Journal of Virological Methods 201 (2014) 24–30
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Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation Dramane Kania a,b,c,d,∗ , Laure Ottomani e , Nicolas Meda b , Marianne Peries c , Pierre Dujols c,d,e , Karine Bolloré c,d , Wendy Rénier c,d , Johannes Viljoen c,f , Jacques Ducos c,d,e , Philippe Van de Perre c,d,e , Edouard Tuaillon c,d,e a
Laboratoire de Virologie, Centre Muraz, Bobo-Dioulasso, Burkina-Faso Unité VIH et Maladies Associées, Centre Muraz, Bobo-Dioulasso, Burkina Faso c INSERM U 1058, Infection by HIV and by agents with mucocutaneous tropism: from pathogenesis to prevention, 34394 Montpellier, France d Université Montpellier 1, 34090 Montpellier, France e CHU Montpellier, Département de Bact´eriologie-Virologie et Département d’Information Médicale, 34295 Montpellier, France f Africa Centre for Health and Population Studies, University of KwaZulu-Natal, Durban, South Africa b
a b s t r a c t Article history: Received 28 September 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 18 February 2014 Keywords: Hepatitis B virus hepatitis B virus diagnosis hepatitis B virus monitoring quantitative PCR
In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a worldwide public health problem. Approximately 5% of the global population are chronically infected with HBV (WHO, 2009; Hwang and Cheung, 2011). As a consequence, almost 400 million chronic carriers are currently exposed to the risk of complications of this persistent infection, such as cirrhosis, liver failure and/or hepatocellular carcinoma. The infection is especially hyper-endemic (prevalence ≥ 8%) in subSaharan Africa and South-East Asia, representing more than 75% of total chronic carriers worldwide (WHO, 2009; Hwang and Cheung,
∗ Corresponding author: Centre Muraz, Avenu Mamadou Konate, ´ 01 BP 390 BoboDioulasso 01, Burkina Faso. Tel.: +226 20 97 01 02; fax: +226 20 97 04 57. E-mail address: [email protected] (D. Kania). 0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2014.01.015
2011). Effective strategies including prevention through vaccination, improved clinical care and biological monitoring for those infected chronically, are required to control the infection and its expansion (WHO, 2010). The presence of HBV DNA in blood reflects virus replication and is a key marker to characterise the course of chronic HBV infection (Hoofnagle, 1990; Kao, 2008; Zacharakis et al., 2008), and together with serum alanine aminotransferase, histological grade, stages of necroinflammation and liver fibrosis, constitutes one of the main criteria currently used for treatment initiation in the developed world (Lok and McMahon, 2009; European, 2012). HBV DNA quantitation in plasma or serum is a standard measure to monitor efficacy or need for initiation of treatment for persons chronically infected with HBV, since sustainable suppression of HBV replication is one of the end points. Improved access to quantitative assays for HBV DNA measurement would be highly effective to inform on the
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status of infection when used in conjunction with other parameters of HBV infection. Several molecular techniques based on target or signal amplification are commercially available, but in addition to becoming increasingly automated, they have the disadvantage of not being freely available in developing countries, mostly due to prohibitive high cost (Hui et al., 2006; Laperche et al., 2006; Pol et al., 2008; Lin et al., 2009; Yang et al., 2009; Caliendo et al., 2011; Ismail et al., 2011). In-house developed real-time PCR (qPCR) techniques are relatively low cost assays and provide an interesting alternative to monitor HBV infection in resource-limited settings (Mendy et al., 2006; Welzel et al., 2006; Daniel et al., 2009; Sitnik et al., 2010). There is a need to compare the performance of variously described methods with commercially available assays. The targeted regions of the HBV genome used for PCR amplification differ according to the various protocols described (S-, C- or X gene). HBV genetic variability is an additional factor that may affect the ability of primers and probes to effectively hybridize, leading to impaired HBV DNA quantitation as a result of a mismatch for a particular circulating genotype (Pol et al., 2008). Most HBV genotypes found in Europe and Africa are known, but with different distribution patterns in the various geographical regions (Pujol et al., 2009). Therefore it is imperative that alternative qPCR methods should be designed and evaluated according to the diversity of circulating genotypes found in a particular setting. The aim of this study was to compare the performance of two qPCR assays targeting the X- and S-genes of HBV, respectively, using an open system (Light Cycler 480), to a fully automated closed system (Roche Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0). Results of HBV DNA quantitation using all three assays were analysed according to HBV genotype. 2. Materials and Methods 2.1. Clinical specimen selection A total of 122 archived clinical serum samples collected between November 2006 and April 2012 from patients monitored for HBV infection and/or liver disease at the Virology Laboratories of Toulouse and Montpellier University Hospitals were selected for HBV DNA quantitation by both qPCRs. Both hospital laboratories used the same commercial real-time PCR assay (Cobas AmpliPrep/Cobas TaqMan [CAP/CTM] HBV Test, version 2.0 [v2.0], Roche Diagnostics GmbH, Mannheim, Germany) to quantify HBV DNA in routine practice, and results produced by this assay were used as the gold standard to which the two qPCR techniques were compared. HBV viral load results obtained by gold standard assay ranged from 1.34 to 7.84 Log10 IU/ml. All samples were positive for hepatitis B surface antigen (HBsAg), with 26.4% being dually positive for HBe antigen. The genotype of HBV was determined using the TRUGENE® HBV Genotyping Kit on the OpenGene® DNA Sequencing System (Siemens Healthcare Diagnostics Inc, Tarrytown, USA) and was available for 68 of 122 samples as follows: A (n = 22), B (n = 3), C (n = 2), D (n = 29), E (n = 11) and G (n = 1). Fifty nine HBV-negative samples, defined as specimens negative for HBsAg, anti-HBc antibodies and HBV DNA, were included in the study to determine specificity of qPCRs. 