and 14 weeks were followed up prospectively in 2015-17. In children, capillary blood was obtained for HBsAg rapid test and dried blood spots to quantify HBV DNA concentrations. Venous blood was also collected from HBsAg-positive children. Mother-to-child transmission was confirmed by whole-genome sequencing. Between Jan 31, 2009, and Dec 31, 2016, 22 243 (66·8%) of 33 309 pregnant women accepted antenatal HBV screening, of whom 3901 (17·5%) were HBsAg positive. 2004 (51·4%) of 3901 children who were born to HBsAg-positive mothers received the HBV birth-dose vaccine, of whom 1800 (89·8%) also completed the three-dose pentavalent vaccine. In total, the current analysis included 607 children who had a follow-up serosurvey. The prevalence of HBsAg was 5·6% in children who received the birth-dose vaccine in less than 24 h, 7·0% in those who received it 24-47 h after birth, and 16·7% in those who received it 48-96 h after birth (ptrend=0·083). 35 (89·7%) of 39 infected children were born to mothers positive for HBV e antigen with high HBV DNA of 5·3 log10 IU/mL or more. Whole-genome sequencing of HBV in infected mother-child pairs confirmed high identity proportions of 99·97-100%. We documented a substantial risk of mother-to-child transmission despite timely administration of the HBV birth-dose vaccine within 24 h after birth. To reach WHO's elimination targets, peripartum antiviral prophylaxis might be required in parts of Africa, in addition to increasing coverage of the HBV birth-dose vaccine. Agence nationale de recherches sur le sida et les hépatites virales (ANRS).

Dried blood spot sampling for hepatitis B virus quantification, sequencing and mutation detection

Hepatitis B virus (HBV) diagnosis is performed on serum samples, but the access to this diagnosis is difficult in low-income regions. The use of dried blood spot (DBS) samples does not require special structure for collection, storage or transport. This study evaluates the use of DBS for detection, quantification and sequencing of HBV DNA using in-house techniques. Two study groups were included: 92 HBsAg + individuals and 49 negative controls. Serum and DBS samples were submitted to quantitative and qualitative in-house PCR for S/pol genes, sequencing and phylogenetic analyses. Total of 84 serum samples were successfully amplified. Of them, 63 paired DBS were also positive in qualitative PCR. Qualitative PCR in DBS presented a sensitivity of 75% and specificity of 100% (Kappa = 0.689). Quantitative PCR in DBS presented a detection limit of 852.5 copies/mL (250 IU/mL), sensitivity of 77.63% and specificity of 100% (Kappa = 0.731). A total of 63 serum samples and 36 DBS samples were submitted to sequencing, revealing the circulation of genotypes A (65.08%), D (4.8%), E (3.2%) and F (27%) with 100% of correspondence between serum and DBS. All sequenced samples displayed polymorphisms in HBsAg gene. An HIV-coinfected patient presented the rtM204V/I-rtL180M double resistance mutation in serum and DBS. In conclusion, DBS is an alternative to detect, quantify and characterize HBV DNA, being a possibility of increasing diagnosis in low-income settings, closing gaps in HBV control.

Entecavir Prevents HBV Reactivation During Direct Acting Antivirals for HCV/HBV Dual Infection: A Randomized Trial

A strategy to prevent hepatitis B virus (HBV) virologic reactivation (HBVr) and clinical reactivation (CR) during direct acting antiviral (DAA) treatment of hepatitis C virus (HCV)/HBV dual infection remains an unresolved issue. Noncirrhotic patients with dual HCV/HBV infection were enrolled and allocated randomly to 1 of 3 groups as follows: 12 weeks of DAA alone (group 1), 12 weeks of DAA plus 12 weeks of entecavir (group 2), or 12 weeks of DAA plus 24 weeks of entecavir (group 3). The entire study duration was 72 weeks. The primary end point was the occurrence of HBVr (defined by an increase of HBV DNA level >10-fold with quantifiable HBV DNA at baseline or the presence of HBV DNA with prior unquantifiable HBV DNA) and CR (defined by serum alanine aminotransferase level >2-fold the upper limit of normal in addition to HBVr). Fifty-six patients were allocated randomly as follows: 20 patients in group 1, 16 patients in group 2, and 20 patients in group 3. In group 1, HBV DNA levels increased significantly as early as 4 weeks after initiation of DAA and persisted until the end of the study. During DAA treatment, HBVr occurred in 50% in group 1 vs 0% in group 2 and 0% in group 3 (P < .001), whereas the majority of HBVr in groups 2 and 3 occurred 12 weeks after cessation of entecavir (cumulative incidence, 93.8% in group 2 and 94.7% in group 3). Three patients (5.4%; 1 in each group) showed CR at week 48 and did not receive entecavir treatment. Twelve weeks of entecavir is suggested to be co-administered with DAA for HCV/HBV dually infected patients. gov no: NCT04405011.

