Evaluation of Serological and Molecular Detection Methods for HBV and its Associated Risk Factors among General Population in the Province of Babil/ Iraq

ABSTRACT


Corresponding Author: Maha Diekan Abbas
Written informed consent was obtained from all of the participants and/or their parents.Designed questionnaires were administered to obtain demographic data from the study group.Negative control were those apparently healthy blood donors who are not suffering from any acute ailment at the time of sampling along with a proper vaccination record.Positive control were those suffering from liver diseases and underlying similar health conditions who have improper vaccination records.

Blood collection:
Each participant's venous blood was drawn in the public health laboratory.For plasma collection, one ml of anticoagulant (EDTA) was combined.The remaining samples were used to collect serum, which is spun at 1000 rpm for 5-10 minutes to separate it, and then it is chilled or frozen at -20 until it is utilized for the appropriate test.Acon-USA's third-generation enzyme immunoassay kit (EIA-3) was used to determine anti-HBsAg levels.The automated VIDAS system's enzyme-linked fluorescence immunoassay (ELFA) (Minividas Kit/HbsAg, Biomerieux-France) was used to confirm the anti-HBsAg reactivity.The viral load was then determined by RT-PCR utilizing the Exi-prepTM viral DNA/RNA kit (Bioneer-Koria) to extract the virus DNA and the RT-PCR Amplification kit (Sacace-Italia) to amplify the viral DNA.

RESULTS AND DISCUSSION
HBV infections start when the immune response that usually clears the virus does not function properly or is insufficiently strong to be effective; as a result, infections are more prevalent among people with low immunity due to poverty (1997, Hoofnagle).As described in table 1, the current study found that the prevalence of anti-HBsAg in the general population was (3.95%).For the age group >45 Yrs, having a greater prevalence of infection (5.6%) than the age group (25-44 Yrs), which had a prevalence of (5.5%).This may support the idea that these age groups are more exposed to the risk factor of infection.This result might be attributed to the exposure of live activities including; sexual activity, work, or travel.Statistical analysis showed a correlation between rate of infection and the age of infected individuals in these age groups (p 0.05).

Table (1): Frequency distribution of anti-HBsAg in general population by ELISA test
The statistical analysis relieved significant difference between the mean of optical density of patients samples and negative control for all age groups (LSD(0.05)1.783).Table 1 showed that the prevalence of anti-HBsAg in the general population was (3.95%), and that out of a total of 14 positive samples, 7 also tested positive for anti-HBc-IgM, a marker for recent or acute HBV infection (Yin and Tong, 2006) .This finding was also supported by (Al-Awady et al., 2008), who explained that the infection might occur primarily at birth due to vertical transmission (from mother to child), the age of presentation is between 25 and 35 years old because the patients are asymptomatic and were unintentionally identified by routine testing while giving blood, getting married, or working, all of which are common activities for people in this age range.According to several risk exposures of ageing groups, such as injections with syringes, blood transfusions, and invasive operations, the incidence of infection in the older age groups can be therefore explained.,2007) found that men predominate over women in all populations of anti-HBsAg carriers, supporting the well-documented fact that higher anti-HBsAg seroprevalence has been reported in male than in female for populations in some Asian countries.Women are more likely than men to clear anti-HBsAg, despite the fact that this is a well-established but poorly understood factor in chronicity (David and Daniel, 2003).These findings support earlier research in this area conducted in Iraq (Husain, 1997) and 2012 (Heim).There were a total of 14 positive anti-HBsAg tests; 8 of them were in urban areas, and the remaining 6 were in rural areas, with a rate of infection of 2:1, as illustrated in Table (2).In this study, the hepatitis B virus distributions by place of residency showed that there was a significantly higher prevalence of the virus in urban than rural areas (p > 0.05).This conclusion could be the result of improved health education in urban than rural regions, which promotes early disease diagnosis.The increased frequency of HBV in urban regions may be attributed to the crowded nature of cities, which may promote HBV and HCV transmission.These findings are consistent with those of earlier research conducted in Iraq by Hussin (1997) and Al-Awady ( 2008) .The distributions of the hepatitis B virus in this study showed a significant difference (p > 0.05) between the prevalence of the virus and the economic status; with high prevalence of the virus in population living with low economic status, compared to medium economic status and the lowest prevalence was found for those living with good economic condition.For HBV, the proportion of those with good, medium, and low economic standing was 1:1:2, respectively.The current study supports earlier research by Mistik and Balik (2001), which revealed that those with lower socioeconomic status and less hygienic living conditions are more likely to contract HBV than other people.
The result of the current study is consistent with the findings of other studies conducted in Japan (Dennis et al., 2005), which demonstrated that lower socioeconomic states have higher rates of HBV prevalence.These results were confirmed by additional findings published by Alter (1993) and Murphy et al. (1994).The distributions of the hepatitis B virus in this study, according to the educational level, showed that patients with low educational level (primary and secondary school education) compared to those with high educational level (graduate and post graduate education), showed a high prevalence of HBV with a significant difference (p >0.05).According to this study's findings, individuals with lower educational levels are more adversely affected by HBV at a ratio of (6:1) than those with higher educational levels.

Table (2):
Frequency distribution of infection ratio with HBV according to residency, economic status and educational level.

Table (3):
Frequency distribution of anti-HBsAg for different age groups by ELISA, Minividas and RT-PCR in general population.
Although the higher mean titers for both ELISA and RT-PCR correlated (2.00 and 1.668*107, respectively), As illustrated in table (3), the index mean of anti-HBsAg produced by ELISA technique does not entirely correlate with viral load of the virus.As shown in table (3), this was also attained at a lower mean titer.This might be attributed to to the fact that all results were obtained automatically by the apparatus which measured the results value depending on Calibrator 1 (represent positive control) and Calibrator 2 (represent negative control).The anti-HBsAg prevalence was also examined by Minividas technique and were more specific compared to ELISA technique.By using the cutting-edge molecular technology RT-PCR, the viral load of the positive specimens was also assessed.This study shows that manual and automated procedures were consistent across all examined blood samples.Since the employed kit of (IgM-HBc) was based on the competitive combination principle, the results in Fig. 3

Figure ( 1 ):
Figure (1): Frequency distribution of anti-HBs-Ag in different age groups of general population by ELISA test.
Figure (2): Frequency distribution of HBV infection according to age and gender among general population.
in the general population group refer to positive correlations with (IgM immunoglobulin) as a component of adaptive immune response and viral load of RT-PCR test.These findings are consistent with a previous study fromHan et al. in 2008, whodiscovered that the IgM anti-HBc and HBV DNA viral load combination has a positive coloring and enhanced diagnostic capability.The high viral load titer in children under 15 who were infected can be attributed to the fact that their mother was either unvaccinated or diseased when they were born, or it could be due to a contaminated device or a hospital-related infection (nasocomial infection).Since the majority of those individuals in this age group were +ve for anti-HBc IgM, the higher viremia observed in the age group (31-40) indicates that there are newly infected individuals in this age group.Higher age virus loads exhibit the gradual reduction.This may be due to the fact that these groups were exposed to the infectious agent before the diagnosis was made and that because of their advanced age, they were more likely to undergo medical procedures and receive blood transfusions.