2.2. HBV DNA detection and quantitation using Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0 All selected specimens were previously tested for HBV DNA in Montpellier or Toulouse laboratory using the CAP/CTM HBV Test, v2.0, which is a fully automated viral nucleic acid extraction and amplification system that targets the HBV precore/core gene (Chevaliez et al., 2010). Samples were processed in
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accordance to the manufacturer’s instructions using 500 l of serum for extraction. The amplification data were analysed with AmpliLink software, version 3.3.5. The lower detection limit (LDL) is 19 IU/ml in serum and the upper detection limit is approximately 1.7 × 108 IU/ml. Specific primers and TaqMan probes were used to detect assay target, and an internal DNA quantitation standard was amplified in each sample. Results obtained by commercial assay on clinical specimens were used as the reference standard to determine correlation, agreement characteristics, clinical sensitivity and specificity of the two developed qPCR techniques. 2.3. DNA extraction for qPCR assays DNA was extracted manually from 200 l of serum with the QIAamp® DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Template DNA was eluted into 50 l of kit elution buffer and either used directly, or stored at -20 ◦ C prior to use. 2.4. HBV DNA detection and quantitation using qPCR assays The two qPCR assays were used with slight modifications targeting the HBV X- and S-genes as previously described by Welzel et al. (2006) and Daniel et al. (2009), respectively. Modifications were specifically directed at the extraction kit, volume of sample used, concentration of master mixture components, PCR apparatus and qPCR2 probe staining. Primer and probe sequences of the two qPCR methods are shown in Table 1. For both qPCR techniques, hydrolysis probes (also called TaqMan probes), were selected. qPCR1 used a primers/probe set targeting a conserved region within the HBV X gene, and qPCR2 a conserved region within the HBV S gene. Alignment of nucleotide sequences within the X and S genes of HBV showed that the primers and probe selected for qPCR1 generated much more frequent mismatches than those employed for qPCR2. Based on 12 published complete HBV genomic sequences from genotypes A to F (Stuyver et al., 2000) and using global gene HBX sequence alignments, we identified 10, 7 and 14 nucleotide mismatches for forward primer, reverse primer and probe of qPCR1, respectively. By contrast, only one mismatch was observed for each primer and probe of qPCR2 using global HBS gene sequence alignments and the HBV complete genomes cited above (see supplementary data). For each qPCR, 10 l of DNA extract was added to 40 l of master mixture incorporating 5 l of forward primer at 10 pmol/l, 5 l of reverse primer at 10 pmol/l, 1 l of dual-labelled fluorogenic probe at 10 pmol/l, 25 l of 2X TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA) and 4 l of RNase/DNase-free water. Each reaction consisted of 2 min at 50 ◦ C, 10 min at 95 ◦ C followed by 50 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C each. The two qPCR assays were performed using the LightCycler 480 II Real-Time PCR Instrument (Roche Diagnostics GmbH, Mannheim, Germany), and amplification data were analysed using the LightCycler 480 Software (Roche Diagnostics). 2.5. Standard and control samples The 2nd World Health Organization (WHO) International Standard for HBV DNA Nucleic Acid Amplification Techniques (NAT), product code 97/750, was obtained from the National Institute for Biological Standards and Control (NIBSC; Hertfordshire, United Kingdom) and used as an external standard for each qPCR assay. The lyophilised material of this standard was reconstituted in 0.5 ml of sterile nuclease-free water and the final concentration obtained was 1,000,000 (6 Log10 ) IU/ml (according to the manufacturer’s instructions). For each experiment, 200 l of the reconstituted standard were extracted together with the clinical samples and serially
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Table 1 Primer and hydrolysis probe sequences used for HBV in-house quantitative real-time PCR assays (qPCR1 and qPCR2). The primers and probes described below are adapted from previous published work (Daniel et al., 2009; Welzel et al., 2006). All the probes were labelled at the 5’ end with the reporter dye (R) FAM (6-carboxyfluorescein) and at the 3’ end with the quencher dye (Q) TAMRA (6-carboxytetramethylrhodamine).
Forward primer Reverse primer Probe
qPCR 1
qPCR 2
HBV-F3: 5’-GGC CAT CAG CGC ATG C-3’ HBV-R3M3: 5’-C [5-NitIdl] GCT GCG AGC AAA ACA-3’ HBV-P3: 5’–CTC TGC CGA TCC ATA CTG CGG AAC TC– 3’
HBV TAQ 1: 5’ -GTG TCT GCG GCG TTT TAT CA- 3’ HBV TAQ 2: 5’-GAC AAA CGG GCA ACA TAC CTT- 3’ HBV TAQ PR: 5’ - CCT CTT CAT CCT GCT GCT ATG CCT CAT C - 3’
