Sample Size Determination And Sampling Procedure
The sample size was estimated by using the formula described by Cochran. A prevalence of HBV of 50% for no previously reported prevalence was used. Additionally, a standard normal deviation corresponding to the critical region of 1.96 at 5% precision was used. After correcting for the 10% loss due to unclear sample, a sample size of 394 was found to be sufficient. However, to raise the statistical power, an overall sample size of 424 participants was used. Purposive sampling was performed and any HBeAg sero-negative hospital attendee after screening was eligible for inclusion in the study.
Hepatitis B Serologic Testing Methods
Luke Dang, MD, Scott Bainbridge, CLS, Nam Tran, PhD
Introduction
Hepatitis B virus was first discovered in 1965. Briefly, HBV is a DNA virus from the Hepadnaviridae family which is spread via contaminated body fluids. After exposure, the virus enters hepatocytes and integrates its circular, partially double-stranded DNA genome to the host cell nucleus as a covalently closed circular DNA intermediate, which acts as a stable nuclear template for viral replication. This mechanism enables the virus to chronically infect the host and reactivate at a later date ). Reverse transcription of the cccDNA then results in assembly and exocytosis of new viral particles. Hepatitis B exhibits geographic variation and is classified into 9 genotypes , with some differences in disease manifestations, although this remains an area of ongoing research.
Clinical Manifestations and Epidemiology
Diagnostic Serologic Testing for Hepatitis B
Hepatitis B screening should be performed on patients with signs and symptoms suggestive of acute or chronic hepatitis as well as asymptomatic individuals with a history of or high risk of exposure or those at high risk of complications .
Table 1. Interpretation of Hepatitis B Serological Results. Adapted from UpToDate. Hepatitis B virus: Screening and diagnosis. Terrault NA, Lok ASF, McMahon BJ, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 2018 67:1560.
Hepatology
References
Diagnosis Of Hepatitis B Infection
Acute hepatitis B is a clinical diagnosis identified by the detection of HBsAg, symptoms, high serum aminotransferases. Usually anti-HBc IgM can be detected and HBV DNA is present. HBeAg can also be identified in most acute phase of infections, but has little clinical importance. The diagnosis of chronic infection is based on the persistence of HBsAg for more than 6 months. Patients with chronic HBV infection are commonly diagnosed by laboratory means but not by clinical presentations. Past HBV infection is defined by the coexistence of anti-HBs and IgG anti-HBc.
Occult HBV infection is defined by persistence of low level of intrahepatic HBV DNA without detectable HBsAg . It is a serological situation defined by the presence of isolated anti-HBc with the absence of HBsAg and anti-HBs antibody . The detection of HBV DNA in the liver is the gold standard of diagnosis for occult HBV infection, since cccDNA remains in the hepatocytes and HBV DNA is occasionally identified in the liver but not in the serum. However, gaining hepatic HBV DNA is difficult in clinical setting since the procedure is invasive. Real-time PCR for serum HBV DNA detection have been shown with adequate sensitivity to identify occult HBV infection in many cases thus, HBV DNA testing is widely used to diagnose occult HBV infection .
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Study Design And Populations
We conducted a cross-sectional retrospective study with quantitative methods of data collection and analysis. Adult participants were recruited from among outpatients coming to Kibuku Health Center IV for HBV screening before immunization during massive screening and vaccination against HBV as part of the government program to reduce HBV infection. These came from Kibuku District and the neighboring districts of Butebo, Budaka, Butaleja, Namutumba and Pallisa. Sampling was done from January 2020 to September 2020.
Serological Markers For Hbv Infection
Serological markers for HBV infection consist of HBsAg, anti-HBs, HBeAg, anti-HBe, and anti-HBc IgM and IgG. The identification of serological markers allows: to identify patients with HBV infection to elucidate the natural course of chronic hepatitis B to assess the clinical phases of infection and to monitor antiviral therapy .
HBsAg is the serological hallmark of HBV infection. After an acute exposure to HBV, HBsAg appears in serum within 1 to 10 weeks. Persistence of this marker for more than 6 months implies chronic HBV infection . Several studies have reported the association between transcription activity of cccDNA in the liver and serum HBsAg levels . Differences in the serum HBsAg levels during the different phases of infection indicate the distribution of cccDNA during the respective phases of the disease. The serum HBsAg titers are higher in patients with HBeAg-positive CHB than in HBeAg-negative CHB . Monitoring of quantitative HBsAg levels predicts treatment response to interferon and disease progression in HBeAg-negative CHB patients with normal serum alanine aminotransferase levels .
