Evaluation of analytical performance of the STANDARDTM M10 MPX/OPX assay for the simultaneous DNA detection and clade attribution of Monkeypox virus.

Monkeypox virus (MPXV) infection confirmation needs reliable polymerase chain reaction (PCR) assays; in addition, viral clade attribution is a key factor in containment measures, considering a more severe syndrome in clade I and the possibility of simultaneous circulation. This study evaluates the performance of all-in-one STANDARD M10 MPX/OPX (SD BIOSENSOR, South Korea - M10). Frozen samples from 205 subjects were selected and stratified according to routine test results (RealStar® Orthopoxvirus PCR Kit 1.0, Altona DIAGNOTICS, Germany - RS; RS-1): in detail, 100 negative skin lesions (SL) and 200 positive samples at the variable stage of infection were analysed. Positive samples were retested with RS (RS-2). Positive and Negative Percent Agreements (PPA, NPA) were calculated. The median (IQR) Ct values of RS and M10 (OPXV target) assays were highly similar. The PPA of M10 compared to RS-1 was 89.5% considering system interpretation, and 96.0% when the operator classified results as positive if any target was detected; NPA was 100%. Comparing the RS-2 run and M10, an overall concordance of 95.3% between assays was found; however, considering operator interpretation, M10 returned more positive results than RS-2. The occurrence of False-Negative results was likely associated with the influence of thawing on low viral concentration; no False-Positive tests were observed. All samples collected at the time of Mpox diagnosis were positive and M10 correctly attributed the clade (West-Africa/II). The M10 MPX/OPX assay demonstrated high reliability in confirming MPXV infection and clade attribution.

Altered TLR7 Expression-Mediated Immune Modulation Is Supportive of Persistent Replication and Intrauterine Transmission of HBV.

Hepatitis B Virus (HBV) is posing as a serious public health threat mainly due to its asymptomatic nature of infection in pregnancy and vertical transmission. Viral sensing toll-like receptors (TLR) and Interleukins (IL) are important molecules in providing an antiviral state. The study aimed to assess the role of TLR7-mediated immune modulation, which might have an impact in the intrauterine transmission of HBV leading to mother to child transmission of the virus. We investigated the expression pattern of TLR7, IL-3, and IL-6 by RT-PCR in the placentas of HBV-infected pregnant women to see their role in the intrauterine transmission of HBV. We further validated the expression of TLR7 in placentas using Immunohistochemistry. Expression analysis by RT-PCR of TLR7 revealed significant downregulation among the Cord blood (CB) HBV DNA positive and negative cases with mean ± standard deviation (SD) of 0.43 ± 0.22 (28) and 1.14 ± 0.57 (44) with p = 0.001. IL-3 and IL-6 expression revealed significant upregulation in the CB HBV DNA-positive cases with p = 0.001. Multinomial logistic regression analysis revealed that TLR7 and IL-3 fold change and mother HBeAg status are important predictors for HBV mother to child transmission. Immunohistochemistry revealed the decreased expression of TLR7 in CB HBV DNA-positive cases. This study reveals that the downregulation of TLR7 in the placenta along with CB HBV DNA-positive status may lead to intrauterine transmission of HBV, which may lead to vertical transmission of HBV.

Establishing a screening strategy for non-discriminatory reactive blood donors by constructing a predictive model of hepatitis B virus infection status from a single blood center in China.