diluted tenfold to obtain 5 different HBV DNA concentrations (1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 and 1 × 102 IU/ml). The standard curve obtained from these concentrations was used to estimate HBV DNA amount in clinical samples in each run of the qPCR assays. In addition, we prepared a low-positive control (LPC) by combining 4 clinical samples with known HBV DNA load, yielding an LPC with an estimated final concentration of approximately 2,500 IU/ml. Negative controls consisted of RNase/DNase-free water. By contrast to the qPCRs using a calibrated curve produced by an external control, the reference assay uses an internal control as HBV quantitation standard (QS). The QS was added to each specimen at a known copy number and was carried through all subsequent steps of specimen preparation, simultaneous PCR amplification and detection. The Cobas TaqMan Analyzer calculates the HBV DNA concentration in the test specimens by comparing the HBV signal to the HBV QS signal for each specimen and control. 2.6. Determination of the analytical performance of both qPCR assays The LDL (or threshold) for each assay was determined using 16 serial concentrations of the WHO International Standard (10; 20; 30; 40; 50; 60; 70; 80; 90; 100; 125; 200; 250; 400;500; 1,000 IU/ml) in at least 10 separate runs. The linearity of each qPCR technique was established by testing in 24 separate PCR-runs, five dilution series (1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 and 1 × 102 IU/ml) of the WHO HBV DNA International Standard. Intra-assay variability of each qPCR assay was assessed by analyzing in a single PCR-run, nine replicates of the LPC at 2500 IU/ml, four replicates of four serial concentrations of the WHO International Standard (1x105 , 1x104 , 1x103 and 1x102 IU/ml) and a triplicate of six clinical samples quantified by means of the Roche Cobas test (503; 2,502; 5,500; 53,400; 564,000; and 6,220,000 IU/ml). The inter-assay variability of each qPCR technique was evaluated (i) by a same operator testing in 10 separate PCR-runs, the LPC and the five serial concentrations of the WHO International Standard; and furthermore, (ii) with two different operators determining five times on separate PCR-runs, the five serial concentrations of the WHO International Standard. qPCR assay specificity was assessed by testing the 59 HBVnegative serum samples including eight HCV-RNA positive samples, four HIV-RNA positive samples and one dual HIV RNA/HCV RNA-positive sample. 2.7. Statistical analysis Data analysis was performed using the SAS version 9.3 software package (SAS Institute, Cary, NC). HBV DNA concentrations obtained were accordingly converted to logarithm of ten (Log10 ) values before performing statistical analysis. The LDL of each qPCR assay was estimated using probit analysis. Coefficient of variation ([(SD/Means) × 100]; SD, the standard deviation) was calculated to estimate intra-assay and inter-assay variability. Continuous variables were described as median and inter-quartile range
(IQR) and were compared using the non-parametric Wilcoxon Mann–Whitney test due to the absence of normal distribution. Chi-square test was used to compare categorical variables. Statistical significance was set at a p value of < 0.05. The Spearman correlation coefficients were calculated to determine the relationship between the HBV DNA levels obtained with the commercial and qPCR assays. Deming regression analysis was used to compare quantitative results obtained between both qPCR and the reference assay. The Bland–Altman method (Bland and Altman, 1995) was used to measure agreement between assays. 3. Results 3.1. Analytical performance of the two qPCR assays Using serial concentrations of the WHO International Standard on repeated runs, the lowest HBV DNA concentration giving 100% detection signal was 125 IU/ml using the qPCR1 assay and 90 IU/ml using the qPCR2. Probit analysis predicts a 95% LDL of 104 IU/ml (95% confidence interval [CI]: 78–176) for qPCR1 and 91 IU/ml (95% CI: 73–292) for qPCR2. Linearity of the measures was observed from 100 to 1,000,000 IU/ml for both assays (Fig. 1). The inter-quartile ranges were increasingly smaller from low concentrations to high concentrations. The coefficient of determination was R2 = 0.998 for qPCR1 and R2 = 0.999 for qPCR2. The mean of within-run and between-run standard deviations for each qPCR assay and its coefficients of variation are represented in Table 2. All 59 samples that were found to be negative for HBV by the commercial assay, were also non-reactive by the two qPCR methods, conferring an analytical specificity of 100% for both assays. Of 122 samples with HBV DNA detected by commercial assay, 121 were confirmed positive using the qPCR1 and 122 using the qPCR2 assay, indicating a clinical sensitivity of 99.2% for qPCR1 and 100% for qPCR 2. The false negative HBV DNA result obtained using the qPCR1 (HBV DNA undetectable), exhibited a low HBV DNA viral load value of 2.87 Log10 IU/ml by reference assay, and 2.67 Log10 IU/ml by qPCR2. The negative result obtained by the qPCR1 was confirmed on a repeated assessment of this sample, classified as HBV genotype D. 3.2. Correlation and agreement of the two qPCR techniques with the Roche CAP/CTM HBV test v2.0 For 122 specimens the three assays yielded a median HBV DNA load of 3.45 Log10 IU/ml (IQR 25-75: 2.83–4.98) for the reference assay, 3.65 Log10 IU/ml (IQR 25-75: 2.87–4.84) for qPCR1 assay and 3.61 Log10 IU/ml (IQR 25-75: 3.05–4.95) for qPCR2 assay. Both qPCR assays were correlated with the reference test (r = 0.834, p < 0.0001 [qPCR1] and r = 0.870, p = 0.0001 [qPCR2]) (Fig. 2). No significant difference was observed between reference and qPCR tests (p = 0.720 and 0.301, respectively) but a visible large dispersion is observed in Fig. 2A, suggesting more discrepant values using the qPCR1 compared to those obtained by qPCR2. There was good agreement by Bland–Altman analysis between qPCR assays and
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Fig. 1. qPCR standard curves established using 5 serial dilutions of the WHO HBV DNA International Standard. The qPCR1 standard curve is represented by (A) and the qPCR2 standard by (B). The cycle threshold (Ct) is the number of cycles before the fluorescence passes a fixed limit (time to positivity). The solid line connects the median Ct values and the 25% and 75% inter-quartile ranges (box plot) for each dilution.
