CHAPTER ONE

INTRODUCTION

1.1 Introduction

Hepatitis B is a viral infection caused by the hepatitis B virus (HBV), an enveloped DNA virus that infects the human liver and causes hepatocellular necrosis and inflammation (Esum et al., 2019). It is transmitted through sexual intercourse, saliva exchange during kissing with an infected person, mother to child during childbirth, through the placenta and breastfeeding (Lok and McMahon 2009). HBV is a global health problem resulting to a remarkable human morbidity and mortality, predominantly through the consequences of chronic infection. As a result, HBV infection was ranked 15th among all causes of human mortality (Lozano et al., 2012). In 2010, 786,000 deaths were attributed to HBV, the vast majority (341,000 deaths) being attributable to liver cancer, 132,200 caused by acute hepatitis B and 312,000 deaths by cirrhosis (Lozano et al. 2012). It was estimated to cause about 2 billion infections with 257 million chronic case of the disease. The African and Western Pacific regions accounted for 68% of those infected and 2.7 million cases are co-infected with HIV (WHO, 2017). Most of the people currently living with HBV infection are persons born before hepatitis B vaccine was widely available and used in infancy. About 350 million people believed to be at risk of developing complications of chronic hepatitis such as cirrhosis and primary hepatocellular carcinoma (HCC) (Ul-Haq et al, 2013). In Africa and Asia, the prevalence of HBV is > 8% and 2 billion people have markers of current or past infection with HBV (Leung, 2009). Approximately 65 million of all chronically infected individuals live in Africa (Kramvis and Kew, 2007).

In Cameroon, the prevalence of HBV ranges from 6-16% (Noubiap et al., 2015). Recent studies report the prevalence of HBV to be as high as 10.1% and 12% among blood donors in hospital blood banks in Cameroon (Noubiap et al., 2013). Esum et al., 2019 reported 5.7% and 7.5% in the Limbe health district and Muyuka health district respectively.

Also Frambo et al., (2014) reported a 9.7% prevalence of HBsAg among pregnant women in the Buea health district. Globally HBV is a potential life-threatening cause of liver diseases resulting to killing of infected liver cells (Doo and Ghany, 2010). This infection can either be acute or chronic and may range from asymptomatic infection or mild disease to severe or rarely fulminant hepatitis. Acute hepatitis B infection is usually a self-limiting disease marked by acute inflammation and hepatocellular necrosis, with a case fatality rate of 0.5-1% (Lavanchy, 2004). Chronic hepatitis B infection encompasses a spectrum of disease and is defined as persistent HBV infection. It implies, the presence of detectable hepatitis B surface antigen (HBsAg) in the blood or serum for longer than six months, with or without associated active viral replication and evidence of hepatocellular injury and inflammation (WHO, 2017). Chronicity is common following acute infection in neonates and young children under the age of 5 years but rarely occurs when infection is acquired in adulthood (McMahon, 2005). Several rapid diagnostic tests (RDTs) or immunoassays have been developed for the detection of HBsAg. Rapid tests developed for screening include solid-phase assays, flow-through, agglutination and lateral-flow.

Most of these test, are immunochromatographic assays. Immunoassays use different methods for detection of HBsAg using polyclonal or monoclonal anti-HBs antibodies.

However, HBV infection may run undetected and delay diagnosis due to unawareness of the infection may lead to HBV related liver diseases (Frambo et al., 2014). Unless people with HBV infection are diagnosed and treated, the number of deaths due to viral hepatitis will continue to increase

