. 52 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 52-56 ReseaRch aRticle Significance of Hepatitis B Virus Diagnosis by Real-time Polymerase Chain Reaction over Serological Markers in Hepatitis B Virus Patients Amer A. Khaleell*, Salah T. Jalall, Saeed G. Hussain2 1Department of Medical Microbiology, College of Health Sciences-Hawler Medical University, Erbil, Iraq, 2Department of Medical Microbiology, College of Medicine-Hawler Medical University, Erbil, Iraq ABSTRACT Hepatitis B virus (HBV) is the leading cause of viral hepatitis, as currently over 2 billion people have HBV infection worldwide. Nucleic acid assay and quantitative hepatitis B surface antigen (HBsAg) have been developed for diagnostic and therapeutic monitoring of patients with HBV infection. These tests might also show correlation between HBV DNA and HBs serostatus. The study aimed to find and analyze the frequency and impact of HBsAg seropositivity among patients revealed HBV DNA negative level through quantitative estimation of both seromarkers. Real-time polymerase chain reaction (RT-PCR) and Elecsys assays were used for quantitative estimation of HBV DNA and HBs antigen, respectively. A total of 256 blood samples were used from patients referred for either diagnostic purpose and/or HBV viral load monitoring after antiviral therapy. Blood profile analysis showed 12.26% HBs antigen seropositivity among patients revealed negative for nucleic acid assay for HBV DNA. Positive HBs antigen titers ranged from 1000–50,000 COI, with seronegative anti-HBs antibody test for all samples tested positive for HBs antigen. This study delineated that negative or undetectable quantitation of HBV DNA level does not exclude HBV infection; as the level might fluctuate in different phases of HBV replication. This gives an impression and raising a question about significance of replacing test for HBsAg with quantitation of HBV DNA PCR assay. Thus, the study refers to a special HBV profile outside the classical pattern. Keywords: Component, hepatitis B surface antigen, hepatitis B virus, hepatitis, quantitative hepatitis B virus DNA INTRODUCTION Hepatitis B virus (HBV) is the leading cause of viral hepatitis, as currently over 2 billion people have HBV infection worldwide. Approximately 600,000 deaths occur every year as a result of the acute and chronic consequences of HBV infection.[1] Chronic HBV affects nearly 350 million patients worldwide and may further progress to cirrhosis and/or hepatocellular carcinoma (HCC) in 15–40% of cases.[2] HBV infection is a dynamic process characterized by replicative and non-replicative phases based on virus-host interaction, which are present in some form of all infected patients.[3] To measure HBV DNA (viral load), a laboratory measures how many HBV DNA units are found in 1 ml of blood. The result is written in international units per ml (IU/ml). High levels of HBV DNA indicate a high rate of HBV replication; however, low or undetectable levels–<2000 IU/ml indicate an “inactive” infection.[4] During HBV infection, markers can be detected in sera samples using immunoassays. For example, patient sera that contain anti-HBs indicates immunity from past infections or vaccinations, and a patient with anti-HBe antibodies in their sera is considered as spontaneous resolution of infection or therapy-induced improvement.[5,6] One of the serological methods using for the diagnosis of HBV is enzyme immune assays. The hepatitis B surface antigen (HBsAg) and HBV DNA levels were considered to be a risk factor of hepatocarcinogenesis in untreated HB patients.[7] The levels of quantified HBsAg are considered as a marker with which to evaluate the host immunological control of infection and HBV replication.[8] Low HBsAg levels in some HBV patients are considered to indicate a high likelihood of HBV clearance and lower hepatitis activity.