This study included 134 patients with chronic HBV infection who were treated at the Viral Hepatitis Clinic Specialized Center of Research in Tropical Medicine in Rondonia (CEPEM). A control group of 30 donors, who all tested negative por ELISA for human immunodeficiency virus (HIV) 1 and 2, HBsAg, anti-HBc and anti-HCV, and who attended the blood bank of the State of Rondonia (FHEMERON) was included in the study. We also included 10 and 26 serum samples from individuals with chronic HCV and co-infection with HBV/HDV respectively.
This study was approved by the Brazilian Institutional Ethics Committee of the Centro de Pesquisa em Medicina Tropical (CEPEM), with process number 107/10. Written, informed consent was obtained from each patient for the publication of this manuscript and any accompanying images.
Viral DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and 200 μl of serum according to the manufacturer’s instructions. Three samples with viral load known were tested: first with high viral load and the others medium and low viral load. After this were diluted with final volume 200 ul, 100 ul and 50 ul. Besides, HBV DNA was extracted from 22 samples of individuals with same profile serological: total isolated anti-HBc. These samples were diluted in 50 uL and 200 uL to optimize the final volume of extraction for samples with low viral load. Subsequently the samples were submitted to reaction for sensitivity analysis. Precipitated DNA was resuspended in elution buffer and stored at −20°C until further use. To avoid false-positive results, we followed strict procedures for nucleic acid amplification .
Primer concentrations were optimized using a concentration gradient ranging from 100–900 nM and SYBR® Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). TaqMan® probe concentrations were similarly optimized using a concentration gradient ranging from 50–300 nM.
Ultra-sensitive real-time PCR
The assay was performed on an ABI 7500 platform (Applied Biosystems) with 30 μl reaction volumes containing 15 μl TaqMan® Universal Master Mix (Applied Biosystems), 3 μl HBVRO1 forward primer (5′-AGGAGGCTGTAGGCATAAATTGG 3′), 3 μl reverse primer (5′-GCACAGCTTGGAGGCTTG-3′), 0.6 μl probe (5′-FAM TCACCTCTGCCTAATC-3′-MGB, 6 μl extracted DNA and 2.4 μl of water.
Construction of the standard curve
To construct the standard curve, we initially used conventional PCR with amplification of a 109 bp fragment in the pre-core region according Kavita 2006 adapted, selecting five samples with known viral load. Approximately 50 ng of DNA was used per reaction with a final volume of 50 μl. Amplification was performed on an ABI Prism 7500 Veriti (Applied Biosystems) with an initial denaturation temperature of 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 58°C for 45 sec, 72°C for 1 min and a final extension of 10 min at 72°C. The selected fragment was purified, ligated to the p-GEM-T Easy® vector (Promega, New York, USA), cloned into a prokaryotic system and subsequent linearization with PstI (Invitrogen™ Life Technologies, Carlsbad, CA, USA). Absolute quantitation was used to determine the exact number of DNA molecules for estimating viral load.
Inter- and intra-assay variation and reproducibility of real-time PCR
To determine intra-experimental variation, we tested the reproducibility of six HBV-positive sera with different viral loads, in duplicate, in the same reaction setup. The same set of samples was used in three experiments performed on different days, to estimate inter-experimental variation in the estimation of viral load. Reproducibility was estimated by calculating the coefficient of variation (CV), which is calculated as the ratio of the standard deviation and the mean of the replicates.
HBV DNA quantification
Calibration curves for HBV DNA were constructed using the OptiQuant® HBV-DNA Quantification Panel, (AcroMetrix Europe BV). Specifically, a serial dilution was prepared from the standards included in the kit, ranging from 2 × 102 – 2 × 106 IU/ml. For our in-house plasmid, pHBVRO, a serial dilution was prepared with a range 2 × 103 – 2 × 109 copies/ml. The concentration was measured spectrophotometrically both by using a NanoDrop® ND-1000 (Thermo Scientific NanoDrop Products, Wilmington, Delaware), and the measurements were recorded in units of nanograms per microliter, which was converted into copies per microliter by using the following equation: ([x ng/μL × 10-9] / [p-GEM-T Easy® vector and 109 HBV DNAbps × 660]) × 6.022e23 = y copies/μL. Using linear regression a standard curve was constructed, which was used to convert copies/ml to standard international units (IU/ml).
To compare the performance of our in-house method (qHBVRO) with that of the commercial kit, OptiQuant® HBV-DNA Quantification Panel (AcroMetrix® Europe BV), we tested 100 serum samples collected from patients chronically infected with HBV.
The correlation between the AcroMetrix® test kit and the qHBVRO assay was calculated using GraphPad 5.0 (GraphPad software) and a two-tailed Pearson′s correlation test with a confidence interval of 95%. The units for measuring viral load (copies/ml and IU/ml) were transformed to log base 10.
We tested 30 samples from blood donors, 10 serum samples from mono-infected HCV patients, 28 samples from patients co-infected with HBV/HDV and 15 samples that were judged indeterminate for HBV surface antigen by the Serology Laboratory of Viral Hepatitis Clinic - IPEPATRO. All samples were submitted to qHBV PCR to determine viral load.
We selected 15 sera, which were considered indeterminate for HBsAg by ELISA when tested in duplicate and which had absorbance values within the gray zone, or ± 10% cut-off confidence interval. These samples were subjected to three separate assays, with each sample performed in duplicate to evaluate the performance of our assay in detecting uncertain samples.