1.2 Literature Review

1.2.1 Treatment of Hepatitis B

The treatment outcomes for chronic hepatitis B (CHB) have improved over the past three decades, first with IFN-alpha and now NAs (Basar et al., 2013). Currently, seven antiviral agents (five NAs: lamivudine, adefovir, entecavir, telbivudine, tenofovir, emtricitabine, as well as standard and two formulations of PEG-IFN) are approved and widely licensed for the treatment of CHB. The pharmacokinetics, inhibitory capacity and resistance patterns of nucleos(t)ide analogue (NAs) and their mechanism of action on HBV polymerase differs. The widespread use of NAs with a low genetic barrier to resistance, such as lamivudine, has led to high rates of resistance in those who have received treatment for CHB. The goal of antiviral therapy for CHB is to reduce (or reverse) necroinflammatory change and hepatic fibrosis leading to progressive liver disease, cirrhosis, decompensated cirrhosis and liver failure, hepatocellular carcinoma (HCC) and death. However, there is still limited evidence from clinical trials of the effect of antiviral therapy on these clinical outcomes. Therefore, surrogate measures of long-term treatment outcomes are used to assess efficacy. These include biochemical measures: normalization of serum alanine aminotransferase (ALT) as a surrogate measure for the resolution of necroinflammation in the liver; and virological markers: a reduction in HBV DNA to undetectable levels by PCR, and hepatitis B e antigen (HBeAg) loss or seroconversion to anti-HBe status or rarely, hepatitis B surface antigen (HBsAg) loss and seroconversion to anti-HBs status. Although NAs are potent inhibitors of HBV DNA replication, they do not result in cure, because antiviral therapy cannot eliminate the covalently closed circular DNA (cccDNA) form in the nucleus, which is the template for transcription of viral RNA. Therefore, long-term (potentially lifelong) NA therapy is required in the majority of persons. Although there are some advantages of IFN therapy, such as a finite duration of therapy, and possibly a higher rate of HBsAg loss. It is less feasible for use in resource limited settings and in infants less than 1 year and pregnant women as it requires administration by injection, is expensive, inconvenient to use, less well tolerated, and requires careful monitoring.

As a priority, all adults, adolescents and children with CHB and clinical evidence of compensated or decompensated cirrhosis (or cirrhosis based on APRI score >2 in adults) should be treated, regardless of ALT levels, HBeAg status or HBV DNA levels. Treatment is recommended for adults with CHB who do not have clinical evidence of cirrhosis (or based on APRI score ≤ 2 in adults), but are aged more than 30 years (in particular), and have persistently abnormal ALT levels and evidence of high level HBV replication (HBV DNA >20 000 IU/mL), regardless of HBeAg status. Where HBV DNA testing is not available, treatment may be considered based on persistently abnormal ALT levels alone, regardless of HBeAg status.

As a first-line antiviral therapies for chronic hepatitis B in all adults, adolescents and children aged 12 years or older in whom antiviral therapy is indicated, the nucleos(t)ide analogues (NAs) which have a high barrier to drug resistance (tenofovir or entecavir) are recommended. Entecavir is recommended in children aged 2–11 years. NAs with a low barrier to resistance (lamivudine, adefovir or telbivudine) can lead to drug resistance and are not recommended. Second-line antiviral therapies for the management of treatment failure in persons with confirmed or suspected antiviral resistance to lamivudine, entecavir, adefovir or telbivudine, a switch to tenofovir is recommended. Lifelong NA therapy to all persons with cirrhosis based on clinical evidence (or APRI score >2 in adults) require lifelong treatment with nucleos(t)ide analogues (NAs), and should not discontinue antiviral therapy because of the risk of reactivation, which can cause severe acute-on-chronic liver injury. Discontinuation of NA therapy may be considered exceptionally in persons without clinical evidence of cirrhosis (or based on APRI score ≤2 in adults), who can be followed carefully long term for reactivation, if there is evidence of HBeAg loss and seroconversion to anti-HBe (in persons initially HBeAg positive), after completion of at least one additional year of treatment, in association with persistently normal ALT levels and persistently undetectable HBV DNA levels (where HBV DNA testing is available). Relapse may occur after stopping therapy with NAs. Retreatment is recommended if there are consistent signs of reactivation (HBsAg or HBeAg becomes positive, ALT levels increase, or HBV DNA becomes detectable again).

1.3 Prevention (Vaccination)

Recombinant DNA-derived vaccines against HBV have been available for more than two decades. The primary hepatitis B immunization series conventionally consists of three doses of vaccine. The Existing recommendations in infants and neonates hepatitis B vaccination involves the administration of a first dose of hepatitis B vaccine as soon as possible after birth, preferably within 24 hours, followed by two or three doses (WHO, 2009). This strategy has resulted in a dramatic decrease in the prevalence of CHB among young children in regions of the world where universal infant vaccination programs have been implemented. A proportion of vaccinated children (5–10%) have a poor response to vaccination and will remain susceptible as adults to acquisition of HBV infection. Motherto-child HBV transmission is also prevented using antiviral therapy. In HBVmonoinfected pregnant women, the indications for treatment are the same as for other adults, and tenofovir is recommended. No recommendation is made on the routine use of antiviral therapy to prevent mother-to-child HBV transmission. In countries with intermediate or low endemicity, a substantial disease burden may result from acute and chronic infection acquired by older children, adolescents and adults. Target groups for catch-up vaccination as well as other preventive strategies include young adolescents; household and sexual contacts of persons who are HBsAg-positive; and persons at risk of acquiring HBV infection, such as men who have sex with men, and persons with multiple sex partners.