Burden and Transmission Risks of Viral Hepatitis in Southern Ethiopia: Evidence Needed for Prevention and Control Measures.

BackgroundHepatitis B virus (HBV) and hepatitis C virus (HCV) are significant causes of liver-associated morbidity and mortality for millions of people globally. Ethiopia is one of the viral hepatitis–endemic countries with no national strategy for surveillance and limited data. As such, this study aimed to investigated the extent and associated risk factors of HBV and HCV among community members in southern Ethiopia.MethodsA community-based cross-sectional study was conducted from January 2020 to August 2020. A structured questionnaire was used to collect behavioral and sociodemographic data. Serum samples were collected and assayed for seromarkers of HBV (HBsAg, anti-HBc, and anti-HBs) and HCV (anti-HCV) using ELISAs. In HBsAg-positive samples, HBV DNA was further quantified using RT-PCR. Data were entered into EpiData 3.1 and analyzed using SPSS 21.0. Descriptive statistics and logistic regression analysis were employed.ResultsThe study included 693 participants. Seromarkers for HBsAg, anti-HCV, anti-HBc, and anti-HBs were found to be 9.5%, 1.4%, 31.1%, and 14.3%, respectively. In 66 HBsAg positives, 57 (86.4%) had quantifiable HBV DNA. Prevalence of current HBV infection (HBsAg+, anti-HBc+, anti-HBs–) and lifetime exposure (positive for either HBsAg or anti-HBc) to HBV were 8.7% and 31.9%, respectively, and 63.1% of participants were vulnerable or had no evidence of prior HBV infection (HBsAg–, anti-HBc–, anti-HBs–). On multivariate logistic regression analysis, multiple sexual contacts, family history of hepatitis infection, alcohol consumption, and khat chewing were significantly associated with HBV. The seroprevalence of HBV was relatively high in this study area.ConclusionThis study showed high prevalence of HBV infection, but low prevalence of HCV. This indicates that HBV is a major health problem in this community. Population-based surveillance, care, and treatment, as well as behavioral change and education programs, should be enhanced to minimize risk exposure.

Managing the Risk of Hepatitis B Virus Reactivation in Patients with B-Cell Lymphomas Treated with Obinutuzumab or Rituximab Immunochemotherapy