Background: When employing the transcription-mediated amplification method for screening blood donors, there are some non-discriminatory reactive results which are screening assay reactive but HBV-DNA discriminatory assay negative. This raises concerns regarding the possibility of false positives among donors, which may lead to permanent deferral of blood donors and affect blood supply. This study aimed to elucidate the infection status of these non-discriminatory reactive blood donors and develop and validate a model to predict individualized hepatitis B status to establish an optimal screening strategy. Methods: Supplementary tests were conducted on initial non-discriminating reactive donations to determine their HBV infection status, including repeat testing, viral load, serological marker detection, and follow-up. Primary clinical variables of the donors were recorded. Based on the Akaike information criterion, a stepwise forward algorithm was used to identify the predictive factors for information and construct a predictive model. The optimal screening strategy was determined through cost-effectiveness analysis. Results: At the Blood Center of Zhejiang Province, 435 cases of initial non-discriminatory reactive donations were collected over two successive periods and sub-categorized through repeated testing into the following three groups: non-repeated positive group, non-discriminated positive group, and non-repeated HBV-DNA positive group. The HBV discriminatory rate increased after repeated testing (110/435, 25.29%). According to supplementary tests, the HBV-DNA positivity rate was 65.52% (285/435), and occult HBV infection was a significantly different among groups (χ2 = 93.22, p < 0.01). The HBV serological markers and viral load in the non-repeated positive group differed from those in the other two groups, with a lower viral load and a higher proportion of false positives. The predictive model constructed using a stepwise forward algorithm exhibited high discrimination, good fit, high calibration, and effectiveness. A cost-effectiveness analysis indicated that utilizing repeated discriminatory testing and the predictive model is an extremely beneficial screening approach for non-discriminatory reactive blood donors. Conclusion: Nearly two-third (65.52%) of the non-discriminatory reactive blood donors were HBV-DNA positive. Our innovative approach of constructing a predictive model as a supplementary screening strategy, combined with repeated discriminatory experiments, can effectively identify the infection status of non-discriminatory reactive blood donors, thereby increasing the safety of blood transfusions.

Improving polymerase chain reaction diagnostic rates for herpes simplex keratitis: results of a pilot study.

Background: Laboratory confirmation is crucial for diagnosis and management of herpes simplex virus (HSV) keratitis. However, the sensitivity of polymerase chain reaction (PCR) in keratitis is low (25%) compared with that of mucocutaneous disease (75%). We developed an educational intervention aimed at improving the diagnostic yield of PCR. Methods: The medical records of keratitis cases seen at the emergency department of a London tertiary ophthalmic referral hospital over two distinct periods, before and after an educational program on swab technique, were reviewed retrospectively. Results: A total of 252 HSV cases were included. Increases in the laboratory-confirmed diagnosis of HSV-1 were observed, in both first presentations (11.1%-57.7%) and recurrent cases (20%-57.6%). The rate of positive HSV-1 PCR in eyes with an epithelial defect increased from 19% pre-intervention to 62% post intervention. Notably, 3% were positive for varicella zoster virus DNA, and there was a single case of Acanthamoeba keratitis. Conclusion: Our results suggest that, with proper swabbing technique, PCR may be more sensitive than previously reported.

Impact of a carrageenan gel on viral load of genital human papillomavirus infections in sexually active women: Findings from the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) trial.

Previous research has shown that women's use of a carrageenan gel reduces the risk of acquiring genital human papillomavirus (HPV) infections but does not help to clear existing ones. Although gel use may not result in complete clearance, it may decrease the viral load of HPV infections. We tested this hypothesis in the Carrageenan-gel Against Transmission of Cervical Human papillomavirus (CATCH) randomized controlled trial. Participants of the CATCH study were selected for viral load testing if they had completed the first four study visits and tested positive for HPV42 or HPV51 in at least one of these visits. HPV42 and HPV51 were chosen as they were among the most abundant low- and high-risk types, respectively, in the study sample. We measured viral load with a type-specific real-time polymerase chain reaction. Results were displayed using summary statistics. Of 461 enrolled participants, 39 were included in the HPV42 analysis set and 56 in the HPV51 analysis set. The median time between visits 1 and 4 was 3.7 months. The viral load (copies/cell) of HPV42 ranged from <0.001 to 13 434.1, and that of HPV51 from <0.001 to 967.1. The net median change in HPV42 viral load over all four visits was -1.04 copies/cell in the carrageenan and -147 copies/cell in the placebo arm (Wilcoxon rank sum test, p = 0.26). There was no net median change in HPV51 viral load over all four visits in either arm (p = 0.45). The use of a carrageenan-based gel is unlikely to reduce the viral load of HPVs 42 or 51.

Epstein-Barr Virus in the Cerebrospinal Fluid and Blood Compartments of Patients With Multiple Sclerosis and Controls.

BACKGROUND AND OBJECTIVES: Epstein-Barr virus (EBV) infection is a major risk factor of multiple sclerosis (MS). We examined the presence of EBV DNA in the CSF and blood of patients with MS and controls. We analyzed whether EBV DNA is more common in the CSF of patients with MS than in controls and estimated the proportions of EBV-positive B cells in the CSF and blood. METHODS: CSF supernatants and cells were collected at diagnostic lumbar punctures from 45 patients with MS and 45 HLA-DR15 matched controls with other conditions, all participants were EBV seropositive. Cellular DNA was amplified by Phi polymerase targeting both host and viral DNA, and representative samples were obtained in 28 cases and 28 controls. Nonamplified DNA from CSF cells (14 cases, 14 controls) and blood B cells (10 cases, 10 controls) were analyzed in a subset of participants. Multiple droplet digital PCR (ddPCR) runs were performed per sample to assess the cumulative EBV positivity rate. To detect viral RNA as a sign of activation, RNA sequencing was performed in blood CD4-positive, CD8-positive, and CD19-positive cells from 21 patients with MS and 3 controls. RESULTS: One of the 45 patients with MS and none of the 45 controls were positive for EBV DNA in CSF supernatants (1 mL). CSF cellular DNA was analyzed in 8 independent ddPCRs: EBV DNA was detected at least once in 18 (64%) of the 28 patients with MS and in 15 (54%) of the 28 controls (p = 0.59, Fisher test). The cumulative EBV positivity increased steadily up to 59% in the successive ddPCRs, suggesting that all individuals would have reached EBV positivity in the CSF cells, if more DNA would have been analyzed. The estimated proportion of EBV-positive B cells was >1/10,000 in both the CSF and blood. We did not detect viral RNA, except from endogenous retroviruses, in the blood lymphocyte subpopulations. DISCUSSION: EBV-DNA is equally detectable in the CSF cells of both patients with MS and controls with ddPCR, and the probabilistic approach indicates that the true positivity rate approaches 100% in EBV-positive individuals. The proportion of EBV-positive B cells seems higher than previously estimated.

miR-3188 inhibits hepatitis B virus transcription by targeting Bcl-2.

Transcription of the covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is subject to dual regulation by host factors and viral proteins. MicroRNAs (miRNAs) can regulate the expression of target genes at the post-transcriptional level. Systematic investigation of miRNA expression in HBV infection and the interaction between HBV and miRNAs may deepen our understanding of the transcription mechanisms of HBV cccDNA, thereby providing opportunities for intervention. miRNA sequencing and real-time quantitative PCR (qRT-PCR) were used to analyze miRNA expression after HBV infection of cultured cells. Clinical samples were analyzed for miRNAs and HBV transcription-related indicators, using qRT-PCR, enzyme-linked immunoassay (ELISA), and Western blot. miRNA mimics or inhibitors were used to study their effects on the HBV life cycle. The target genes of miR-3188 and their roles in HBV cccDNA transcription were also identified. The expression of 10 miRNAs, including miR-3188, which was significantly decreased after HBV infection, was measured in clinical samples from patients with chronic HBV infection. Overexpression of miR-3188 inhibited HBV transcription, whereas inhibition of miR-3188 expression promoted HBV transcription. Further investigation confirmed that miR-3188 inhibited HBV transcription by targeting Bcl-2. miR-3188 is a key miRNA that regulates HBV transcription by targeting the host protein Bcl-2. This observation provides insights into the regulation of cccDNA transcription and suggests new targets for anti-HBV treatment.

Effect of pre-emptive rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder in pediatric kidney transplant recipients: A case series from the pediatric nephrology research consortium.

BACKGROUND: There are scant data on the effect of rituximab on EBV DNA levels and prevention of post-transplant lymphoproliferative disorder (PTLD) in pediatric kidney transplant recipients with EBV DNAemia. METHODS: Kidney transplant recipients with EBV DNAemia treated with rituximab to prevent PTLD between 7/1999 and 7/2019 at five pediatric centers were included. Those with confirmed PTLD at the onset of rituximab were excluded. Primary outcomes included percentage change in EBV DNAemia and occurrence of PTLD post rituximab. RESULTS: Twenty-six pediatric kidney transplant recipients were included. Median age at transplant was 4 years (IQR 2.1-10.3). EBV DNA load monitoring by qPCR was performed at 1-3 month intervals. EBV DNAemia onset occurred at a median of 73 days post-transplant (IQR 52-307), followed by DNAemia peak at a median of 268 days (IQR 112-536). Rituximab was administered at a median of 9 days post peak (IQR 0-118). Rituximab regimens varied; median dose 375 mg/m2 (IQR 375-439) weekly for 1-4 doses per course. Following rituximab, EBV DNA load decreased to <10% of baseline at 120 days in 20/26 patients; however, only 30% achieved complete resolution at last follow-up (median 2094 days post-transplant [IQR 1538-3463]). Two (7%) developed PTLD at 915 and 1713 days post rituximab. All recipients had functioning grafts. One death occurred in a child with PTLD following remission due to unrelated reasons. CONCLUSIONS: In the largest pediatric kidney transplant recipient case series with EBV DNAemia given rituximab to prevent PTLD, rituximab achieved a short-term reduction in DNA load; however, recurrent DNAemia is common.

A quantitative assay for the assessment of cutaneous human papillomaviruses and polyomaviruses over time: A proof-of-concept in two patients with atopic dermatitis and psoriasis.

The human skin virome, unlike commensal bacteria, is an under investigated component of the human skin microbiome. We developed a sensitive, quantitative assay to detect cutaneous human resident papillomaviruses (HPV) and polyomaviruses (HPyV) and we first used it to describe these viral populations at the skin surface of two patients with atopic dermatitis (AD) and psoriasis (PSO). We performed skin swabs on lesional and non-lesional skin in one AD and one PSO patient at M0, M1 and M3. After extraction, DNA was amplified using an original multiplex PCR technique before high throughput sequencing (HTS) of the amplicons (named AmpliSeq-HTS). Quantitative results were ultimately compared with monoplex quantitative PCRs (qPCRs) for previously detected viruses and were significantly correlated (R2 = 0.95, ρ = 0.75). Fifteen and 13 HPV types (mainly gamma and beta-HPVs) or HPyV species (mainly Merkel Cell Polyomavirus (MCPyV)) were detected on the skin of the AD and PSO patients, respectively. In both patients, the composition of the viral flora was variable across body sites but remained stable over time in non-lesional skin samples, mostly colonized with gamma-papillomaviruses. In lesional skin samples, beta-papillomaviruses and MCPyV were the major components of a viral flora more prone to vary over time especially with treatment and subsequent clinical improvement. We believe this method might be further used in extensive studies to further enhance the concept of an individual cutaneous viral fingerprint and the putative role of its alterations through various skin diseases and their treatments.

The effect of intrahepatic cholestasis in pregnancy combined with different stages of hepatitis B virus infection on pregnancy outcomes: a retrospective study.

BACKGROUND AND AIMS: To investigate the impact of intrahepatic cholestasis of pregnancy (ICP) with hepatitis B virus (HBV) infection on pregnancy outcomes. METHODS: We selected 512 pregnant women, collected the data including maternal demographics, main adverse pregnancy outcomes and maternal HBV infected markers HBeAg and HBV-DNA loads status, then have a comparative analysis. RESULTS: There were 319 solitary ICP patients without HBV infection (Group I) and 193 ICP patients with HBV infection. Of the latter, there were 118 cases with abnormal liver function(Group II) and 80 cases with normal liver function(Group III). All HBV-infected pregnant women with ICP were divided into hepatitis Be antigen (HBeAg)-positive group (102 cases) and HBeAg-negative group (91 cases), according to the level of the serum HBeAg status; and into high viral load group (92 cases), moderate viral load group (46 cases) and low viral load group (55 cases) according to the maternal HBV-DNA level. Group II had a higher level of serum total bile acids, transaminase, bilirubin as well as a higher percentage of premature delivery, neonatal intensive care unit (NICU) admission and meconium-stained amniotic fluid (MSAF) compared with the other two groups(P < 0.05), but there were no significant differences in the above indicators between the Group I and Group III. Among the HBV-infected patients with ICP, HBeAg-positive group had a higher level of serum transaminase, bilirubin and bile acid as well as earlier gestational weeks of delivery, lower birth weight of new-borns and a higher rate of NICU admission than HBeAg-negative group (P < 0.05). Those with a high viral load (HBV-DNA > 106 IU/ml) had a higher level of transaminase, bilirubin, and bile acid as well as shorter gestational weeks of delivery, lower birth weight of new-borns and a higher rate of NICU admission compared with those with a low or moderate viral load (P < 0.05). CONCLUSION: HBV-infected pregnant women with ICP combined with abnormal liver function have more severe liver damage, a higher percentage of preterm birth and NICU admission. HBeAg-positive status and a high HBV-DNA load will increase the severity of conditions in HBV-infected pregnant women with ICP. HBV-infected patients with ICP who have abnormal liver function, HBeAg-positive or a high viral load should be treated more actively.

[Analysis of clinical characteristics of persistent HBeAg positivity in patients with chronic hepatitis B treated with nucleos(t)ide analogues].

Objective: To explore the clinical characteristics of persistent HBeAg positivity in patients with chronic hepatitis B treated with nucleos(t)ide analogues. Methods: A retrospective analysis was performed according to different data types. An independent sample t-test, Mann-Whitney U test, chi-square test, or Fisher's exact probability method were used. Chronic hepatitis B patients followed up for four years were collected from the follow-up case database of the Department of Infectious Diseases of Zhongshan Third Hospital from January 2009 to December 2018 and were divided into two groups, A and B, with 87 and 145 cases respectively, according to the duration of HBeAg-negativity≤ 3 and persistent positivity >3 years. Statistical analysis was conducted on the age, gender, family history, baseline, follow-up visit duration, liver function, and other data among the two patient groups. Results: There were no statistically significant differences in gender, age, family history of liver cirrhosis, family history of liver cancer, liver cirrhosis condition before treatment, fatty liver disease combined condition before treatment, baseline HBsAg, anti-HBc, alanine aminotransferase, albumin, or total bilirubin between the two groups of patients (P > 0.05). HBV DNA and HBeAg were significantly higher in group B than those in group A at baseline, with P≤0.001. Aspartate aminotransferase and γ-glutamyl transferase were significantly higher in group A than those in group B at baseline. The proportion of family history of hepatitis B was significantly higher in group B (69.0%) than that in group A (50.6%) among the two groups of patients, and the difference was statistically significant (P = 0.005). The proportion of mothers with hepatitis B was significantly higher in group B (25.5%) than in group A (11.5%), P = 0.010. During the treatment process, the HBV DNA quantification was significantly higher in group B than that in group A at 0.5 and 1 years (P≤0.002). The proportion of HBV DNA <100IU/ml was also significantly different at six months and one year (χ(2)=30.327, P < 0.001 and χ(2)=11.779, P = 0.001). The HBsAg level was higher in group B than that of group A in the second and fourth years, P < 0.05. During the entire treatment process, the HBeAg level was significantly higher in group B than that in group A (P < 0.001). A total of seven cases developed liver cirrhosis or cancer during follow-up, including three cases in group A and four cases in group B (P > 0.05). Conclusion: HBeAg-positive patients with chronic hepatitis B have persistent HBeAg positivity when treated with long-term nucleos(t)ide analogues. Accordingly, a greater proportion of this kind of patient family and mothers have a remarkable history of hepatitis B and a reduced HBV DNA relapse rate in the early stages (within a year or less).

Origin and dispersal history of Hepatitis B virus in Eastern Eurasia.

Hepatitis B virus is a globally distributed pathogen and the history of HBV infection in humans predates 10000 years. However, long-term evolutionary history of HBV in Eastern Eurasia remains elusive. We present 34 ancient HBV genomes dating between approximately 5000 to 400 years ago sourced from 17 sites across Eastern Eurasia. Ten sequences have full coverage, and only two sequences have less than 50% coverage. Our results suggest a potential origin of genotypes B and D in Eastern Asia. We observed a higher level of HBV diversity within Eastern Eurasia compared to Western Eurasia between 5000 and 3000 years ago, characterized by the presence of five different genotypes (A, B, C, D, WENBA), underscoring the significance of human migrations and interactions in the spread of HBV. Our results suggest the possibility of a transition from non-recombinant subgenotypes (B1, B5) to recombinant subgenotypes (B2 - B4). This suggests a shift in epidemiological dynamics within Eastern Eurasia over time. Here, our study elucidates the regional origins of prevalent genotypes and shifts in viral subgenotypes over centuries.