1.4 Diagnosis

Chronic hepatitis B infection is defined by the detection of HBsAg on two occasions six months apart. The most important marker for the diagnosis of hepatitis B infection that may require treatment is the detection of hepatitis B surface antigen (HBsAg). The selection of EIA or RDTs should not be mutually exclusive. Choice of appropriate technology can be complex but can usually be distilled down to three main factors: performance, cost and accessibility. The different methods of diagnosis are discussed below.

Point-of-care tests (POC)

In the past 20 years, POC tests for diabetes, anemia, pregnancy, HIV and malaria have become common diagnostic tools in both high- and low- to middle-income areas. They have significantly improved the quality of care within the framework of patient-centered approaches (Omi, 2008). In the setting of infectious diseases, most existing POC tests consist of immunoassays providing qualitative and sometimes, quantitative determination of various markers of infection, including antigens and antibodies, within a limited amount of time. For this reason, they are also called ‘‘rapid diagnostic tests” (John and Price, 2014). Non-immunological POC tests that detect and sometimes quantify pathogen nucleic acids are at earlier stages of development. POC tests can use whole blood, serum or plasma collected by venipuncture. However, the main focus is on the potential use of alternative matrices, such as tiny amounts of finger stick capillary whole blood or oral fluid. Finger stick capillary whole blood collection uses a manual or automated lancet device that punctures the finger (or the heel in infants in order to prevent hitting the bone).

Similarly, oral fluid can be simply, safely and cheaply collected and tested (McKie et al., 2002). POC tests have been designed for use at the sites of patient care, including physicians’ offices, outpatient clinics, intensive care units, emergency rooms, medical laboratories, or even patients’ homes for self-testing. In low- to middle-income countries, they are widely used in these settings, as well as in blood banks (Prugger et al., 2016). POC tests enable the delivery of results at the time of testing without the need for a follow-up visit, thereby increasing the proportion of individuals informed of their status (Molitor et al., 1999). POC tests have the potential to reduce emergency admissions, hospitalizations or the length of hospital stay, while enabling care to be delivered closer to patients’ homes (St John, 2010).

Immunological point-of-care tests (Rapid diagnostic tests)

The principle of RDTs is to capture the antigens or antibodies being investigated on a solid surface, before attaching molecules to them that enable their detection with the naked eye or a dedicated reader. An ideal RDT must meet the World Health Organization (WHO) ‘‘ASSURED” criteria. A few basic technologies are offered by manufacturers, including: lateral-flow tests, also called immunochromatographic strips or striptests; flowthrough tests, usually supplied as individual cassettes; tests based on the agglutination of particles; and tests based on solid-phase assays (so-called ‘‘dipsticks”), allowing for the detection of one or multiple parameters with a single assay.

Many laboratories in resource-limited settings may not have access to specialized equipment and process few specimens, per day. Advancement in viral hepatitis testing and RDTs now include a same-day point-of-care assays for nucleic acid testing (NAT) and core antigen (HBsAg), which avoid the need for expensive laboratory processing. This may make the rapid diagnostic tests (RDTs), to be more appropriate. In general, RDTs do not require cold storage and may be tested using capillary (finger-stick) whole blood. The results of RDTs are read visually. Some RDTs used oral fluid making them valuable in cases where collection of venous or capillary whole blood is challenging and are also adequate sensitivity and specificity. They can be performed by readily trained nonmedical practitioners and can be used in outreach programs (e.g., prison services, substance use or treatment services) to increase the uptake of hepatitis screening. The expansion of their use depends on their performance and operational characteristics in the setting of intended use, ultimately with the aim being to reach resource-limited settings and offer cost-efficient testing services as an alternative to assays that require specific laboratory infrastructure and staff skills to perform. However, the sensitivity and specificity of some of the latest generation of RDTs across a wide range of settings and different populations can be comparable to those of EIAs. It is the simplest and most widely used tests for specific qualitative or semi-quantitative detection of HBV antigens or antibodies (de Puig et al., 2017). However, the quality of assays is variable. A variety of RDTs are under evaluation and/or are currently in use in low- and middle income countries for screening, diagnostic and surveillance purposes (Lesmana et al., 2011).