Introduction: Reactivation of hepatitis B virus (HBV) is an identified risk associated with immunochemotherapy for non-Hodgkin lymphoma (NHL) in patients (pts) with resolved HBV infection. This study aimed to evaluate the risk of HBV reactivation and explore risk factors in NHL pts with resolved HBV infection who received anti-CD20 antibody (obinutuzumab or rituximab)-containing immunochemotherapy in the phase III GOYA and GALLIUM studies (involving pts with previously untreated diffuse large B-cell lymphoma [DLBCL] and indolent NHL, respectively). Methods: Pts were randomized to receive induction with either obinutuzumab (1000 mg on days 1, 8, and 15 of cycle 1, and day 1 of cycles 2‒8) or rituximab (375 mg/m2 on day 1 of each cycle) in combination with CHOP (GOYA and GALLIUM), CVP (GALLIUM), or bendamustine (GALLIUM) for 6-8 cycles. In GALLIUM, induction was followed by obinutuzumab or rituximab maintenance. Baseline screening was performed locally for hepatitis B surface antigen (HBsAg) and antibody against hepatitis B core antigen (anti-HBc). Pts positive for HBsAg were excluded, while those with resolved HBV infection (HBsAg-negative but anti-HBc-positive) could be enrolled if they had baseline HBV DNA Results: Of 2797 evaluable pts (GOYA, 1407; GALLIUM, 1390), 326 pts were seropositive for anti-HBc (207 were seropositive for antibodies against HBsAg) and 11 had detectable but non-quantifiable HBV DNA (10 to Conclusions: HBV DNA monitoring-guided preemptive NAT was effective in preventing HBV-related hepatitis during treatment with obinutuzumab- or rituximab-containing immunochemotherapy. Antiviral prophylaxis was effective in preventing HBV reactivation and may be an appropriate option for pts with resolved HBV infection and multiple risk factors. Disclosures Kusumoto: Chugai: Honoraria, Other: GALLIUM and GOYA are sponsored by F. Hoffmann-La Roche Ltd. Third-party medical writing support, under the direction of Shigeru Kusumoto, was provided by Cheryl Wright of Gardiner-Caldwell Communications, and was funded by F. Hoffmann-La Roche Ltd, Research Funding. Arcaini: Celgene, Roche, Sandoz: Consultancy; Gilead: Research Funding; Pfizer, Celgene, Bayer, Roche: Membership on an entity9s Board of Directors or advisory committees. Kim: JJ Takeda: Research Funding; Celltrion, Inc: Consultancy, Honoraria; Novartis: Research Funding; Roche: Research Funding; Donga: Research Funding; Mundipharma: Research Funding; Kyowa-Kirin: Research Funding. Peters: Genentech, Merck: Honoraria; Roche: Honoraria. Tanaka: BMS: Honoraria, Research Funding; Chugai: Honoraria, Research Funding; GSK: Honoraria, Research Funding. Zelenetz: Celgene: Consultancy; Amgen: Consultancy. Kuriki: Chugai: Employment. Fingerle-Rowson: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Nielsen: F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ueda: Chugai: Employment. Piper-Lepoutre: Roche: Employment. Sellam: Roche: Employment. Tobinai: Daiichi Sankyo Co., Ltd: Consultancy, Honoraria; AbbVie: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; HUYA Bioscience: Honoraria; Chugai: Honoraria, Research Funding; Eisai: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen: Honoraria, Research Funding; Kyowa Hakko Kirin: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Servier: Research Funding; Takeda: Honoraria, Research Funding; Zenyaku Kogyo: Honoraria.

Discovery of Novel Pyrimidine-Based Capsid Assembly Modulators as Potent Anti-HBV Agents.

Core assembly modulators of viral capsid proteins have been developed as an effective treatment of chronic hepatitis B virus (HBV) infection. In this study, we synthesized novel potent pyrimidine derivatives as core assembly modulators, and their antiviral effects were evaluated in in vitro and in vivo biological experiments. One of the synthesized derivatives, compound 23h (R1 = MeSO2, R2 = 1-piperidin-4-amine, R3 = 3-Cl-4-F-aniline) displayed potent inhibitory effects in the in vitro assays (52% inhibition in the protein-based assay at 100 nM and an IC50 value of 181 nM in the serum HBV DNA quantification assay). Moreover, treatment with compound 23h for 5 weeks significantly decreased serum levels of HBV DNA levels (3.35 log reduction) in a human liver-chimeric uPA/SCID mouse model, and these effects were significantly increased when 23h was combined with tenofovir, a nucleotide analogue inhibitor of reverse transcriptase used for the treatment of HBV infection.

Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings

ObjectiveA massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-limited countries. We have developed and evaluated the loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by the WHO (≥20 000 and ≥ 200 000 IU/mL). MethodsPan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively. ResultsOur primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40 and 400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI 0.93–0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20 000 IU/mL; and 0.98 (0.97–0.99), 98.0% and 94.6% for ≥200 000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93–0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200 000 IU/mL. The assay was not adapted to discriminate low-level viraemia. DiscussionWe have developed a simple, rapid (60 min), and inexpensive (US$8/assay) alternative to PCR to diagnose high viraemia ≥200 000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries.