1. INTRODUCTION:

Hepatitis B virus (HBV) affects approximately two billion people worldwide and more than 240 million people in the world are currently chronic carrier that could develop serious complications in the future, like liver cirrhosis and hepatocellular carcinoma (Dusheiko et al.,2017).

The world is divided into three endemic regions according to the prevalence of HBV infection; The high (8%–15%), intermediate (2%–7%) and low prevalence (<2%) regions (Margolis et al.,1991). Iraq is among the intermediate HBV endemic countries, because the carrier rate of HBV is between (3%-4.5%) among normal population and (2%-3%) among healthy blood donors (Al –Hamdani et al.,2012).

The HBV virus is a DNA virus belongs to the Hepadnaviridae family. The HBV -DNA is a circular, partial double strand genome. All coding information is on the minus DNA strand and it is organized into five open reading frames (Hansi et al.,2017).In the past, Iraq is considered as one of the intermediate endemic countries, and many studies have been conducted since 1973; the results of these studies shows the prevalence of HBsAg in Al-Rusafa sector of Baghdad according to Iraqi CDC (Center for Disease Control and Prevention) from 2002 to 2008. Of the countries with which Iraq has frontiers, Turkey, KSA and UAE have an intermediate endemicity, while Iran, Kuwait and Bahrain are areas of a low endemicity (Andre, 2000). In Iraq, the carrier rate of HBV between 3-4.5% among normal population and 2-3% among healthy blood donors. A lower rate was detected among Iraqi blood donors 1%- 2% and HBsAg seroprevalence among normal population 1.6% (Omer and Al-Salmani, 2005), An important determinant of epidemiology of HBV is the age of acquisition of the virus. In areas with high endemicity, most infections occur in children (4-8 years), and in area with intermediate endemicity the infection occur in adolescents and adults, while in areas with low endemicity, most infections occur in adolescents and young adults (15-29 years) (Huo et al., 2005). In India, it was found that the peak prevalence of HBsAg is after the second decade of life (Chowdhury, 2004), whereas in Pakistan, the peak of HBsAg is in persons with 21- 40 years of age (high prevalence) (Alam et al., 2007), while in Indonesia, the peak of HBV in adult persons was noticed (Achwan et al., 2007). In Iraq, the normal population prevalence of HBsAg was found to be higher in males than females according to many studies.Drug resistant virus mutants emerge as a function of at leastfour factors: the viral mutation frequency, the intrinsic mutabilityof the antiviral target site, the selective pressure exertedby the drug and the magnitude and rate of virus replication.

The current study aims to determine the distribution of patients with chronic hepatitis B and the percentage of antiviral drugs resistance.

2. MATERIALS AND METHODS:

2-1- Study groups:

The present study was conducted in Hepatology and Gastroenterology Teaching Hospital and Central Public Health Laboratories/ HIV/AIDS and Hepatitis Referral Laboratory (Baghdad), from April 2016 through February 2017. A total of 230 patients with CHB who had attended the Hepatology and Gastroenterology Teaching Hospital in Baghdad, all of them were HBsAg positive for more than six months. 18 were HBeAg positive and 212 were HBeAg negative. 130 males and 100 females with ranging from (11-78) year.

2-2- Collection of serum specimens:

From each individual in this study, 5 ml of blood was drawn by vein puncture using disposable syringes. The blood was placed in plastic disposable tubes; it was left to stand at room temperature (20-25Cₒ) to allow it to clot, then the sera was separated by centrifugation 10000 r.p.m for 5 minutes and stored at -20 ₒC till examination. The specimens were transferred to the Virology Unit for detection of HBsAg,HBeAg, anti-HBe and anti-HBc in serum specimens by ELISA test. All sera and reagents were allowed to stand at room temperature before use in the test.

2-3- Detection of serological markers:

HBsAg (serum) ELISA test:

This test was performed using commercially available kit (DRG.ELISA). Reactive results were indicated by the absorbance reading of 0.03 and above, while the non-reactive results were indicated by the absorbance reading less than 0.03.

Anti-HBe (serum) ELISA test:

This test was performed using commercially available kit (DRG.ELISA). Reactive results were indicated by the absorbance reading of 0.1 and above, while the non-reactive results were indicated by the absorbance reading less than 0.1.

HBeAgELISA test:

This test was performed using commercially available kit (DRG.ELISA). Reactive results were indicated by the absorbance reading of 0.2 and above, while the non-reactive results were indicated by the absorbance reading less than 0.2.

Anti-HBc (serum) ELISA test:

2-4- HBV viral load:

determine the HBV DNA level was a real-time polymerase chain reaction (PCR) assay, in which HBV DNA was extracted from serum by using spin columns (QIAamp DNA mini columns; Qiagen), and their concentrations were determined by using a programmable DNA high-speed Thermal Cycler (LightCycler; Roche Diagnostics). The oligonucleotide sequences of primers corresponding to the HBV S gene

The PCR conditions were 4 mM MgCl₂, 3 pmol of each hybridization probe, 20 pmol of 2 PCR primers, 2 mL of hybridization probe mix (LightCycler Fast Start DNA Master probe mix; Roche Diagnostics), and 10 mL of HBV DNA samples, in a total volume of 20 mL. The PCR program consisted ofan initial cycle (95°C for 10 minutes) followed by 45 amplificationcycles (94°C for 15 seconds, 50°C for 30 seconds, and 72°C for 30seconds). The instrument detected and recorded the fluorescencespectrum of each reaction tube during the annealing phase.An external standard was constructed by ligation of a 1.3-kb wildtypeHBV plasmid (genotype D) into the pBlueBac plasmid vector(kind gift from Dr. Delaney, Hershey Medical Center, Hershey, PA).Quantification of the DNA concentration of the plasmid was determinedby spectrophotometry. Duplicates of serial 10-fold dilutionsof the plasmid ranging from 108 copies/mL to 100 copies/mL wereincluded in each run to generate a standard curve. The copy numberin each experimental reaction was determined by interpolation of thederived threshold cycle (CT), as previously described.

3. RESULTS AND DISCUSSION:.

3-1- Age distribution of CHB patients:.

Table 3.1 shown the distribution of CHB patients according to age. It was found that the age ranged between 11-79 year with a mean of (36.11).It was also found that most of the patients were located within third and fourth decade 20-39 year with a percentage of 45.5% and it was found that the eight decade 70-79 year constitutes the least percentages 6%. While the results showed the most frequent age group's of CHB patients have drug resistant are (20-29) and (30-39) in percent 32.5% and 20.5% Respectively.

Table 3.1 The distribution of CHB patients according to the age.

Age group (years)	Total CHB			Drug resist. CHB
	total	HBeAg(-) CHB	HBeAg(+) CHB	total	HBeAg(-) CHB	HBeAg(+) CHB
	No (%)	No (%)	No (%)	No (%)	No (%)	No (%)
10-19	31 (13.5%)	28 (13.2%)	3 (16.7%)	10 (12%)	10 (12.5%)	0 (0%)
20-29	60 (26%)	55 (26%)	5 (27.9%)	27 (32.5%)	25 (31.2%)	2 (66.6%)
30-39	45 (19.5%)	42 (19.8%)	3 (16.6%)	17 (20.5%)	16 (20%)	1 (33.3%)
40-49	36 (15.8%)	34 (16%)	2 (11.1%)	10 (12,1%)	10 (12.5%)	0 (0%)
50-59	23 (10%)	21 (9.9%)	2 (11.1%)	8 (9.6%)	8 (10%)	0 (0%)
60-69	21 (9%)	19 (9%)	2 (11.1%)	7 (8.4%)	7 (8.8%)	0 (0%)
70-79	14 (6%)	13 (6.1%)	1 (5.5%)	4 (4.9%)	4 (5%)	0 (0%)
total	230 (100%)	212 (100%)	18 (100%)	83 (100%)	80 (100%)	3 (100%)

The distribution of HBV serological markers among HBe(+) CHB and HBe(-) CHB patients groups and the comparison between them.

HBe(-) CHB group showed the prevalence of Anti-HBc (+) 90% of patient while CHB drug resistance group showed 87.5% of patient. Anti-HBe Ag showed in 50.9% CHB patient while appear 12.5% in CHB drug resistance group.

in the HBe(+) CHB group, the result showed the prevalence of Anti-HBc (+) 83%, Anti-HBe Ag showed in 0% of patient while CHB drug resistance group, The prevalence of anti-HBe 0 % and 33.3% for Anti-HBc.

All patients of this study were HBsAg positive for more than 6 months. The term HBeAg negative CHB defines the condition of disease caused by strains of HBV that are not producing HBeAg (Hadziyannis,1995).

In clinical practice, the term HBeAg negative CHB is appropriate for patients with chronic HBV infection who test negative for HBeAg, are usually positive for anti-HBe, have increased alanine aminotransferase (ALT) levels and display detectable HBV DNA in serum by classical hybridization techniques (Hadziyannis,1995).

Despite the fact that the identification of the underlying mutations in the HBV genome responsible for HBeAg negativity and the infecting HBV genotype are not routinely performed in clinical laboratories, mutation analysis should be done whenever possible and reported in a standardized way (Lok et al., 2001).

Regarding the anti-HBc marker which was detects the presence of both IgM and IgG antibody to hepatitis B core antigen and is indicated as an aid in the diagnosis of ongoing or previous hepatitis B viral infection.

It is increased in the serum of infected patients early in the disease and last for many years after infection resolved, so anti-HBc is present even in the window period when the HBsAg has disappeared and yet anti-HBs is not detectable.

The percentage of HBcAb were 90% in CHB, , 83% in HBe(+) CHB and 51% in HBe(-) CHB and there was no difference between these groups except for HBe(+) CHB show a slightly lower percentage. These results coinsided with the studies in Iraq as Al-Waysi (2005) who found 93.75% positive anti-HBc among CHB and Ono-Nita et al. (2004) who found 90% positive anti HBc among Asian CHB patients. Also Saied (2011) who found 99% among blood donor.

The degree of infectivity and viral replication in CHB patients was also studied by the detection of HBeAg and anti-HBe. The HBeAg percentage were 7.8 % in CHB. Anti-HBe were 79% in CHB patients, 0% in HBe(+) CHB and 80% in HBe(-) CHB. These results are in accordence with the results of Rassam et al (1988) found (17.6%) positive HBeAg and 54% positive anti-HBe among patients with chronic liver disease in Iraq. Saied (2011) who found 7.6% HBeAg and 92.3% anti-HBe among blood donor. Mohammed (1986) who found that only 4.9% of pregnant wemen had HBeAg.

While Al-Salmani (1987) found HBeAg positive in 10.4% of the carriers and anti-HBe was detected in 70.1% of them. Studies in Saudi Arabia done by Sheath and Godwin (1986) who detected HBeAg in 9% of HBsAg positive individuals admitted to king Faisal specialist hospital for other diseases.Al-Waysi (2005) who found 35.93% positive HBeAg and 56.25% positive anti-HBe. Youssif (1998) who found a higher results for HBeAg 54.2% and lower results of anti-HBe 44.58% among 83 patients with chronic active hepatitis (CAH). The difference of the present results and those of the previous Iraqi studies may be due to either sample size or due to the increased existence of HBe-negative mutant HBV strains in our country.

3-4- Viral load:

Table 3.4 shows the distribution of patient numbers based on viral load

Where the category (<10,000) showed the highest frequency by 36% from CHB patient have drug resistance, followed by the category (≥1,000,000) by 30% percent.

Table 3.4 shows the distribution of patient numbers based on viral load.

Category (IU/ml)	No. of Patients (%)	HBe Ag (-)	HBe Ag (+)
≥ 1,000,000	25 (30%)	24 (30%)	1 (33.3%)
100,000 – 1,000,000	8 (10%)	8 (10%)	0 (0%)
10,000 -100,000	20 (24%)	19 (24%)	1 (33.3%)
< 10,000	30 (36%)	29 (36%)	1 (33.3%)
Total	83 (100%)	80 (100%)	3 (100%)

Nevertheless, Feld, et al. (2007). carried out a prospective study and showed that HBV-DNA levels > 2,000 copies/mL in HBeAg-negative patients with normal ALT levels was a relevant predictor of elevated ALT levels at future follow-up visits. Considering the evidence that viral load levels > 2,000 copies/mL are associated with an increasing probability of cirrhosis of the liver and HCC, ( Chan et al., 2008).

Some study showed viral load levels were lower in the population of HBeAg-negative patients compared to the HBeAg-positive patients, this study has shown that HBV-DNA levels are significantly higher in HBeAg-positive individuals (Shao et al., 2007).

HBeAg was positive in 15.3% of individuals for whom these results were available, a proportion that is almost identical to that observed in the REVEAL-HBV study.

We believe that there is growing evidence that patients with high HBV-DNA levels and low ALT levels should be considered to be harboring the active infection and to be at an increased risk of complications.

However, when our subset analysis focused on such individuals, a relatively large proportion was found to have HBV-DNA levels > 2,000 copies/mL. This threshold was used since the effect of treatment in this population is unknown, making it impossible to distinguish those patients in whom low HBV-DNA levels are a result of therapy and seroconversion from those patients in whom the disease is inactive. By excluding those individuals with low viral load levels from this analysis, the prevalence of a high viral load and to evaluate ALT, since it would be expected that most patients with high ALT levels would have the active disease.

In Brazil, tests for the evaluation of HBV-DNA levels are not generally available within the public healthcare system; therefore, in HBeAg-negative patients with normal ALT levels the disease may be wrongly classified as inactive. Report that patients with elevated HBV-DNA levels are more prone to disease progression. For example, Yuen, et al., (2009) showed that age, gender, core promoter mutation, HBV-DNA and cirrhosis, but not ALT levels, are predictive of the development of HCC. Nevertheless, Feld, et al. (2007) Carried out a prospective study and showed that HBV-DNA levels > 10,000 copies/mL in HBeAg-negative patients with normal ALT levels was a relevant predictor of elevated ALT levels at future follow-up visits.

HBsAg titers were preserved relative to HBV replication. HBeAg correlated with viral load, but emerging BCP/ PC variants were observed to independently reduce HBeAg titer. Clinical interpretation of HBsAg and HBeAg titers might therefore be refined by considering the phase of disease, as well as quasispecies diversity (Lim et al., 2007)

The BCP/PC variants are the most common to be associated with HBeAg- negative CHB. The mechanisms of reduced or abolished HBeAg production have been defined in vitro. The ntA1762T and G1764A BCP mutations are thought to alter the binding of transcriptional proteins to reduce CP-directed transcription, decreasing pre-C mRNA levels and causing a relative reduction in HBeAg (Laras et al., 2002).

The G1896A PC mutation encodes for a stop codon that prevents translation of the PC protein from PC mRNA (Carman et al., 1989). The emergence of these variants would therefore be expected to be associated with a decline in HBeAg titer independent of viral replication.

We concluded from the current study that males are more infected by CHB than females and that the age group (20-30) is the most prevalent category in which the infection was confirmed. 25% of the patients who were resistant to antiviral drugs had a viral load of more than 1 million virus.

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Received on 05.02.2018 Modified on 29.03.2018

Research J. Pharm. and Tech 2018; 11(8): 3503-3508.

DOI: 10.5958/0974-360X.2018.00648.0

gender	Total CHB			Drug resist. CHB
	total	HBeAg(-) CHB	HBeAg(+) CHB	total	HBeAg(-) CHB	HBeAg(+) CHB
	No (%)	No (%)	No (%)	No (%)	No (%)	No (%)
Male	130 (57%)	120 (57%)	10 (55.5%)	50 (60.2%)	48 (60%)	2 (66.6%)
Female	100 (43%)	92 (43%)	8 (44.5%)	33 (39.8%)	32 (40%)	1 (33.3%)
Total	230 (100%)	212 (100)	18 (100%)	83 (100%)	80 (100%)	3 (100%)
M / f ratio	1.33:1	1.33:1	1.25:1	1.51:1	1.5:1	2:1

Marker	Total CHB			Drug resist. CHB
	total	HBeAg(-) CHB	HBeAg(+) CHB	total	HBeAg(-) CHB	HBeAg(+) CHB
	No (%)	No (%)	No (%)	No (%)	No (%)	No (%)
HBs Ag	230 (100%)	212 (100%)	18 (100%)	83 (100%)	83 (100%)	3 (100%)
HBe Ab	118 (51.3%)	108 (50.9%)	0 (0%)	10 (12%)	10 (12.5%)	0 (0%)
HBc Ab	207 (90%)	190 (90%)	15 (83%)	71 (85.5%)	70 (87.5%)	1 (33.3%)
Total	230 (100%)	212 (100%)	18 (100%)	83 (100%)	80 (100%)	3 (100%)