[7,9] The aim of this study was to estimate HBV-DNA positivity states among serologically screened patients and to compare the sensitivity of molecular and serological methods for the diagnosis of HBV infections. Cihan University-Erbil Scientific Journal (CUESJ) Corresponding Author: Amer A. Khaleel, College of Health Sciences-Hawler Medical University, Erbil, Iraq. E-mail: ameralikhaleel@yahoo.com Received: Mar 31, 2019 Accepted: Apr 17, 2019 Published: Jan 20, 2020 DOI: 10.24086/cuesj.v4n1y2020.pp52-56 Copyright © 2020 Amer Amer A. Khaleel, Salah T. Jalal, Saeed G. Hussain. This is an open-access article distributed under the Creative Commons Attribution License. https://creativecommons.org/licenses/by-nc-nd/4.0/ https://creativecommons.org/licenses/by-nc-nd/4.0/ Khaleel, et al.: Significance of HBV diagnosis by RT-PCR 53 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 52-56 MATERIALS AND METHODS For this cross-sectional study, a total of 256 blood samples were collected from HBV patients referred to Erbil Central Laboratory, from June 2017 to September 2017. Both sexes were included (154 males and 102 females), with age range between 20 and 60 years. Real-time polymerase chain reaction (RT-PCR) as a molecular and Elecsys assay as a serological method were used for the diagnosis and comparing their sensitivity purposes. For RT-PCR technique, 5 ml of venous blood were collected using EDTA tubes and centrifuged at 1000 rpm for 5 min and patients’ plasma was separated. Viral DNA was extracted from a 200 µl aliquot of serum using a Qiagen mini blood kit (Qiagen, Hilden, Germany) according to the manufacture instructions. Viral loads of HBV DNA were estimated by RT-PCR (HBV RG PCR artus, Germany). The amplification was performed in a 50 µl reaction mixture containing 30 µl of mixture of HBV RG Master mix (buffer, dNTP, primer, probe, and enzymes) and HBV RG inhibition control mixed with 20 µl of DNA template to each reaction. The primers HBV-Taq I (forward primer): CAA CCT CCA ATC ACT CAC CAAC and HBV-Taq 2 (reverse primer): ATA TGA TAA AAC GCC GCA GAC AC were used from the sequences of the conserved regions of the HBV surface gene used previously by Weinberger et al.[10] For performing RT-PCR, Rotorgene 3000 (a machine used routinely in Erbil Central Laboratory for detection of variety of DNAs and RNAs) was used following manufacture’s instructions. The RT-PCR cycling parameters consisted of denaturation at 95°C for 15 s, 55°C for 30 s, and 72°C for 15 s. The results were considered positive if a signal was detected in Cycling A FAM, whereas no signal detection indicated as negative results. The quantitation of HBV DNA was performed for all blood samples and the results were considered positive and significant if viral load was more than 1×105 viral copies/ ml. Titers <50 IU were considered as negative for HBV DNA. The screened results were categorized according to positive HBV DNA titers into inactive, gray zone, and active carriers. Quantitation of HBsAg was measured using the Elecsys HBsAg quantitative assay. Detection of HBsAg by Elecsys utilizes a sandwich principle and this assay has two stages: First, a complex is formed with two monoclonal HBsAg- specific antibodies, one of which is biotinylated and the other labeled with a ruthenium complex. This complex joins to the solid phase through the interaction of biotin and streptavidin after the attachment of streptavidin-coated microparticles.[11] The mixture is subsequently aspirated into a measuring cell, where application of a voltage induces chemiluminescent emission, which is measured by a photomultiplier. All serum samples were tested at a dilution of 1:400. The Elecsys HBsAg quantitative assay is calibrated to give results in IU/ml.