Differentiation of exhausted CD8+ T cells after termination of chronic antigen stimulation stops short of achieving functional T cell memory.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8+ T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory.

Early Transcriptional Divergence Marks Virus-Specific Primary Human CD8+ T Cells in Chronic versus Acute Infection.

Distinct molecular pathways govern the differentiation of CD8+ effector T cells into memory or exhausted T cells during acute and chronic viral infection, but these are not well studied in humans. Here, we employed an integrative systems immunology approach to identify transcriptional commonalities and differences between virus-specific CD8+ T cells from patients with persistent and spontaneously resolving hepatitis C virus (HCV) infection during the acute phase. We observed dysregulation of metabolic processes during early persistent infection that was linked to changes in expression of genes related to nucleosomal regulation of transcription, T cell differentiation, and the inflammatory response and correlated with subject age, sex, and the presence of HCV-specific CD4+ T cell populations. These early changes in HCV-specific CD8+ T cell transcription preceded the overt establishment of T cell exhaustion, making this signature a prime target in the search for the regulatory origins of T cell dysfunction in chronic viral infection.

Seroprevalence of hepatitis B, C, and D and associated factors in the semi-isolated Yanomami Amazonian indigenous community.

BACKGROUND: Viral hepatitis is a significant health concern among indigenous population in the Americas. In Brazil, reports find high endemicity of HBV and HDV infections has been reported in several indigenous groups. However, few studies have documented the prevalence of HBV, HCV and HDV in the Yanomami. In this study, the prevalence of hepatitis B, C, and D serological markers and potential risk factors were investigated to provide guidance for the development of strategies aimed at reducing viral transmission in the Yanomami indigenous villages. METHODS: This cross-sectional study was carried out in March 2015 and included 430 individuals from four Yanomami villages: Alapusi (n = 78), Castanha/Ahima (n = 126), Gasolina (n = 105), and Taibrapa (n = 121). A rapid test was used for detection of HBsAg and anti-HCV and chemiluminescent immunoassay for anti-HBs, anti-HBc, and anti-HDV antibodies. RESULTS: HBsAg, anti-HBc, and anti-HBs were detected in 8.8, 45.5, and 49.4% of the participants, respectively. The estimated HBV status: current infection 9.6% (38/395); resolved infection 43.3% (171/395); vaccine immunity 20.5% (81/395), and susceptible to HBV 26.6% (105/395). Gasolina presented the lowest prevalence of HBV infection (6.5%) and the highest prevalence of vaccine immunity (26.9%). Children < 15 years old were highly susceptible to infection, as 53.1% did not have antibodies to HBV, while more than 80% of individuals over 45 years of age had been exposed to HBV. The markers for HDV were founded among 12.5% (4/32) of the HBsAg carriers. Anti-HCV was identified in all villages, with the highest prevalence in Alapusi (5.1%). Possible risk factors such as the use of piercings, tattoos, and contact with prospectors showed no statistical difference between the groups. CONCLUSIONS: Viral hepatitis B and serological markers for HCV and HDV were found to be widely distributed among the Yanomami indigenous community, while the prevalence of vaccine immunity to HBV was low. This finding reinforces the importance of promoting systematized diagnostic and vaccination strategies in indigenous communities. Our data confirm that isolated and difficult-to-reach indigenous communities lack appropriate access to diagnosis, treatment, and vaccination.

The Inflammatory Cytokine Profile Associated With Liver Damage Is Broader and Stronger in Patients With Chronic Hepatitis B Compared to Patients With Acute Hepatitis B.

Liver damage in hepatitis B is immune driven and correlates with inflammatory markers in patient serum. There is no comparison of these markers to determine if inflammatory profiles are distinct to different types of liver damage across patients at different stages of disease. We measured 25 inflammatory markers in patients with acute hepatitis B and chronic hepatitis B with hepatitis B e antigen seroconversion and chronic patients stopping nucleoside analogue therapy. Myeloid markers dominated the inflammatory profile in all stages of hepatitis B. More inflammatory markers were detectable in chronic patients, including elevated concentrations of cytotoxic effectors Fas ligand, TRAIL, and TNF-α.

Hepatitis B virus-specific CD4 T cell responses differentiate functional cure from chronic surface antigen+ infection.