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Molecular Methods For Hbv Infection
HBV DNA is a direct measurement of the viral load, which reveals the replication activity of the virus. It is detectable at the early stage of infection and increases up to peak level approximately 3 months after the exposure to HBV and then gradually diminishes in chronic infection or disappears at the recovery from HBV infection.
As the prevalence of serologically negative HBV infection has increased, HBV-DNA detection has obtained more awareness in clinical medicine . The detection of HBV DNA is a reliable marker of replication activity, and higher titers of HBV DNA are related to the more rapid disease progression and higher incidence of HCC . Furthermore, HBV DNA testing is useful in routine clinical setting to determine patients who need antiviral therapy and monitor them for suitable treatment .
There are two principles of techniques to identify and quantify HBV DNA: signal amplification such as hybrid capture and branched DNA technology target amplification such as polymerase chain reaction . Real-time PCR can detect wide dynamic range of viral load . For this reason, it has come to be the standard method to detect and quantify HBV DNA in clinical setting. Furthermore, it can be fully automated and does not generate carry-over contamination . Table 1 displays the comparison of assays for quantitative measurement of HBV DNA.
Demographic Characteristics And Risk Factors For Infection With Hepatitis B Virus
As presented in Table , we had more female and married participants aged 30 years and above .
Table 1 Summary of the sociodemographic characteristics of the study participants.
A total of 1273 reported for screening for HBsAg as a prerequisite for immunization against HBV over a period of 9 months at Kibuku Health center IV, of which 624 were below the age of consent and 182 turned down subsequent recruitment in the study. Forty-three participants were HBsAg seropositive representing a prevalence of 9.2% and 424 were HBsAg seronegative. There was 100% concordance between the two screening tests .
The HBsAb, HBeAb, HBcAb seropositivity was detected among 48, 73 and 45 participants respectively. In contrast, 327 of the participants did not present with any marker of past exposure to HBV as an infcetion or as a vaccine. .
Figure 2
Representative results of the one step hepatitis B virus combo test cassette for the HBV serological markers among the HBsAg negative participants.
Interestingly, 3, 26 and 3 were simultaneously positive for the two markers of HBsAb/HBeAb, HbeAb/HBcAb and HBsAb/HBcAb respectively. In addition, 12 were concomitantly positive for the three markers of HBsAb/HBeAb/HBcAb .
Table 2 Outline of the HBV serological profile of HBsAg seronegative participants screened from Kibuku Health center IV.Table 3 Socio-demographic factors associated with HBsAb serostatus among HBsAg seronegative hospital attendees screened for HBV before immunization.
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Diagnosis Of Hepatitis B
Jeong Eun Song, Do Young Kim
Department of Internal Medicine, Yonsei University College of Medicine, Seoul, Korea
Contributions: Conception and design: DY Kim Administrative support: None Provision of study materials or patients: None Collection and assembly of data: None Data analysis and interpretation: None Manuscript writing: All authors Final approval of manuscript: All authors.
Correspondence to:
Abstract: Hepatitis B virus infection is a major global health problems leading to severe liver disease such as cirrhosis and hepatocellular carcinoma . HBV is a circular, partly double-stranded DNA virus with various serological markers: hepatitis B surface antigen and anti-HBs, anti-HBc IgM and IgG, and hepatitis B e antigen and anti-HBe. It is transmitted by sexual, parenteral and vertical route. One significant method to diminish the burden of this disease is timely diagnosis of acute, chronic and occult cases of HBV. First step of HBV diagnosis is achieved by using serological markers for detecting antigens and antibodies. In order to verify first step of diagnosis, to quantify viral load and to identify genotypes, quantitative or qualitative molecular tests are used. In this article, the serological and molecular tests for diagnosis of HBV infection will be reviewed.
Keywords: Hepatitis B virus serology molecular diagnosis
Submitted Aug 01, 2016. Accepted for publication Aug 28, 2016.
doi: 10.21037/atm.2016.09.11
Hepatitis B Dna Viral Load
Of 308 patients with a positive HBsAg result, 270 with available samples were tested for HBV DNA viral load. A total of 56 had undetectable DNA, 38 had DNA detectable but below the level of quantification, and 176 had a quantifiable level of DNA. The detection of HBV DNA viral load was strongly linked to HBeAg status, 96% of HBeAg-positive and 70% of HBeAg-negative participants having detectable levels .