Table 2 Intra-run and inter-run variability. For intra-run variability, WHO HBV DNA International Standard serial dilutions, low-positive control (LPC) and selected clinical samples were tested at least 3 times in the same run using each in-house qPCR assay. For inter-run variability, LPC and WHO International Standard serial dilutions were tested at least 5 times in different runs using each in-house qPCR assay and performed by two different operators. In-house qPCR1
Intra-run Sample 1 (2.70 Log10 IU/ml) Sample 2 (3.74 Log10 IU/ml) Sample 3 (4.73 Log10 IU/ml) Sample 4 (5.75 Log10 IU/ml) Sample 5 (6.79 Log10 IU/ml) LPC (3.40 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (4.11 Log10 IU/ml) Inter-run Operator 1 LPC (3.40 Log10 IU/ml) STD 1 (6.00 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (3.90 Log10 IU/ml) Operator 2 STD 1 (6.00 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (4.00 Log10 IU/ml)
In-house qPCR2
Mean (Log10 IU/ml)
SD (Log10 IU/ml)
%CV
Mean (Log10 IU/ml)
SD (Log10 IU/ml)
%CV
2.43 2.44 4.47 5.82 6.64 3.05 4.99 3.98 3.07 1.96 3.89
0.07 0.15 1.01 0.01 0.00 0.11 0.04 0.02 0.07 0.05 0.05
2.80 6.18 0.31 0.19 0.06 3.66 0.80 0.46 2.40 2.36 1.38
2.31 3.64 4.50 5.79 6.52 3.24 5.00 4.00 2.99 2.00 4.00
0.24 0.06 0.02 0.01 0.01 0.05 0.00 0.01 0.02 0.01 0.04
10.18 1.58 0.34 0.10 0.22 1.51 0.08 0.21 0.71 0.66 1.06
3.54 5.98 5.00 4.00 3.02 1.95 3.92
0.14 0.04 0.03 0.04 0.05 0.13 0.07
3.96 0.60 0.59 0.92 1.70 6.75 1.81
3.58 5.99 5.01 3.99 3.03 1.98 3.93
0.15 0.04 0.04 0.03 0.07 0.05 0.06
4.14 0.61 0.84 0.79 2.34 2.41 1.60
5.99 5.01 4.00 2.99 2.25 4.05
0.02 0.01 0.02 0.05 0.47 0.11
0.26 0.23 0.56 1.58 20.81 2.79
5.92 5.06 4.02 3.00 1.97 4.00
0.19 0.18 0.07 0.07 0.03 0.11
3.15 3.60 1.81 2.18 1.64 2.70
LPC, low-positive control; STD, standard; SD, standard deviation; CV, coefficient of variation; IU, international unit; ml, milliliter; Log10, logarithm of ten; qPCR, quantitative real-time polymerase chain reaction.
reference assay (Fig. 3). The mean difference (bias ± SD) between the qPCR and reference tests was + 0.07 ± 0.77 Log IU/ml for qPCR1, and + 0.13 ± 0.52 Log IU/ml for qPCR2. Out of the 122 HBV positive samples, 113 (92.6%) of the qPCR1 results, and 114 (93.4%) of the qPCR2 results, were within 95% limit of agreement. As shown in Fig. 3A, a proportional bias was observed using qPCR1 in which HBV DNA concentrations tended to be underestimated in samples exhibiting a low viral load level, and overestimated when the values
were high. qPCR1 gave HBV DNA measurements equivalent (i.e., difference in HBV DNA < 0.5 Log10 IU/ml) to the reference assay results in 73 out of 122 (59.8%) samples. In contrast, equivalent HBV DNA results were obtained in 87 out of 122 (71.3%) samples using the qPCR2. A trend for a higher concordance between the reference assay and the qPCR2 assay was observed when compared to qPCR1 (p = 0.059). Among the discrepant results with ≥ 0.5 Log10 IU/ml difference, qPCR values were lower than reference values in
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Fig. 2. Correlation between Cobas TaqMan v2.0 test and the in-house qPCR assays for HBV DNA quantitation: (A) Cobas TaqMan v2.0 test versus qPCR1; and (B) Cobas TaqMan v2.0 test versus qPCR2. The fitted regressions are represented with solid lines.
Fig. 3. Bland–Altman representations for agreement between Cobas TaqMan v2.0 test and in-house qPCR assays: (A) Cobas test versus qPCR1; and (B) Cobas test versus qPCR2. The mean differences and the 95% limits of agreement are represented by horizontal solid lines. For symbols, white circles represent HBV genotype A (n = 22), black circles genotype B (n = 3), black triangles genotype C (n = 2), white triangles genotype D (n = 29), white squares genotype E (n = 11), black squares genotype G (n = 1) and asterisks non-defined genotypes (n = 54). Discontinuous oblique line in Fig. 3A indicates the proportional bias observed between the qPCR1 and the Cobas TaqMan.
21 out of 49 cases (42.9%) for qPCR1, and 10 out of 35 cases (28.6%) for qPCR2.
The main characteristics of the three assays (Roche CAP/CTM, qPCR1 and qPCR2) were described in the Table 3.
3.3. Impact of HBV genotypes on DNA quantification by the Roche CAP/CTM HBV test v2.0 and qPCR assays
4. Discussion
Available HBV genotype (n = 68), was classified into 6 genotypes as follows: A (22), B (3), C (2), D (29), E (11) and G (1). The impact of HBV diversity was explored in the three most frequent genotypes (A, D and E). Similar HBV DNA levels were obtained by reference and qPCR assays for genotypes A (p = 0.782), D (p = 0.923) and E (p = 0.795). Discrepant results (difference in HBV DNA ≥ 0.5 Log10 IU/ml), were found in 7/29 (D), 4/11 (E) and 2/22 (A), genotyped samples by qPCR1, whereas, qPCR2 yielded discrepant results in 4/29 (D), and 1/11 (E) genotyped samples. Frequent discrepant results between the commercial and qPCR assays were observed for genotype B (2/3) and G (1/1), with lower HBV DNA values found in 2 cases for qPCRs (Fig. 3). However, due to the low number of those genotypes this observation did not permit a clear conclusion.
In this study, two qPCR techniques were compared to a commercial assay using a large number of clinical samples with different HBV genotypes. The two qPCR techniques showed high analytical performance for HBV DNA quantitation in serum. A notable linearity was observed between qPCR and reference assays over a broad quantitative range with the ability to detect DNA values over 107 Log10 IU/ml. The intra-assay and inter-assay variations were also very good and close to those reported for other commercial molecular assays (Laperche et al., 2006; Ismail et al., 2011). The coefficients of variation were excellent over a 3 Log10 IU/ml range with variability below 5%. As expected, technique variability was greatest near the LDL of each assay (20.8% for qPCR1 and 10.2% for qPCR2).