BACKGROUND & AIMS: With or without antiviral treatment, few individuals achieve sustained functional cure of chronic hepatitis B virus (HBV) infection. A better definition of what mediates functional cure is essential for improving immunotherapeutic strategies. We aimed to compare HBV-specific T cell responses in patients with different degrees of viral control. METHODS: We obtained blood from 124 HBV-infected individuals, including those with acute self-limiting HBV infection, chronic infection, and chronic infection with functional cure. We screened for HBV-specific T cell specificities by ELISpot, assessed the function of HBV-specific T cells using intracellular cytokine staining, and characterized HBV-specific CD4 T cells using human leukocyte antigen (HLA) class II tetramer staining, all directly ex vivo. RESULTS: ELISpot screening readily identified HBV-specific CD4 and CD8 T cell responses in acute resolving infection compared with more limited reactivity in chronic infection. Applying more sensitive assays revealed higher frequencies of functional HBV-specific CD4 T cells, but not CD8 T cells, in functional cure compared to chronic infection. Function independent analysis using HLA multimers also identified more HBV-specific CD4 T cell responses in functional cure compared to chronic infection, with the emergence of CD4 T cell memory both after acute and chronic infection. CONCLUSIONS: Functional cure is associated with higher frequencies of functional HBV-specific CD4 memory T cell responses. Thus, immunotherapeutic approaches designed to induce HBV functional cure should also aim to improve CD4 T cell responses. LAY SUMMARY: Immunotherapy is a form of treatment that relies on harnessing the power of an individual's immune system to target a specific disease or pathogen. Such approaches are being developed for patients with chronic HBV infection, in an attempt to mimic the immune response in patients who control HBV infection spontaneously, achieving a so-called functional cure. However, what exactly defines protective immune responses remains unclear. Herein, we show that functional cure is associated with robust responses by HBV-specific CD4 T cells (a type of immune cell).

Immunological and virological aspects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hepatitis C virus.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can generate a systemic inflammatory response, characterized by a cytokine storm and associated with an exaggerated release of proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-17, all of which can affect the liver. Here, we aimed to evaluate the cytokine profiles of patients suffering from coronavirus disease (COVID)-19 and/or hepatitis. We subjected 87 patients to serology and/or polymerase chain reaction analysis for the hepatitis C virus. They were also tested for TNF-α, IL-6, and IL-17 using commercial immunoassay kits. The test results of the COVID-19/hepatitis C patients (n = 8) were compared with that of the negative controls (n = 28), hepatitis C patients (n = 29), and COVID-19 patients (n = 22). All COVID-19 patients (mono- and coinfected) expressed high levels of cytokines. The COVID-19/hepatitis patients exhibited higher levels of IL-6 (6.33 ± 3.9 pg/ml) and IL-17 (102.23 ± 2.7 pg/ml); however, TNF-α values were lower (68.08 ± 15.88 pg/ml), as compared with that of the hepatitis patients (p < 0.001), and lower than that of the COVID-19 patients and exceptionally for TNF-α (p < 0.05). These data highlight the importance of monitoring patients with hepatitis and COVID-19.

Cytokines profile in patients with acute and chronic hepatitis B infection.

Hepatitis B virus (HBV) is one of the leading causes of acute and chronic hepatitis and represents a serious public health threat. Cytokines are important chemical mediators that regulate the differentiation, proliferation, and function of immune cells, with accumulating evidence indicating that the inadequate immune responses are responsible for the elimination or persistence of HBV. This study aimed to determine the cytokine profiles (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-10, and IL-17A) during HBV infection and investigate their association with genotypes. A total of 66 plasma samples, 19 from patients with acute and 47 with chronic hepatitis B infection, were subjected to biochemical tests, nested-PCR, and real-time PCR, with cytokines evaluated using a commercial BD Cytometric Bead Array Human Th1/Th2/Th17 Cytokine Kit. Healthy controls (10 individuals) were selected from blood donors with no history of liver diseases. No correlation was found between genotypes, viral load, and cytokines analyzed. All cytokines showed higher levels of production among infected individuals when compared with the control group. A positive correlation classified as moderate to strong was found between cytokines IFN-γ, TNF, IL-10, IL-6, IL-4, and IL-2 through the Spearman correlation coefficient. TNF (P = 0.009), IL-10 (P < 0.001), and IL-6 (P < 0.001) levels were higher in acute individuals compared with chronic and control groups. Theses cytokines could be involved in the elimination of virus and protection against chronicity.