Figure Figure22 shows the distribution of HBV DNA viral loads, by country and HBeAg status, in terms of the percentage of samples that exceed a given concentration. A vertical line is drawn at 2000IU/ml, the threshold for initiating anti-HBV treatment according to some guidelines . Overall, 92% of HBeAg-positive and 28% of HBeAg-negative participants had HBV DNA viral load more than 2000IU/ml. As expected, HBV DNA levels were generally high among HBeAg-positive patients irrespective of clinical site, 53% having a value greater than 1×108IU/ml. Values were spread more uniformly among HBeAg-negative patients and appeared on average to be lower in Zimbabwean than in Ugandan patients.
Hepatitis B DNA viral load by hepatitis B e antigen status.
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Deterrence And Patient Education
Patient education remains one of the most important components in preventative measures regarding HBV infection.
Education should be provided to expecting parents about the importance of vaccination and to clarify erroneous beliefs about vaccinations.Patient education should also include counseling about the avoidance of risky behaviors that predispose an individual to be infected, including promiscuous sexual activity or intravenous drug abuse. They should also be advised not to share items such as shaving razors, toothbrushes, or hair combs due to possible transmission via mucosal contact or through microtrauma to protective barriers.
Enhancing Healthcare Team Outcomes
As hepatitis B infection is highly transmissible via accidental needlesticks, healthcare providers involved in taking care of a patient with HBV should exercise caution and practice proper preventative measures such as vaccination. Patient education should also include counseling about HBV transmission. The interprofessional team’s role is crucial in ensuring the best patient outcomes.
The vaccination rate is low in many developing countries, and the majority of patients are undiagnosed. Educational programs and improved awareness among the general public and healthcare providers are necessary to improve the identification of the patients, reduce transmission of the disease, and reduce the complications of hepatitis B infection.
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Pearls And Other Issues
Hepatitis D has been long associated with HBV infections and cannot exert pathological influence without the presence of HBV infection. Two forms of infection exist coinfection and superinfection . Superinfection tends to be more severe than coinfection. Due to the preexisting hepatitis B infection, anti-HBcAg IgM is undetectable in superinfection states but can be noted in coinfection.
Diagnosis Of Acute And Chronic Hepatitis B
HBsAg is the first serologic marker to appear and may be detected within 1 to 2 weeks after exposure. It precedes the development of symptoms by an average of 4 weeks.106 The presence of HBsAg indicates ongoing infection. Qualitative but not quantitative methods are used by most clinical laboratories because the amount of antigen does not correlate with disease activity or with the presence of an acute or chronic infection.28 Some symptomatic patients may have self-limited, acute HBV infection without detectable HBsAg. These patients, up to 9% in some studies, have other detectable markers of infection.106 HBeAg appears virtually simultaneously, peaks, and then declines in parallel with HBsAg. It usually disappears before HBsAg does. Adult patients who remain persistently positive for HBeAg for more than 10 weeks are likely to become chronically infected. HBeAg indicates a high level of viral replication and infectivity. Most patients with nondetectable HBeAg have resolving, minimal, or no active liver disease.28 Precore mutants of HBV do not express HBeAg they may be responsible for a more severe course and, in some cases, fulminant disease. Serum aminotransferase levels become elevated but are nonspecific. They begin to increase just before the development of symptoms and then peak with the development of jaundice.
Howard C. Thomas, Jennifer A. Waters, in, 1998
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Interpreting The Hepatitis B Serologic Panel
The hepatitis B blood tests are collectively known as the serologic panel. This set of tests can accurately diagnose current and past hepatitis B infection. Since there are a number of markers and at least six interpretations of the various results, determining their meaning can be challenging. To help clarify, below are the six interpretations organized in a table from these hepatitis B markers.
Data Presentation And Statistical Analysis
Categorical data are presented as frequencies and percentages. Univariate analysis was used to determine the crude odds ratio , whereas multinomial logistic regression analysis was used to determine the adjusted odds ratio . All analyses were performed at the 95% level of significance, and p< 0.05 was considered to be statistically significant. Data were analyzed using SPSS version 26.