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Table 3 Main characteristics of the two in-house real-time PCR assays (qPCR1 and qPCR2) compared to the Roche Cobas AmpliPrep/Cobas TaqMan test, version 2.0 for HBV DNA measurement. Characteristics
CAP/CTM
qPCR1
qPCR2
Target region Probe technology Extraction method Amplification system Amplification software Input volume for extraction (l) Lower detection limit (IU/ml) Upper detection limit (IU/ml) Linearity (R2 ) Intra-run %CV Inter-run %CV
pre-Core/Core gene TaqMan Cobas AmpliPrep Cobas TaqMan Amplilink v3.3.5 500a 19 [11-107]a 170000000a 0.9987a 14.50 [10.00-27.00]a 14.83 [10.00-28.00]a
X-gene TaqMan Qiagen manual LightCycler 480 LC 480 software 200 104 [78-176] 100000000 0.9979 1.38 [0.06-6.18] 2.30 [0.23-20.81]
S-gene TaqMan Qiagen manual LightCycler 480 LC 480 software 200 91 [73-292] 100000000 0.9995 1.06 [0.08-10.18] 2.15 [0.61-4.14]
a Data obtained from the document of procedure instructions of the Roche Cobas® AmpliPrep/Cobas TaqMan HBV kit (package insert informations); qPCR1, in-house real-time PCR assay 1; qPCR2, in-house real-time PCR assay 2; R2 , Coefficient of determination; %CV, coefficient of variation.
The two generic tests presented a LDL of almost 100 IU/ml which was higher when compared to the Roche assay (19 IU/ml in serum). However, this cut-off value is better than the LDL of the commercial signal amplification techniques from Bayer (347 IU/ml for the bDNA v3.0 VERSANT assay) (Yang et al., 2009). These levels of detection of HBV DNA load obtained by our qPCR assays would enable routine clinical management and are sufficient for detection of active HBV replication in patients with DNA levels more than 2,000 IU/ml (Shi and Shi, 2009; Chakravarty, 2012; European, 2012). Such displayed sensitivity could also be adequate for routine monitoring of response to antiviral therapy, although ultra-sensitive methods are more desirable to detect suboptimal suppression of viral replication in patients receiving nucleotide/nucleoside analogues. Likewise, the two qPCR assays may fail to detect serum HBV DNA in case of occult HBV infection, but this situation is infrequent (Zheng et al., 2011). Theoretically, it remains possible to improve the LDL of qPCR assays by increasing the quantity of serum used for the DNA extraction phase. In our study, a 2.5 fold less serum volume was used in the qPCR assays by comparison to the reference assay (200 l for qPCRs vs. 500 l for Roche). In addition, we only used 20% of total available eluted nucleic acid per sample in our qPCR reaction. Therefore, by increasing the amount of eluted nucleic acids, the LDL of the qPCR methods could potentially be set as low as 40 IU/ml when using an input volume of serum similar to those used in the commercial assay. Our study, based on a large number of samples with different genotypes, confirms the potential value of these assays. For each qPCR assay, good agreement and correlation were obtained with the reference test. Welzel et al. reported a good correlation using the primers and probe of the qPCR1 in comparison to the Bayer VERSANT HBV DNA 3.0 on HBV DNA-positive clinical specimens, however, discrepant results (differences of HBV DNA ≥ 0.5 Log10 IU/ml) were frequent (Welzel et al., 2006). Daniel et al. also reported a good correlation between a PCR using the primers and probe of the qPCR2 and the Artus HBV RG PCR assay (Daniel et al., 2009). Genotypes were taken into account in the first study, but not in the second one, and both explored a small number of samples, 29 and 30 HBV DNA positive samples, respectively. The mean nucleotide divergence is estimated at 8% between the eight distinct HBV genotypes denoted from A to H. (Norder et al., 1993; Stuyver et al., 2000). Geographic distribution of HBV variants differs in populations around the globe. Genotypes E, D and A are the most prevalent in sub-Saharan Africa (Vray et al., 2006; Hubschen et al., 2008; Mansour et al., 2012), whereas, genotypes A, D and G are overrepresented in European countries (Huy and Abe, 2004; Pujol et al., 2009). Polymorphisms within the primers/probe and the three different HBV regions targeted by the assays (C, X and S-genes) may affect accuracy of viral load measurements differently. Although we confirmed the good performance obtained with the qPCR1, we also
observed a lower agreement with the commercial assay, a proportional bias, and one HBV genotype D sample with a low DNA viral load that was not detected by this assay. These results suggest that the ability of the qPCR2 technique may be superior to the qPCR1 test for HBV DNA detection and quantitation, irrespective of genotype. In conclusion, this study evaluating two HBV DNA qPCR assays on an open real-time PCR system showed that both assays were reliable for accurate HBV detection and quantitation. The approximately 10-fold cost-saving implication of these assays, could assist in the implementation of national programmes in low- to middleincome countries for diagnosing and monitoring HBV infection, and encourage provision of access to anti-HBV therapy. Further development should validate automated extraction methods using small and robust open apparatus in order to standardize, facilitate and improve the performance of these assays. Lastly, validation of such assays on dried blood spots would render accessible HBV DNA quantitation for those where access to venous sample collection is not freely available, as found in most developing countries (WHO, 2010). Competing interests None declared. Acknowledgements We would like to thank the Inserm Unit 1058 and the Virology Laboratory of Lapeyronie University Hospital in Montpellier, France for their financial and technical supported. We would also like to thank the Virology Laboratory and the Purpan Federative Biology Institute of Toulouse University Hospital, France for supplying the HBV genotype. We acknowledge the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS) site and the Service de Coopération et d’Action Culturelle of French Embassy in Burkina Faso for their constant support. Thanks to Cara-Chan Manville for her assistance with the English proofreading. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet. 2014.01.015. References Bland, J.M., Altman, D.J., 1995. Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 346, 1085–1087.
