Evaluation of HBsAg Quantification as Surrogate to HBV DNA Viral Load in Hepatitis B Infected Patients in Anambra State, Nigeria

Hepatitis B is an infectious disease of great public health importance. Nigeria is one of the countries with the highest incidence of Hepatitis B Virus (HBV) infection worldwide. However, the accessibility and affordability of HBV DNA quantification (viral load) assay is the key laboratory test for therapy initiation, and monitoring is a challenge to HBV management. This study aimed at determining the relationship between HBV DNA quantification and routine haemato-serological parameters in order to develop a more cost-effective diagnostic algorithm for Hepatitis B management. Cross sectional study design was used with a total of 264 subjects comprising of 88 HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects on antiviral therapy as case subjects and 88 age-matched apparently healthy HBsAg seronegative individuals were recruited as control subjects. Hepatitis B Virus DNA assay was performed using real time PCR technique while ELISA technique was used for Hepatitis B surface antigen quantification. HBsAg quantification showed strong positive correlation with HBV DNA viral load both in treatment and non-treatment groups (r = 0.673; p < 0.001). However, the Receiver Operation Characteristics curve indicated a very poor performance characteristics (AUC = 0.537, p = 0.002). The non-treatment group has higher viral load (M = 805.50 IU/ml) compared with treatment group (M = 65.50 IU/ml) (p 0.001). There was a significant difference in HBV DNA levels among the four serological patterns observed in the study (p 0.001). This study has revealed that HBsAg quantification has strong correlation with HBV viral load but might not be efficient in clinical practice as a predictor of serum HBV viral load due to its poor performance characteristics in identifying high positive viral load.

Hepatitis B is an infectious disease of great public health importance. Nigeria is one of the countries with the highest incidence of Hepatitis B Virus (HBV) infection worldwide. However, the accessibility and affordability of HBV DNA quantification (viral load) assay is the key laboratory test for therapy initiation, and monitoring is a challenge to HBV management. This study aimed at determining the relationship between HBV DNA quantification and routine haemato-serological parameters in order to develop a more cost-effective diagnostic algorithm for Hepatitis B management. Cross sectional study design was used with a total of 264 subjects comprising of 88 HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects on antiviral therapy as case subjects and 88 age-matched apparently healthy HBsAg seronegative individuals were recruited as control subjects. Hepatitis B Virus DNA assay was performed using real time PCR technique while ELISA technique was used for Hepatitis B surface antigen quantification. HBsAg quantification showed strong positive correlation with HBV DNA viral load both in treatment and non-treatment groups (r = 0.673; p < 0.001). However, the Receiver Operation Characteristics curve indicated a very poor performance characteristics (AUC = 0.537, p = 0.002). The non-treatment group has higher viral load (M = 805.50 IU/ml) compared with treatment group (M = 65.50 IU/ml) (p < 0.001). There was a significant difference in HBV DNA levels among the four serological patterns observed in the study (p < 0.001). This study has revealed that HBsAg quantification has strong correlation with HBV viral load but might not be efficient in clinical practice as a predictor of serum HBV viral load due to its poor performance characteristics in identifying high positive viral load.

Introduction
Hepatis B is a top health priority worldwide and a devastating cause of morbidity and mortality [1]. Hepatitis B is an infectious disease of great public health importance caused by hepatitis B virus (HBV). It is an enveloped DNA virus that infects the liver and causes hepatocellular necrosis and inflammation [2]. This virus belongs to the family hepadnaviridae and genus orthohepadnavirus and it is the only hepadnavirus that causes infection in humans [3]. It is 100 times more infectious than the most dreaded human immunodeficiency virus infection and 10 times more infectious than hepatitis C virus [4]. Hepatitis B virus is one of several viruses known to cause viral hepatitis and continues to be the major cause of viral hepatitis in the developing and underdeveloped world. In addition to causing chronic liver disease and cirrhosis, it has a formidable track record of being linked to primary hepatocellular carcinoma. It is estimated that HBV and HCV are the root cause of about 80% of all hepatocellular carcinomas (HCC) by promoting cirrhosis which significantly reduced the life expectancy of the infected patients [5].
About 257 million people are chronically infected annually and about 2 in 3 people with Hepatitis B do not know they are infected [6]. Recent statistics indicate that not less than 23 million Nigerians are estimated to be infected with the HBV, making Nigeria one of the countries with the highest incidence of HBV infection in the world [7]. A national study done in Nigeria in 2016 shows a prevalence rate of 12.4 percent [8]. This worldwide burden of hepatitis B mandates accurate and timely diagnosis of patients infected with HBV and the use of treatment strategies derived from evidence-based guidelines. Most hepatitis B patients are asymptomatic in the early stage as specific clinical symptoms often occur at advanced disease stages, which are usually irreversible. Hence, the prognosis of the infection to liver disease is very crucial. The presence of derangement in specific laboratory analytes at the early stage of infection may signal a risk of fibrosis, cirrhosis and ultimately HCC.
Among all the several clinical diagnostic tests which have been developed for the detection of HBV infections, the serum HBV DNA level is a key factor affecting the initiation of antiviral therapy and evaluation of its efficacy [9]. Evaluation of the relationship between the serum HBV DNA levels and hepatic pathology is a current hotspot in the diagnosis and treatment of CHB [10]. Quantification of the HBsAg levels has received renewed attention because of its diagnostic potential in predicting the response to antiviral treatment and identifying the infection status of an individual [11]. Determination of the circulating levels of HBsAg could provide crucial information that could complement the mea-