Development of a High-throughput Sequencing Platform for Detection of Viral Encephalitis Pathogens Based on Amplicon Sequencing.

Objective: Viral encephalitis is an infectious disease severely affecting human health. It is caused by a wide variety of viral pathogens, including herpes viruses, flaviviruses, enteroviruses, and other viruses. The laboratory diagnosis of viral encephalitis is a worldwide challenge. Recently, high-throughput sequencing technology has provided new tools for diagnosing central nervous system infections. Thus, In this study, we established a multipathogen detection platform for viral encephalitis based on amplicon sequencing. Methods: We designed nine pairs of specific polymerase chain reaction (PCR) primers for the 12 viruses by reviewing the relevant literature. The detection ability of the primers was verified by software simulation and the detection of known positive samples. Amplicon sequencing was used to validate the samples, and consistency was compared with Sanger sequencing. Results: The results showed that the target sequences of various pathogens were obtained at a coverage depth level greater than 20×, and the sequence lengths were consistent with the sizes of the predicted amplicons. The sequences were verified using the National Center for Biotechnology Information BLAST, and all results were consistent with the results of Sanger sequencing. Conclusion: Amplicon-based high-throughput sequencing technology is feasible as a supplementary method for the pathogenic detection of viral encephalitis. It is also a useful tool for the high-volume screening of clinical samples.

[Effect of HBV DNA load on the safety and prognosis of systematic therapy in advanced hepatocellular carcinoma].

Objective: To study the effect of hepatitis B virus (HBV) infection on the occurrence of liver damage, HBV reactivation (HBVr) and the influence of HBVr on the prognosis of patients with advanced hepatocellular carcinoma (HCC) receiving systemic therapy. Methods: The clinical data of 403 patients with HBV-related HCC at the Department of Infectious Diseases, The Third Affiliated Hospital of Sun Yat-Sen University et al, from July 2018 to December 2020 were collected. The incidence of liver damage and HBVr during systematic therapy, and the influence of HBVr on survival prognosis were analyzed. Results: Of the 403 patients, 89.1% were male (n=359), with a median age of 51 years (51.5±12.1). Before propensity score matching (PSM), the proportion of patients with cirrhosis, TNM and advanced BCLC stage was higher in high HBV-DNA (baseline HBV-DNA>1000 U/ml, n=147) group comparing with the low HBV-DNA (baseline HBV DNA≤1000 u/ml, n=256) group (P<0.05). There was no significant difference in baseline indexes between the two groups after PSM. In 290 patients after PSM, there was no significant difference in the incidence of liver damage and HBVr between high HBV-DNA group and low HBV-DNA group (P>0.05). Survival analysis was performed on 169 patients with survival data, the median overall survival (OS) was found to be 11.49 months (95%CI: 7.77-12.89) and 16.65 months (95%CI: 10.54-21.99, P=0.008) in the high and low HBV-DNA groups, respectively. And median progression-free survival (PFS) was 7.41 months (95%CI: 5.06-8.67) and 10.55 months (95%CI: 6.72-13.54, P=0.038), respectively, with a statistically significant difference. There were no differences in overall survival (OS) and progression-free survival (PFS) between patients with and without HBVr and those with or without liver damage (P>0.05). Conclusions: HBV-DNA levels above 1 000 U/ml before systemic therapy do not increase the risk of liver damage or HBVr during systemic therapy in patients with HBV-related hepatocellular carcinoma, and such patients can safely receive systemic therapy.

The occurrence of immune-related adverse events is an independent risk factor both for serum HBsAg increase and HBV reactivation in HBsAg-positive cancer patients receiving PD-1 inhibitor combinational therapy.