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Caliendo, A.M., Valsamakis, A., Bremer, J.W., Ferreira-Gonzalez, A., Granger, S., Sabatini, L., Tsongalis, G.J., Wang, Y.F., Yen-Lieberman, B., Young, S., Lurain, N.S., 2011. Multilaboratory evaluation of real-time PCR tests for hepatitis B virus DNA quantification. J. Clin. Microbiol. 49, 2854–2858. Chakravarty, R., 2012. Role of molecular diagnostics in the management of viral hepatitis B. Expert Opin Med Diagn 6, 395–406. Chevaliez, S., Bouvier-Alias, M., Laperche, S., Hezode, C., Pawlotsky, J.M., 2010. Performance of version 2.0 of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay for hepatitis B virus DNA quantification. J. Clin. Microbiol. 48, 3641–3647. Daniel, H.D., Fletcher, J.G., Chandy, G.M., Abraham, P., 2009. Quantitation of hepatitis B virus DNA in plasma using a sensitive cost-effective “in-house” real-time PCR assay. Indian J. Med. Microbiol. 27, 111–115. European, 2012. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J. Hepatol. 57, 167–185. Hoofnagle, J.H., 1990. Alpha-interferon therapy of chronic hepatitis B. Current status and recommendations. J. Hepatol. 11 (Suppl 1), S100–S107. Hubschen, J.M., Andernach, I.E., Muller, C.P., 2008. Hepatitis B virus genotype E variability in Africa. J. Clin. Virol. 43, 376–380. Hui, C.K., Bowden, S., Zhang, H.Y., Wong, A., Lewin, S., Rousseau, F., Mommeja Marin, H., Lee, N.P., Luk, J.M., Locarnini, S., Leung, N., Naoumov, N.V., Lau, G.K.K., 2006. Comparison of real-time PCR assays for monitoring serum hepatitis B virus DNA levels during antiviral therapy. J. Clin. Microbiol. 44, 2983–2987. Huy, T.T., Abe, K., 2004. Molecular epidemiology of hepatitis B and C virus infections in Asia. Pediatr. Int. 46, 223–230. Hwang, W.E., Cheung, R., 2011. Global Epidemiology of Hepatitis B Virus (HBV) Infection. N. A. J. Med. Sci. 4, 7–13. Ismail, A.M., Sivakumar, J., Anantharam, R., Dayalan, S., Samuel, P., Fletcher, G.J., Gnanamony, M., Abraham, P., 2011. Performance characteristics and comparison of Abbott and artus real-time systems for hepatitis B virus DNA quantification. J. Clin. Microbiol. 49, 3215–3221. Kao, J.H., 2008. Diagnosis of hepatitis B virus infection through serological and virological markers. Expert Rev. Gastroenterol. Hepatol. 2, 553–562. Laperche, S., Thibault, V., Bouchardeau, F., Alain, S., Castelain, S., Gassin, M., Gueudin, M., Halfon, P., Larrat, S., Lunel, F., Martinot-Peignoux, M., Mercier, B., Pawlotsky, J.M., Pozzetto, B., Roque-Afonso, A.M., Roudot-Thoraval, F., Saune, K., Lefrere, J.J., 2006. Expertise of laboratories in viral load quantification, genotyping, and precore mutant determination for hepatitis B virus in a multicenter study. J. Clin. Microbiol. 44, 3600–3607. Lin, Y.Y., Huang, J.F., Liu, S.F., Yu, M.L., Tsai, C.H., Yang, J.F., Lin, I.L., Dai, C.Y., Lin, Z.Y., Chen, S.C., Chang, W.Y., Chuang, W.L., 2009. Performance characteristics of two real-time PCR assays for quantification of hepatitis B virus DNA. Scand. J. Infect. Dis. 41, 614–618. Lok, A.S., McMahon, B.J., 2009. Chronic hepatitis B: update 2009. Hepatology 50, 661–662. Mansour, W., Malick, F.Z., Sidiya, A., Ishagh, E., Chekaraou, M.A., Veillon, P., Ducancelle, A., Brichler, S., Le Gal, F., Lo, B., Gordien, E., Lunel-Fabiani, F., 2012. Prevalence, risk factors, and molecular epidemiology of hepatitis B and hepatitis
delta virus in pregnant women and in patients in Mauritania. J. Med. Virol. 84, 1186–1198. Mendy, M.E., Kaye, S., van der Sande, M., Rayco-Solon, P., Waight, P.A., Shipton, D., Awi, D., Snell, P., Whittle, H., McConkey, S.J., 2006. Application of real-time PCR to quantify hepatitis B virus DNA in chronic carriers in The Gambia. Virol. J. 3, 23. Norder, H., Hammas, B., Lee, S.D., Bile, K., Courouce, A.M., Mushahwar, I.K., Magnius, L.O., 1993. Genetic relatedness of hepatitis B viral strains of diverse geographical origin and natural variations in the primary structure of the surface antigen. J. Gen. Virol. 74 (Pt 7), 1341–1348. Pol, J., Le Pendeven, C., Beby-Defaux, A., Rabut, E., Jais, J.P., Pilloux, M., Osada, C., Zatla, F., Assami, H., Grange, J.D., Kremsdorf, D., Nicolas, J.C., Soussan, P., 2008. Prospective comparison of Abbott RealTime HBV DNA and Versant HBV DNA 3.0 assays for hepatitis B DNA quantitation: Impact on HBV genotype monitoring. J. Virol. Methods 154, 1–6. Pujol, F.H., Navas, M.C., Hainaut, P., Chemin, I., 2009. Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett. 286, 80–88. Shi, Y.H., Shi, C.H., 2009. Molecular characteristics and stages of chronic hepatitis B virus infection. World J. Gastroenterol. 15, 3099–3105. Sitnik, R., Paes, A., Mangueira, C.P., Pinho, J.R., 2010. A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes. Rev. Inst. Med. Trop. Sao Paulo 52, 119–124. Stuyver, L., De Gendt, S., Van Geyt, C., Zoulim, F., Fried, M., Schinazi, R.F., Rossau, R., 2000. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J. Gen. Virol. 81, 67–74. Vray, M., Debonne, J.M., Sire, J.M., Tran, N., Chevalier, B., Plantier, J.C., Fall, F., Vernet, G., Simon, F., Mb, P.S., 2006. Molecular epidemiology of hepatitis B virus in Dakar, Senegal. J. Med. Virol. 78, 329–334. Welzel, T.M., Miley, W.J., Parks, T.L., Goedert, J.J., Whitby, D., Ortiz Conde, B.A., 2006. Real-time PCR assay for detection and quantification of hepatitis B virus genotypes A to G. J. Clin. Microbiol. 