Feasibility of dried blood spot for hepatitis C diagnosis in vulnerable subjects and people living in remote areas from Brazil.

BACKGROUND: Agile, accessible and cheap diagnosis of hepatitis C virus (HCV) infection is essential to achieve the elimination of this infection, worldwide, as mandated by the World Health Organzation as part of its strategy for 2030. Dried blood spots (DBS) can be an attractive alternative for sample collection among people living in remote areas and vulnerable populations due to the less invasive collection, its biosafety, and storage & transportation of samples at room temperature. DESIGN: This study aims to estimate the usefulness of dried blood spot samples for the diagnosis and the assessment of HCV infection rates in three different settings in Brazil. Cross-sectional analysis of a sample collection from different populations, aiming to assess the performance of the testing algorithms and respective procedures among different populations with diverse background infection rates. METHODS: We reported the evaluation of DBS as alternative samples for detecting anti-HCV in different groups in real life conditions: (I) Vulnerable subjects living in remote areas of Southeast, North and Northeast Brazil (n = 1464); (II) Beauticians (n = 288); (III) People who use non-injectable drugs (n = 201); (IV) patients referred to outpatient care (n = 275). RESULTS: General assay accuracy was 99%, with a weighted kappa value of 0.9, showing an excellent performance. Sensitivities ranged from 87.5% to 100.0% between groups and specificities were above 99.2%. A total of 194 individuals had HCV RNA in serum and concordance of anti-HCV detection in DBS was 98.4%. CONCLUSIONS: DBS samples could be used for anti-HCV detection in different populations recruited in real life conditions and ambulatory settings, with a high overall sensitivity and specificity.

A Systematic Evaluation of IgM and IgG Antibody Assay Accuracy in Diagnosing Acute Zika Virus Infection in Brazil: Lessons Relevant to Emerging Infections.

Accurate diagnostics underpin effective public health responses to emerging viruses. For viruses, such as Zika virus (ZIKV), where the viremia clears quickly, antibody-based (IgM or IgG) diagnostics are recommended for patients who present 7 days after symptom onset. However, cross-reactive antibody responses can complicate test interpretation among populations where closely related viruses circulate. We examined the accuracy (proportion of samples correctly categorized as Zika positive or negative) for antibody-based diagnostics among Brazilian residents (Rio de Janeiro) during the ZIKV outbreak. Four ZIKV enzyme-linked immunosorbent assays (ELISAs; IgM and IgG Euroimmun, IgM Novagnost, and CDC MAC), two dengue ELISAs (IgM and IgG Panbio), and the ZIKV plaque reduction neutralization test (PRNT) were evaluated. Positive samples were ZIKV PCR confirmed clinical cases collected in 2015-2016 (n = 169); negative samples (n = 236) were collected before ZIKV was present in Brazil (≤2013). Among serum samples collected ≥7 days from symptom onset, PRNT exhibited the highest accuracy (93.7%), followed by the Euroimmun IgG ELISA (77.9%). All IgM assays exhibited lower accuracy (<75%). IgG was detected more consistently than IgM among ZIKV cases using Euroimmun ELISAs (68% versus 22%). Anti-dengue virus IgM ELISA was positive in 41.1% of confirmed ZIKV samples tested. The Euroimmun IgG assay, although misdiagnosing 22% of samples, provided the most accurate ELISA. Anti-ZIKV IgG was detected more reliably than IgM among ZIKV patients, suggesting a secondary antibody response to assay antigens following ZIKV infection. Antibody ELISAs need careful evaluation in their target population to optimize use and minimize misdiagnosis, prior to widespread deployment, particularly where related viruses cocirculate.

Applicability of oral fluid samples for tracking hepatitis B virus mutations, genotyping, and phylogenetic analysis.