[11] Statistical Analysis Statistical analysis was done using SPSS 23.0. HBV titers expressed as means ± standard error. Comparisons between active and inactive carriers use t-tests. Comparisons between multiple groups were performed by ANOVA. P ≤ 0.05 was considered as statistically significant. RESULTS AND DISCUSSION Of the 256 cases, 154 (60%) were male and 102 (40%) were female, with age range between 20 and 60 years. RT-PCR results showed that 163 of 256 samples were HBV DNA negative, from them, 20 samples showed positive results for HBsAg quantitation [Table 1]. The Asian Pacific Association for the Study of the Liver, European Association for the Study of the Liver, and the guidelines of the American Association for the Study of Liver Diseases refer that the disappearance of HBsAg is the ideal and cornerstone during natural course of HBV infection.[12-14] From the clinical standpoint of view, the progression from chronic hepatitis B to cirrhosis is increasingly signified through the HBV viral load.[8] Meanwhile, quantitative HBsAg level was regarded as marker for clearance, hepatitis activity, and immunological host response to control HBV replication and the likelihood of inactive virus carrier.[9,7,15] However, little is known about correlation between HBV replication and low levels of HBsAg, as reported in some patients with chronic HBV infection.[16-18] The results of the present study showed 12.26% HBs seropositivity (range 1000–5000 COI) among patients seronegative for HBV DNA [Table 2], with highly significant difference (P ≤ 0.001) between mean of HBV DNA seropositive Table 1: Baseline patient’s characteristics Variables All patients (n=256) Gender Male 154 (60%) Female 102 (40%) HBV DNA IU/ml 256 <50 (negative) 163 (63.67%) 101–500 22 (24.44%) 501–1000 10 (11.11%) 1001–2000 10 (11.11%) 2001–20,000 20 (22.22%) >20,000 31 (34.44%) qHBsAg tested 163/256 (63.67%) <10 COI 143 (55.85%) 1000–2000 COI 3 (15%) 2001–3000 COI 4 (20%) 3001–4000 COI 8 (40%) 4001–5000 COI 5 (25%) Anti-HBsAb tested 20 Positive 0.00 Negative 20 (100%) HBs positive, HBV DNA negative 20/163 qHBsAg: Quantitative hepatitis B surface antigen, HBV: Hepatitis B virus Table 2: Frequencies of HBV DNA seropositivity among patients negative for HBV DNA assay Number screened HBV DNA PCR +ve HBV DNA PCR−ve (n=163) HBs +ve HBs −ve n=256 93 (36.32%) 20 (12.26%) 143 (87.73%) HBV: Hepatitis B virus, PCR: Polymerase chain reaction Khaleel, et al.: Significance of HBV diagnosis by RT-PCR 54 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 52-56 and negative titers [Table 3]. Regarding to the gender positivity for PCR test, in total of 154 male patients 65 (42%) were positive and of 102 Female patients only 28 (27%) were positive [Table 4]. The seromarkers profile among patients negative for HBV DNA revealed anti-HBs antibody seronegative among the total 20 patients positive for HBsAg [Table 5]. However, results of anti-HBs titers among patients negative for HBV DNA showed 20 samples were positive showing different titers [Table 6]. During the immune-tolerant phase, HBsAg level is the highest one, and the level decreases progressively from immune tolerance to inactive phase that reflects a low replicative phase of immune control.[19] The detection of HBV infection is vital for diagnosis our group published a paper that delineated the impact of PCR DNA assay for the diagnosis of HBV infection.[20] The data of this study are agree with other studies[21] for the serologic profile of HBsAg seropositivity among patients screened negative for HBV DNA. They reported 6% of their samples gave HBsAg positive among donations negative for HBV DNA level, others[8] referred that in nine patients with HCC; HBV DNA was undetectable but the level of HBsAg was ≥2000 COI. Others reported 31.4% HBsAg positive among patients negative for HBV DNA.[22] However, data from another study showed that 100 of 140 patients were seronegative for HBV DNA.[23] Another study[24] reported that 69% of confirmed HBsAg-positive donations had undetectable HBV DNA levels. However, others[25,26] found that none of the HBsAg-positive donors with very low or negative for HBV DNA were anti-HBs antibody positive.