Background: Previous studies have suggested the potential of PD-1/PD-L1 inhibitors in the treatment of chronic HBV infection. However, since phase III clinical trials have not yet been announced, additional clinical insights may be obtained by observing changes in serum hepatitis B surface antigen (HBsAg) and HBV-DNA levels in cancer patients undergoing PD-1 inhibitor therapy. Objective: To explore the effects of PD-1 inhibitor combinational therapy on serum HBsAg and HBV-DNA levels, investigate the incidence of HBsAg loss, HBV reactivation (HBVr), and immune-related adverse events (irAEs), and identify the risk factors associated with significant HBsAg fluctuations and HBVr. Methods: A retrospective study including 1195 HBsAg-positive cancer patients who received PD-1 inhibitors between July 2019 and June 2023 was conducted, and 180 patients were enrolled in this study. Serum HBsAg levels before and after PD-1 inhibitor administration were compared across different subgroups. The Pearson χ2 or Fisher exact test was performed to investigate the relationships between categorical variables. Univariable and multivariable analysis were performed to identify the risk factors associated with significant HBsAg fluctuations and HBVr. Results: With the concurrent use of antiviral agents, serum HBsAg levels decreased (Z=-3.966, P < 0.0001) in 129 patients and increased (t=-2.047, P=0.043) in 51 patients. Additionally, 7 patients (3.89%) achieved serum HBsAg loss. Virus replication was suppressed in most of the enrolled patients. When divided patients into different subgroups, significant HBsAg decreases after PD-1 inhibitor administration were discovered in lower baseline HBsAg group (Z=-2.277, P=0.023), HBeAg-seronegative group (Z=-2.200, P=0.028), non-irAEs occurrence group (Z=-2.007, P=0.045) and liver cancer group (Z=-1.987, P=0.047). Of note, 11 patients and 36 patients experienced HBVr (6.11%) and irAEs (20%), respectively, which could lead to discontinuation or delayed use of PD-1 inhibitors. After multivariable analysis, HBeAg-seropositive (OR, 7.236 [95% CI, 1.757-29.793], P=0.01) and the occurrence of irAEs (OR, 4.077 [95% CI, 1.252-13.273], P=0.02) were identified as the independent risk factors for significant HBsAg increase, the occurrence of irAEs (OR, 5.560 [95% CI, 1.252-13.273], P=0.01) was identified as the only independent risk factor for HBVr. Conclusion: PD-1 inhibitors combined with nucleos(t)ide analogues (NAs) may exert therapeutic potential for chronic HBV infection in cancer patients. However, attention also should be paid to the risk of significant elevation in HBsAg levels, HBVr, and irAEs associated with PD-1 inhibitor combinational therapy.

The significance of detecting HBV pgRNA and HBcrAg in HBV patients treated with NAs.

The value of detecting hepatitis B virus (HBV), pregenomic RNA (pgRNA), and hepatitis B core-related antigen (HBcrAg), both separately and jointly, in the management of HBV patients undergoing treatment with Nucleotide Analog was investigated. A total of 149 HBV patients who were being treated with Nucleotide Analog were enrolled in this study. The quantitative levels of HBV pgRNA and HBcrAg in the sera of these patients were determined, aiming to comprehend their replication levels and expression during the course of antiviral therapy. The patients were separated into 3 groups based on treatment duration: treatment time ≤ 12 months, treatment time ranging from 12 months to <60 months, and treatment time ≥ 60 months. Significantly different levels of HBcrAg and HBV pgRNA were observed among 3 groups (P < .05). In the group of patients with positive hepatitis B e antigen, both HBcrAg and pgRNA levels were higher compared to the group with negative hepatitis B e antigen, and this difference between the 2 groups was found to be statistically significant. Stratified analysis based on levels of hepatitis B surface antigen (HBsAg) revealed that the group with HBsAg levels < 100 IU/mL had lower levels of both HBcrAg and pgRNA compared to the group with HBsAg levels ≥ 100 IU/mL (P < .001). Following antiviral therapy, various degrees of transcription of covalently closed circular DNA continue to exist within the liver of HBV patients. The levels of serum HBcrAg and HBV pgRNA vary among patients with different treatment durations, indicating their efficacy in evaluating disease conditions during antiviral therapy.

Significance of hepatitis B virus capsid dephosphorylation via polymerase.

BACKGROUND: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.