Little is known about the usefulness of saliva samples for hepatitis B virus (HBV) genotyping and mutation analysis. The aim of this study was to evaluate the usefulness of oral fluid samples to determine HBV genotype distribution, S/polymerase mutations, and HBV subpopulation diversity among chronically HBV-infected individuals. Serum and oral fluid samples were obtained from 18 individuals for PCR and nucleotide sequencing of the HBV surface antigen gene. Biochemical analysis of liver enzymes (ALT, AST, GGT) and HBV, HCV, and HIV serological tests were also performed. All serum samples were HBsAg (+), anti-HBc (+), and anti-HBs (-); 55.6% were HBeAg (+)/anti-HBe (-), and 11.1% were anti-HIV (+). The mean HBV DNA viral load was 6.1 ± 2.3 log IU/mL. The HBV genotype distribution was as follows: A, 72.2%; D, 11.1%; E, 5.6%; F, 11.1%. A concordance of 100% in genotype classification and 99.8% in sequence similarity between paired oral fluid and serum samples was observed. HBsAg mutations were detected in all samples, but no resistance mutations were found in the polymerase gene. This study demonstrates that oral fluid samples can be used reliably for tracking HBV mutations, genotyping, and phylogenetic analysis. This could be important for molecular epidemiology studies with hard-to-reach populations.

Analysis of Humoral Immune Responses in Chikungunya Virus (CHIKV)-Infected Patients and Individuals Vaccinated With a Candidate CHIKV Vaccine.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu-like symptoms. The acute symptoms disappear after 1 week, but chronic arthralgia can persist for years. In this study, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed. METHODS: Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors, and antibody epitope mapping was performed with a peptide array. RESULTS: The greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies and their binding avidity and cross-reactivity with other alphaviruses increased over time. Chikungunya virus and o'nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2- to 6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV, and the E2 ASR1 was the major antibody target. CONCLUSIONS: These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy.

Phenotype and function of HBV-specific T cells is determined by the targeted epitope in addition to the stage of infection.

OBJECTIVE: Chronic HBV infection affects more than 250 million people worldwide and remains a global healthcare problem in part because we lack curative treatment. Sustained viral control requires HBV-specific T cells, but these become functionally impaired in chronic infection. Clinical evidence indicates that functional cure of HBV infection by the host immune response is feasible. Developing T cell-based therapies able to achieve functional cure will require identification of the requirements for a successful T cell response against HBV and the relative contribution of individual T cell specificities to HBV control. DESIGN: The phenotype and function of HBV-specific T cells were studied directly ex vivo using fluorochrome-labelled multimers. We studied multiple HBV-specific T cell specificities targeting different HBV proteins in individuals with either an acute self-limiting or chronic HBV infection. RESULTS: We detected strong T cell responses targeting multiple HBV viral proteins in acute self-limiting and low-frequency core and polymerase-specific T cells in chronic infection. Expression of the T cell inhibitory receptor PD-1, as well as T cell differentiation, T cell function and T cell regulation differed by stages and outcomes of infection. In addition, these features differed significantly between T cells targeting different HBV specificities. CONCLUSION: HBV-specific T cells with different target specificities are characterised by distinct phenotypical and functional profiles. These results have direct implications for the design of immunological studies in HBV infection, and are potentially relevant for informing immunotherapeutic approaches to induce functional cure.

Utility of oral fluid samples for hepatitis B antibody detection in real life conditions.

BACKGROUND: Hepatitis B virus (HBV) testing in oral fluid samples may provide advantages in diagnosis, screening or prevalence studies, especially among individuals with venous access difficulties. This study aims to optimize one commercially available assay for detecting total anti-HBc marker in oral fluid samples and to evaluate its utility under real life conditions in different settings for the purposes of prevalence and diagnostic studies. METHODS: Oral fluid was collected using a Salivette device and some parameters were initially evaluated: type of elution buffer and sample volume. Thereafter, the utility of oral fluid samples for detection of anti-HBc was evaluated in real life conditions in which, 1296 individuals gave serum and oral fluid samples. All serum samples were submitted to commercial EIAs to detect total anti-HBc, according to the manufacturer's instructions and oral fluid samples according to previous optimization. RESULTS: In optimization evaluation, PBS/BSA 0.5% and 100 µL of oral fluid (volume was two-fold increased compared to serum in EIA) were chosen as transport buffer and sample volume. In the field study, anti-HBc was detected in 211 out of 1296 serum samples giving overall oral fluid sensitivity of 52.6% and specificity of 96%. Concordance was higher in ambulatory setting (67.7) compared to general population (31.8). Mean ± standard deviation values of optical density/cutoff (OD/CO) in serum samples were higher in false-negative oral fluid samples than those seen in true positive samples. Sensitivity was higher in those presenting active infection compared to anti-HBc isolate and past infection. Sensitivity also increased in the ambulatory group when HCV individuals were excluded. CONCLUSIONS: It was possible to optimize a commercial EIA for detecting anti-HBc in oral fluid samples and where the highest concordance was found in ambulatory settings and among individuals with active infection.