44, 3325–3333. WHO, 2009. Hepatitis B vaccines. Wkly. Epidemiol. Rec. 84, 405–419. WHO, 2010. Viral hepatitis. WHA63.18. Sixty-third World Health Assembly. Available at: http://www.who.int/gb/ebwha/pdf files/WHA63/A63 R18-en.pdf. Accessed April 4, 2013. Yang, J.F., Lin, Y.Y., Huang, J.F., Liu, S.F., Chu, P.Y., Hsieh, M.Y., Lin, Z.Y., Chen, S.C., Wang, L.Y., Dai, C.Y., Chuang, W.L., Yu, M.L., 2009. Comparison of clinical application of the Abbott HBV PCR kit and the VERSANT HBV DNA 3.0 test to measure serum hepatitis B virus DNA in Taiwanese patients. Kaohsiung J. Med. Sci. 25, 413–422. Zacharakis, G., Koskinas, J., Kotsiou, S., Tzara, F., Vafeiadis, N., Papoutselis, M., Maltezos, E., Sivridis, E., Papoutselis, K., 2008. The role of serial measurement of serum HBV DNA levels in patients with chronic HBeAg(-) hepatitis B infection: association with liver disease progression. A prospective cohort study. J. Hepatol. 49, 884–891. Zheng, X., Ye, X., Zhang, L., Wang, W., Shuai, L., Wang, A., Zeng, J., Candotti, D., Allain, J.P., Li, C., 2011. Characterization of occult hepatitis B virus infection from blood donors in China. J. Clin. Microbiol. 49, 1730–1737.
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Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Performance of two real-time PCR assays for hepatitis B virus DNA detection and quantitation Dramane Kania a,b,c,d,∗ , Laure Ottomani e , Nicolas Meda b , Marianne Peries c , Pierre Dujols c,d,e , Karine Bolloré c,d , Wendy Rénier c,d , Johannes Viljoen c,f , Jacques Ducos c,d,e , Philippe Van de Perre c,d,e , Edouard Tuaillon c,d,e a
Laboratoire de Virologie, Centre Muraz, Bobo-Dioulasso, Burkina-Faso Unité VIH et Maladies Associées, Centre Muraz, Bobo-Dioulasso, Burkina Faso c INSERM U 1058, Infection by HIV and by agents with mucocutaneous tropism: from pathogenesis to prevention, 34394 Montpellier, France d Université Montpellier 1, 34090 Montpellier, France e CHU Montpellier, Département de Bact´eriologie-Virologie et Département d’Information Médicale, 34295 Montpellier, France f Africa Centre for Health and Population Studies, University of KwaZulu-Natal, Durban, South Africa b
a b s t r a c t Article history: Received 28 September 2013 Received in revised form 22 January 2014 Accepted 24 January 2014 Available online 18 February 2014 Keywords: Hepatitis B virus hepatitis B virus diagnosis hepatitis B virus monitoring quantitative PCR
In-house developed real-time PCR (qPCR) techniques could be useful conjunctives to the management of hepatitis B virus (HBV) infection in resource-limited settings with high prevalence. Two qPCR assays (qPCR1 and qPCR2), based on primers/probes targeting conserved regions of the X and S genes of HBV respectively, were evaluated using clinical samples of varying HBV genotypes, and compared to the commercial Roche Cobas AmpliPrep/Cobas TaqMan HBV Test v2.0. The lower detection limit (LDL) was established at 104 IU/ml for qPCR1, and 91 IU/ml for qPCR2. Good agreement and correlation were obtained between the Roche assay and both qPCR assays (r = 0.834 for qPCR1; and r = 0.870 for qPCR2). Differences in HBV DNA load of > 0.5 Log10 IU/ml between the Roche and the qPCR assays were found in 49/122 samples of qPCR1, and 35/122 samples of qPCR2. qPCR1 tended to underestimate HBV DNA quantity in samples with a low viral load and overestimate HBV DNA concentration in samples with a high viral load when compared to the Roche test. Both molecular tools that were developed, used on an open real-time PCR system, were reliable for HBV DNA detection and quantitation. The qPCR2 performed better than the qPCR1 and had the additional advantage of various HBV genotype detection and quantitation. This low cost quantitative HBV DNA PCR assay may be an alternative solution when implementing national programmes to diagnose, monitor and treat HBV infection in low- to middle-income countries where testing for HBV DNA is not available in governmental health programmes. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) infection is a worldwide public health problem. Approximately 5% of the global population are chronically infected with HBV (WHO, 2009; Hwang and Cheung, 2011). As a consequence, almost 400 million chronic carriers are currently exposed to the risk of complications of this persistent infection, such as cirrhosis, liver failure and/or hepatocellular carcinoma. The infection is especially hyper-endemic (prevalence ≥ 8%) in subSaharan Africa and South-East Asia, representing more than 75% of total chronic carriers worldwide (WHO, 2009; Hwang and Cheung,
∗ Corresponding author: Centre Muraz, Avenu Mamadou Konate, ´ 01 BP 390 BoboDioulasso 01, Burkina Faso. Tel.: +226 20 97 01 02; fax: +226 20 97 04 57. E-mail address: [email protected] (D. Kania). 0166-0934/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2014.01.015
2011). Effective strategies including prevention through vaccination, improved clinical care and biological monitoring for those infected chronically, are required to control the infection and its expansion (WHO, 2010). The presence of HBV DNA in blood reflects virus replication and is a key marker to characterise the course of chronic HBV infection (Hoofnagle, 1990; Kao, 2008; Zacharakis et al., 2008), and together with serum alanine aminotransferase, histological grade, stages of necroinflammation and liver fibrosis, constitutes one of the main criteria currently used for treatment initiation in the developed world (Lok and McMahon, 2009; European, 2012). HBV DNA quantitation in plasma or serum is a standard measure to monitor efficacy or need for initiation of treatment for persons chronically infected with HBV, since sustainable suppression of HBV replication is one of the end points. Improved access to quantitative assays for HBV DNA measurement would be highly effective to inform on the
