Usefulness of in-house PCR methods for hepatitis B virus DNA detection
Introduction
Hepatitis B virus (HBV) is distributed worldwide, with an estimated 2 billion people infected with this disease (WHO, 2014). HBV is a circular, partially double-stranded DNA virus belonging to the hepadnavirus family (Takahashi et al., 1976). HBV is the principal etiological agent of acute or chronic liver disease, which eventually evolves into hepatocarcinoma and liver cirrhosis (Alexopoulou and Karayiannis, 2014).
The detection of HBV DNA is useful for characterizing the course of chronic hepatitis B infection and demonstrating the effects of antiviral drugs on HBV replication (EASL, 2012). Furthermore, the introduction of nucleic acid amplification tests has significantly reduced the diagnostic window period for screening blood and plasma donors (Enjalbert et al., 2014). Serum HBV DNA detection using highly sensitive methods is essential to facilitate early diagnosis, identify the presence of occult hepatitis in HBsAg-negative patients (Gerlich et al., 2010) or identify patients with mutations in the precore region where HBeAg is typically not detected (Valsamakis, 2007).
Several commercial assays have been developed for the detection and quantitation of HBV DNA in serum or plasma samples. These assays are based on the amplification of DNA targets using HBV-specific primers (COBAS Amplicor HBV Monitor); branched-DNA signal amplification, such as the VERSANT HBV DNA 3.0 assay; the hybridization of a chemiluminescent probe as hybrid capture; or a specific fluorescent probe in real-time PCR (Abbott Real-time HBV or Cobas TaqMan HBV test) (Ciotti et al., 2008, Morris et al., 2013, Shi et al., 2008, Yao et al., 2004, Yuan et al., 2004). In Brazil, HBV DNA testing is commonly conducted to monitor HBV treatment, but commercial assays are generally used. Recently, Santos et al. (2014) developed an in-house real-time PCR method for HBV DNA detection during the NAT screening of hepatitis B donors, but the cost of this assay is not available.
The costs of commercial assays are too high for use in developing countries, such as Brazil, which also have much heavier HBV burden than developed countries. The aim of the present study was to evaluate the clinical performance of three qualitative methods for HBV DNA detection in serum samples. PCR was applied to analyze the serum of three panels of samples from individuals referred to the Brazilian Reference Center for Viral Hepatitis and compared with the results obtained using the COBAS Amplicor HBV Monitor System (Roche Diagnostics) and Cobas TaqMan HBV Test v2.0 (Roche Diagnostics).
Section snippets
Clinical specimens
Three panels of sera samples were obtained from HBsAg-reactive individuals referred to the Viral Hepatitis Laboratory at Oswaldo Cruz Institute (Oswaldo Cruz Foundation, FIOCRUZ). These individuals did not receive antiviral treatment, and information regarding the clinical phase was not available.
The first panel comprised 50 samples tested using four protocols (one-round PCR to amplify the core region, one-round PCR to amplify the surface region, semi-nested PCR to amplify the surface region
Populations studied
Three panels of samples were used in the present study. Panel I comprised 50 samples from HBsAg-reactive individuals, including 15 females and 35 males, with a mean age of 40.3 years (±9.5). Panel II comprised 87 HBsAg positive samples, including 43 females and 44 males, with a mean age of 45.8 years (±13.4). The serological information is summarized in Table 2. Panel III comprised two individuals, where one individual provided seven serum samples for seven months after the initial onset of
Discussion
Molecular techniques are important for the diagnosis and management of HBV infection (EASL, 2012). Because HBV DNA is detectable in serum prior to biochemical evidence of hepatitis and persists at variable levels throughout the course of this chronic disease, sensitive and accurate HBV DNA tests are required. Furthermore, reliable but inexpensive HBV DNA assays are extremely important for the control and treatment of HBV infection in low-income countries, such as Brazil.
In the present study, we
Conclusion
In-house semi-nested PCR presented the best performance among the in-house methods evaluated in the present study and is a promising alternative for HBV DNA detection in low-resource settings. Under these conditions, in-house semi-nested PCR could be employed for the initial screening of HBV DNA-reactive samples.
Conflict of interest
The authors disclose no actual or potential conflicts of interest, including any financial, personal or other relationships with people or organizations that could inappropriately influence the results obtained in the present study.
Acknowledgments
The authors would like to thank Juliana Custódio Miguel and Elisangela Ferreira da Silva for technical assistance with blood processing. This research was financially supported through grants from the Foundation for Supporting Research at Rio de Janeiro State (FAPERJ), Brazilian National Counsel of Technological and Scientific Development (CNPq) and the Oswaldo Cruz Foundation (FIOCRUZ).
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