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Understanding Your Test Results
Understanding your hepatitis B blood tests can be confusing. It is important to talk to your health care provider so you understand your test results and your hepatitis B status. Are you infected? Protected? Or at risk? The Hepatitis B Panel of blood tests includes 3 tests and all three results must be known in order to confirm your status.
Below is a chart with the most common explanation of the test results, but unusual test results can occur. Please note that this chart is not intended as medical advice, so be sure to talk to your health care provider for a full explanation and obtain a printed copy of your test results. In some cases, a person could be referred to a liver specialist for further evaluation.
More Detailed Information About Hepatitis B Blood Tests
An acute hepatitis B infection follows a relatively long incubation period – from 60 to 150 days with an average of 90 days. It can take up to six months, however, for a person to get rid of the hepatitis B virus. And it can take up to six months for a hepatitis B blood test to show whether as person has recovered from an acute infection or has become chronically infected .
The following graphic from the U.S. Centers for Disease Control and Prevention represents the typical course of an acute hepatitis B infection from first exposure to recovery.
According to the CDC, a hepatitis B blood test result varies depending on whether the infection is a new acute infection or a chronic infection.
Study Area And Population
The study was conducted in Kaduna Metropolis, Kaduna State, Nigeria between August and December 2011. The state covers an area of approximately 45,567 km2, and has an estimated population of more than 6 million people, with three senatorial zones and 23 Local Government Areas. The climatic conditions in Kaduna State are tropical with well-defined wet and dry season. The study population consisted of 800 pregnant women at various trimesters of pregnancy seen at the point of booking to attend ante-natal care in four selected hospitals in Kaduna Metropolis. The hospitals included 44 Nigerian Army Reference Hospital, St. Gerard Hospital, Barau Dikko Specialist Hospital, and Yusuf Dantsoho Memorial Hospital. Informed consent was obtained from each participant and ethical approval was obtained from the Ethical Committee of each of these hospitals. A self-designed semi-structured questionnaire was used to collect demographic data, clinical information, and risk factors associated with HBV infection. A questionnaire was administered to each pregnant woman prior to sample collection.
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Laboratory Findings And Diagnostic Tests
Serum HBsAg and anti-HBs are the most useful screening tests for chronic HBV infection or immunity to HBV. HBsAg is present in most chronically infected persons. Lack of anti-HBs in an unvaccinated HBsAg-negative person indicates susceptibility to HBV infection.3
Screening for HBsAg is recommended at the first prenatal visit for all pregnant women.3,109 Women in labor without HBsAg test information should have HBsAg serology on arrival. In addition, pretested women who have a history of certain risk factors should be retested at the time of admission to the hospital for delivery.3,110
Fabrizio Fabrizi MD, … Paul Martin MD, in, 2017
Data Collection And Blood Sampling
Demographic characteristics and predictors of HBV infection were collected by using a close-ended questionnaire administered by a nurse. In addition to the demographic characteristics of age, sex and marital status, the participants were asked whether they had ever transfused blood, consumed alcohol or had lived with HBV-infected persons before. The questionnaire was administered by a nurse or research assistant on site. For laboratory investigations, 4 mL of blood was drawn by vein puncture into anticoagulant vacutainers from which serum was obtained. The serum was kept in sterile viols and stored at 20 °C until further use.
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Interpretation Of Diagnostic Tests
Hepatitis B surface antigen is the first marker of HBV detectable in serum in acute infection. By the time clinical and biochemical hepatitis is present after an incubation period of up to 140 days, other serologic markers of HBV infection appearâincluding antibody to HBV core antigen . Hepatitis B core antigen, a marker of viral replication found in infected hepatocytes, does not circulate in serum. However, its corresponding antibody does. Documented HBsAg positivity in serum for 6 or more months suggests chronic HBV with a low likelihood of subsequent spontaneous resolution. Chronic HBV is diagnosed by the absence of IgM anti-HBc antibody. IgM anti-HBc antibody is a marker of acute or recent acute hepatitis B and is detectable for 6 months after infection, whereas IgG anti-HBc is lifelong. If acute HBV resolves, neutralizing antibody against HBsAg develops. If HBV infection becomes chronic, other HBV markersâincluding HBV viremia and hepatitis e antigen âshould be sought. Both of these markers imply viral replication and thus greater infectivity, although any patient who is HBsAg positive is potentially infectious.
Rima Fawaz, Maureen M. Jonas, in, 2021