[24,27] Among explanation for such unusual HBV serologic profile, the patients may be chronically infected with HBV particularly; they are in the non-replicative phase as part of the natural course of infection.[22] The question might be raised here is about such serologic profile of HBsAg positive among patients negative for HBV DNA, are these patients infectious and transmissible for community,[21] referred for the need of larger sample volume (5–10 ml) necessary for extraction and concentration to determine the real HBV DNA status among samples negative for DNA. In this study, Pearson’s correlation analysis revealed no significant correlation between serum HBV DNA and HBsAg levels (r = 0.331; P > 0.3) [Figure 1]. Different studies reported discrepant results regarding correlation between HBV DNA and HBsAg levels, our data were disagree with other studies who reported positive correlation.[28-34] Meanwhile, our results agree with others for no correlation between HBV DNA and HBsAg levels.[35,36] Such correlation is only weak in patients negative for HBsAg.[19,30,37,38] This is because the level of HBV DNA fluctuates from undetectable to >2,000,000 IU/ml.[12] This is why the level of HBsAg does not fall in the same proportion or level when correlated with HBV DNA level as a marker for Table 3: Mean titers of HBV DNA among patients screened Number screened HBV DNA PCR +ve (n=93) HBV DNA PCR −ve (n=163) P-value n=256 18,582,661.24±5,934,488.97 31.12±4.45 0.000 HBV: Hepatitis B virus, PCR: Polymerase chain reaction Figure 1: Pearson correlation between quantitative hepatitis B virus DNA level and hepatitis B surface antigen. R = 0.229, P = 0.331 Table 5: HBsAg and anti-HBs seromarkers profile among patients negative for HBV DNA Serological profile HBs Anti-HBs (n=20) >10 COI +ve <0.9 COI −ve <10 IU/ml −ve >10 IU/ml +ve HBV DNA PCR negative (n=163) 20 (12.27%) 143 (87.73%) 20 (100%) 0.00 HBV: Hepatitis B virus, PCR: Polymerase chain reaction, HBsAg: Hepatitis B surface antigen Table 4: Frequencies of HBV DNA positivity according to the gender Gender Positive PCR Negative PCR Total P-value Male 65 (42.20%) 89 (57.79%) 154 ≤0.001 Female 28 (27.45%) 74 (72.54%) 102 Total 93 (36.3232%) 163 (63.67%) 256 HBV: Hepatitis B virus, PCR: Polymerase chain reaction Table 6: Distribution of HBsAg and anti-HBs titers among patients negative for HBV DNA HBs titers COI Anti-HBsAb IU/ml Non-reactive <0.9 COI 1000–2000 2001–3000 3001–4000 4001–>5000 Non-reactive <10 Reactive >10 0.00 3 (15%) 4 (20%) 8 (40%) 5 (25%) 20 (100%) 0.00 HBV: Hepatitis B virus, PCR: Polymerase chain reaction, HBsAg: Hepatitis B surface antigen Khaleel, et al.: Significance of HBV diagnosis by RT-PCR 55 http://journals.cihanuniversity.edu.iq/index.php/cuesj CUESJ 2020, 4 (1): 52-56 disease progression.[4] Martinot-Peignoux et al.[39] referred that HBV DNA diminished with time from seroclearance; however, covalently closed circular DNA as integrated HBV DNA remained detectable in liver biopsies. It is thus critical for clinicians to be aware of the significance of HBV DNA serial measurement and lifelong follow-up to establish and confirm that inactive carrier condition is maintained.[4] Among major guidelines, if HBV DNA is undetectable, either prophylaxis with lamivudine or strict follow-up every 1–3 months for ALT and HBV DNA level is indicated.[13] CONCLUSION Serology will undoubtedly continue to be widely used in the detection of HBV infection; however, HBV DNA assays can serve as an important tool, especially in detecting low levels of HBV DNA and in patients with the past HBV infection. Data from the present study also concluded that negative or undetectable quantitation of HBV DNA level does not exclude HBV infection; as the level might fluctuate in different phases of HBV replication. This gives an impression and raising a question about significance of replacing test for HBsAg with quantitation of HBV DNA PCR assay. 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