Assessing hepatitis B immunity using dried blood spot samples from HIV+ individuals.

This study aims to evaluate the utility of an optimized enzyme immunoassay (EIA) to detect and quantify antibodies against hepatitis B surface antibody (anti-HBs) in dried blood spots (DBSs) within the context of human immunodeficiency virus (HIV) status. Serum and DBS samples were obtained from 56 HIV+ and 99 HIV- patients and subjected to EIA for the detection of anti-HBs, where sample volume and cut off value were modified for DBS testing. Sensitivities of anti-HBs detection in DBS were 79.8% and 76.8% in HIV- and HIV+ subjects, respectively. Concordant results for anti-HBs in serum and DBS presented high mean CD8 cell counts, HIV viral load and optical density (OD) values of anti-HBs. Anti-HBs titers were significantly higher in serum, whether or not anti-HBs titers were detected in DBS. It was possible to detect anti-HBs in DBS as low as 17.4 and 27.3 IU/mL among HIV+ and HIV- subjects, respectively. In conclusion, DBS can be used to detect and quantify anti-HBs in HIV-infected individuals, which could increase access to diagnosis and vaccination.

Hepatitis D infection in Brazil: Prevalence and geographical distribution of anti-Delta antibody.

In Brazil, the Amazon Basin is endemic for hepatitis D virus (HDV) infection; however, studies in other regions of the country are scarce. This study aims to map the seroepidemiological situation of anti-Delta antibodies in chronic hepatitis B carriers in all five Brazilian geographic regions. Serum samples from 1240 HBsAg positive individuals (55.4% men; mean age 43.1 ± 13.4 years) from 24 of 26 Brazilian states were tested for the presence of anti-Delta antibodies using a commercial immunoassay. Anti-Delta antibodies were detected in 40 samples (3.2%; 52.5% female; mean age of 38.1 ± 13.8 years). Age less than 20 years was significantly associated with anti-HDV positivity (P < 0.001). The distribution of anti-Delta differed markedly in the diverse regions of the country. The highest prevalence of anti-HDV was found in the North (8.5%; P < 0.001), followed by Central West (2.5%), Southeast (1.7%), Northeast (0.8%), and South (0.0%). Anti-Delta antibodies were detected in 12 states, but more than 60% of the positive cases were observed in two states, Amazonas and Acre, located in the western portion of the Amazon region. The overall HDV prevalence of 3.2% emphasizes that HDV is far from being a disease under control in Brazil. Despite the low HDV prevalence in non-endemic regions, this infection persists as a major concern in two states (Acre and Amazonas) in the north of the country, indicating that a continuous epidemiological surveillance program should be implemented in all Brazilian regions.

Detection of occult hepatitis B in serum and oral fluid samples.

In occult hepatitis B infection (OBI), hepatitis B virus DNA (HBV DNA) can be detected in serum samples; however, oral fluid collection for detection of HBV DNA has not yet been explored, despite the availability of collection devices. Serum and oral fluid samples from 45 hepatitis B core antibody (anti-HBc)-positive patients were collected for the amplification of the HBV polymerase gene. HBV DNA was detected in five serum and four oral fluid samples (the detection limit for oral fluid was 1.656 log IU/mL in paired serum). In conclusion, simple methodologies of sample collection and in-house polymerase chain reaction (PCR) allowed detection of HBV DNA, and these could be used to improve the diagnosis of OBI, especially in locations with limited resources.

Comparison of the performance of enzyme immunoassays for hepatitis B and C detection in dried blood spot.

Dried blood spots (DBSs) could be an alternative to serum for hepatitis B virus (HBV) and hepatitis C virus (HCV) diagnosis. This study aims to evaluate two enzyme immunoassays (EIAs) for HBsAg and anti-HCV detection using DBS. Serum was tested using commercial EIA. DBS was tested using optimized EIA developed for serum and commercial EIA developed for DBS (Imunoscreen). Concordances between DBS and serum samples for both markers and EIAs were higher than 97%. Both EIAs demonstrated good performance for HBsAg and anti-HCV detection using DBS, and these methods could be used unchangeably increasing the access for HBV and HCV diagnosis.

Performance of ANTI-HCV testing in dried blood spots and saliva according to HIV status.