Study Area
The study was carried out at the Gastroenterology Unit of Nnamdi Azikiwe

University Teaching Hospital (NAUTH), a tertiary institution in South Eastern
Nigeria which is a referral center for Hepatitis B care in Nigeria.

Study Design
Cross sectional study design was used in the study. A total of 264 subjects were recruited which comprised of 88 HBsAg seropositive treatment naïve subjects, 88 HBsAg seropositive subjects on antiviral therapy (Tenofovir 300 mg daily/Entecavir 0.5 mg daily or Pegylated interferons 180 μg weekly) as case subjects and 88 age-matched apparently healthy HBsAg seronegative individuals were recruited as control subjects.

Sample Size Determination
Using G* Power software version 3.0.10, power analysis for a one-way ANOVA with three groups was conducted to determine a sufficient sample size using an alpha of 0.05, a power of 0.96, and a medium effect size (f = 0.25). Based on the aforementioned assumptions, the calculated sample size of 264, with 88 subjects per group has 96% power to detect a difference of 0.25 at significance level of 0.05 [13].

Sampling Technique
Purposive sampling technique was employed in selecting the participants based on the inclusion criteria. Patients that gave their consent who also met the selection criteria were recruited as they come to the clinic until the sample size was completed.

Sample Collection
After obtaining informed consent, 10 ml of venous blood was collected from the fore arm of each subjects using a disposable syringe; 7 ml was dispensed to a ste-

Inclusion Criteria
Participants included in this study were hepatitis B seropositive subjects aged 18 to 65 years attending Gastroenterology Clinic of NAUTH, Nnewi and apparently healthy HBsAg seronegative individuals (NAUTH staff, students and others) who gave their consent.

Exclusion Criteria
Those patients who were co-infected with HIV, HDV and HCV including HBsAg seronegative subjects who have received HBV vaccination were excluded from the study with those less than 18 years and above 65 years.

Statistical Analysis
Data obtained were analyzed using Statistical Package for Social Sciences (SPSS) version 20) software. Data were expressed as mean ± SD and median. The significance of differences in mean values among groups was analyzed using one-way ANOVA for normally distributed variables, while Kruskall Wallis was used to analyze the significant differences in median values among different groups for variables not normally distributed. Mann-Whitney was also used to analyze significant differences between groups. Spearman's correlation coefficient was used to assess the levels of relationship between two variables. Regression analysis and receiver Operators curve were also used appropriately. The level of significance was considered at p < 0.05.

Results
Median serum levels of HBV-DNA concentration and HBsAg Titers of the hepatitis B subjects   (Table 2).

Discussion
This study hypothesized that quantification of HBsAg could be used to predict  [26] gave a similar report. Considering the modes of transmission of HBV, the high sexual activity of individuals within these age brackets might explain this high frequency. The age group < 20 had the least frequency both among males and females (3.9 both).
The >50 age group were the second to the least of the subjects (9% females and 12% for females). This finding is in agreement with that of Yewande et al., (2018) [24]. The children (<20) and the aged (>50) are less prone to risky behaviours than the middle aged (21 -30, 31 -40).

Conclusion
This study has revealed that HBsAg quantification has strong correlation with HBV viral load but might not be efficient in clinical practice as a predictor of serum HBV viral load due to its poor performance characteristics in identifying high positive viral load.