Limitations of RDTs

The use of RDTs may be limited by the following weaknesses: a cost that sometimes exceeds that of traditional testing methods; only qualitative, non-quantitative ‘‘yes or no” result. There is subjective interpretation that may be inadequate, especially in patients with a low amount of antigen or antibodies. RDT is low throughput methods, lack of sensitivity compared to existing reference level laboratory tests, particularly when oral fluid or finger stick whole blood are used. The HBsAg Rapid Diagnostic Test strip is for professional in vitro diagnosis use only and it only indicates the presence of HBsAg in the specimen. The HBsAg Rapid Test strip cannot detect less than 1ng/ml of HBsAg in specimen. If the test is negative and clinical symptoms persist, additional follow-up testing using other clinical methods is suggested. A negative result at any time does not preclude the possibility of hepatostis B infection.

Immunoassays (laboratory-based)

The most widely used HBsAg assays are laboratory-based immunoassays. This can be in the form of an enzyme immunoassay (EIA), electrochemiluminescence immunoassay (ECL) or chemiluminescence immunoassay (CLIA). These are best suited to settings with high throughput of specimens and where infrastructure (electricity, cold storage, climatecontrolled rooms) and skilled staff are consistently available. Other simple assays such as agglutination assays are also available for detection of HBsAg but these generally require serum/plasma specimens and cold storage.

The HBsAg EIA test kit is a solid phase quantitative enzyme immunoassay based on a sandwich principle for the detection of HBsAg in human serum or plasma. The microwell is plate with monoclonal antibodies specific to various subtype of HBsAg. During testing the specimrn and the enzyme conjugated HBsAg antibodies are added to the antibody coated microcell plate and the incubated. If the specimen contains HBsAg, it will bind to the antibodies coated on the microwell plate and simultaneously bind to the conjugate to form immobilized antibody- HBsAg-conjugate complexes. If the specimen does not contain HBsAg, the complexes will not be formed. After initial incubation, the microcell plate is washed to remove unbound materials. Substrate A and substrate B are added and the incubated to produce a blue color, indicating the amount of HBsAg present in the specimen. Sulfuric acid solution is added to the microcell plate to stop the reaction which produce a color change from blue to yellow. The color intensity, which corresponds to the

amount of HBsAg present in the specimen, is measured with a microplate reader at 450/630-700 nm or 450 nm.

Limitations of Immunoassays (laboratory-based)

The limitations of Immunoassays (laboratory-based) for the detection of HBV virus are as follows; a cold chain is needed, interference from sample matrices during analysis is required, equipment such as centrifuges which makes it unable to perform the test in poor equipped laboratories and in areas with no electricity. Also the assay development time is longer and has a poor suitability for multi-analyte applications. False positive results may occur due to high tires of heterophilic anti mouse antibodies. False negative results may occur if the quantity of HBsAg present during the stage of disease when the specimen was collected. Erroneous result may also be due to fibrin particles and microbial concentration.

The positive control in the test kit is not to be used to qualify assay sensitivity, it is used to verify that the test kit components are capable of detecting the reactive specimen

 

1.5 Rationale

A variety of RDTs are under evaluation and/or are currently in use in low- and middle income countries for screening, diagnostic and surveillance purposes. It is therefore necessary to evaluate and compare sensitivity and selectivity of some selected RDTs to ELISA as reference test in the diagnosis of the disease in Cameroon. This study will be helpful to understand the efficiency and accuracy of some selected RDTs in the diagnosis HBV. Results from this study will be useful for better diagnosis for an effective treatment and management of the disease.

1.6 Hypothesis

HEXAGON HBsAg and Diaspot for the detection of hepatitis B virus are more sensitive

than Foresight Enzyme linked Immunosorbent Assay.

1.7 Objectives

1.7.1 Main objective

To evaluate the quality of rapid diagnostic test (Hexagen HBsAg and Diaspot) in the detection of Hepatitis B virus in infected samples using foresight ELISA are reference test.

1.7.2 Specific objective

1. To compare the sensitivity of Hepatitis B diagnostic tools.
2. To compare the selectivity of Hepatitis B diagnostic tools.
3. To determine the prevalence of Hepatitis B in field samples.