The use of saliva and dried blood spots (DBS) could increase access to HCV diagnosis for high-risk populations, such as HIV-infected individuals, but the performance of these assays has not been well established in this group. This study aims to evaluate HIV status, particularly TCD4+ cell count and viral load, in the performance of anti-HCV testing using DBS and saliva. A total of 961 individuals classified as HCV+, HIV+, or HIV/HCV+, as well as negative controls, donated serum, DBS, and saliva samples for anti-HCV testing using a commercial enzyme immunoassay. Sample volume was modified for DBS and saliva, and an ROC curve was used for cut-off determination in saliva. Anti-HCV sensitivities were greater than 93% using DBS and saliva in the HCV+ group, while they were 83.3% and 95.6% for HCV/HIV+ individuals for DBS and saliva assays, respectively. Specificity varied from 91.7% to 100% using saliva and DBS in HIV monoinfected and control subjects. When only anti-HCV/HCV RNA+ serum samples, that is, true positives, were considered, the sensitivities were 98.3% and 100% for DBS and saliva, respectively, in the HCV+ group and 91.6% and 94.8% for DBS and saliva, respectively, in the HIV/HCV+ group. High absorbance values were observed among those presenting with HCV RNA in serum and low HIV viral load (less than 50 copies/mL). In conclusion, DBS and saliva samples could be used for anti-HCV detection, particularly to identify active HCV cases, but low sensitivity was observed for anti-HCV testing using DBS in the HIV/HCV+ group.

Detection and molecular characterisation of a diagnosis escape variant associated with occult hepatitis B virus in Brazil.

BACKGROUND: Many studies have identified mutations in the hepatitis B surface antigen (HBsAg) as important factors limiting the ability of commercial serological assays to detect this viral antigen. However, an association between mutations in the HBsAg gene and the occurrence of occult HBV infection (OBI) in patients has not been established. OBJECTIVES: To detect hepatitis B virus (HBV) DNA in patients with anti-HBc as a unique serological marker, a previously published, cost-effective TaqMan-based real-time polymerase chain reaction (PCR) test with minor groove binding probes was adapted for use in this study. The current study also aimed to investigate HBsAg mutations and genotypes of HBV in OBI at the Viral Hepatitis Ambulatory Clinic in Rio de Janeiro to determine any possible association. METHODS: Intra-assay and inter-assay reproducibility were determined, and the mean coefficient of variation values obtained were 2.07 and 3.5, respectively. Probit analysis indicated that the 95% detection level was 25 IU/mL. The prevalence of OBI was investigated in 35 serum samples with an 'anti-HBc alone' profile from individuals who attended our clinic between 2011 and 2013. FINDINGS: HBV DNA was detected in only one sample, resulting in an OBI rate of 2.9%. Nucleotide sequencing of the pre-S/S region was performed to genotype and analyse mutations within the HBsAg gene of this HBV DNA. The HBV in the OBI case was classified as sub-genotype A1, and a sequence analysis of the small S gene revealed 12 mutations in the major hydrophilic region compared to the consensus A1 sequence. Most of these mutations occurred in amino acid residues that have been reported as clinically relevant because they have been implicated in vaccine escape and/or inability to detect HBsAg by commercial serological assays. MAIN CONCLUSIONS: Our study suggests the importance of specific HBsAg mutations, different from those in D, B, and C genotypes, in sub-genotype A1 HBV associated with OBI.

Poor sensitivity of rapid tests for the detection of antibodies to the hepatitis B virus: implications for field studies.

Rapid tests (RTs) can be used as an alternative method for the conventional diagnosis of hepatitis B virus (HBV). This study aims to evaluate antibodies to HBsAg (anti-HBs) and antibodies to HBeAg (anti-HBe) RTs under different Brazilian settings. The following three groups were included: GI: viral hepatitis outpatient services; GII: low resource areas; and GIII: crack users and beauticians. Imuno-rápido anti-HBsAg™ and Imuno-rápido anti-HBeAg™ RTs were evaluated and showed specificities greater than 95% in all groups. The sensitivity values to anti-HBs were 50.38%, 51.05% and 46.73% and the sensitivity values to anti-HBe were 76.99%, 10.34% and 11.76% in the GI, GII and GIII groups, respectively. The assays had a low sensitivity and high specificity, which indicated their use for screening in regions endemic for HBV.