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status of infection when used in conjunction with other parameters of HBV infection. Several molecular techniques based on target or signal amplification are commercially available, but in addition to becoming increasingly automated, they have the disadvantage of not being freely available in developing countries, mostly due to prohibitive high cost (Hui et al., 2006; Laperche et al., 2006; Pol et al., 2008; Lin et al., 2009; Yang et al., 2009; Caliendo et al., 2011; Ismail et al., 2011). In-house developed real-time PCR (qPCR) techniques are relatively low cost assays and provide an interesting alternative to monitor HBV infection in resource-limited settings (Mendy et al., 2006; Welzel et al., 2006; Daniel et al., 2009; Sitnik et al., 2010). There is a need to compare the performance of variously described methods with commercially available assays. The targeted regions of the HBV genome used for PCR amplification differ according to the various protocols described (S-, C- or X gene). HBV genetic variability is an additional factor that may affect the ability of primers and probes to effectively hybridize, leading to impaired HBV DNA quantitation as a result of a mismatch for a particular circulating genotype (Pol et al., 2008). Most HBV genotypes found in Europe and Africa are known, but with different distribution patterns in the various geographical regions (Pujol et al., 2009). Therefore it is imperative that alternative qPCR methods should be designed and evaluated according to the diversity of circulating genotypes found in a particular setting. The aim of this study was to compare the performance of two qPCR assays targeting the X- and S-genes of HBV, respectively, using an open system (Light Cycler 480), to a fully automated closed system (Roche Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0). Results of HBV DNA quantitation using all three assays were analysed according to HBV genotype. 2. Materials and Methods 2.1. Clinical specimen selection A total of 122 archived clinical serum samples collected between November 2006 and April 2012 from patients monitored for HBV infection and/or liver disease at the Virology Laboratories of Toulouse and Montpellier University Hospitals were selected for HBV DNA quantitation by both qPCRs. Both hospital laboratories used the same commercial real-time PCR assay (Cobas AmpliPrep/Cobas TaqMan [CAP/CTM] HBV Test, version 2.0 [v2.0], Roche Diagnostics GmbH, Mannheim, Germany) to quantify HBV DNA in routine practice, and results produced by this assay were used as the gold standard to which the two qPCR techniques were compared. HBV viral load results obtained by gold standard assay ranged from 1.34 to 7.84 Log10 IU/ml. All samples were positive for hepatitis B surface antigen (HBsAg), with 26.4% being dually positive for HBe antigen. The genotype of HBV was determined using the TRUGENE® HBV Genotyping Kit on the OpenGene® DNA Sequencing System (Siemens Healthcare Diagnostics Inc, Tarrytown, USA) and was available for 68 of 122 samples as follows: A (n = 22), B (n = 3), C (n = 2), D (n = 29), E (n = 11) and G (n = 1). Fifty nine HBV-negative samples, defined as specimens negative for HBsAg, anti-HBc antibodies and HBV DNA, were included in the study to determine specificity of qPCRs. 2.2. HBV DNA detection and quantitation using Cobas AmpliPrep/Cobas TaqMan HBV Test, version 2.0 All selected specimens were previously tested for HBV DNA in Montpellier or Toulouse laboratory using the CAP/CTM HBV Test, v2.0, which is a fully automated viral nucleic acid extraction and amplification system that targets the HBV precore/core gene (Chevaliez et al., 2010). Samples were processed in
25
accordance to the manufacturer’s instructions using 500 l of serum for extraction. The amplification data were analysed with AmpliLink software, version 3.3.5. The lower detection limit (LDL) is 19 IU/ml in serum and the upper detection limit is approximately 1.7 × 108 IU/ml. Specific primers and TaqMan probes were used to detect assay target, and an internal DNA quantitation standard was amplified in each sample. Results obtained by commercial assay on clinical specimens were used as the reference standard to determine correlation, agreement characteristics, clinical sensitivity and specificity of the two developed qPCR techniques. 2.3. DNA extraction for qPCR assays DNA was extracted manually from 200 l of serum with the QIAamp® DNA Blood Mini kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Template DNA was eluted into 50 l of kit elution buffer and either used directly, or stored at -20 ◦ C prior to use. 2.4. HBV DNA detection and quantitation using qPCR assays The two qPCR assays were used with slight modifications targeting the HBV X- and S-genes as previously described by Welzel et al. (2006) and Daniel et al. (2009), respectively. Modifications were specifically directed at the extraction kit, volume of sample used, concentration of master mixture components, PCR apparatus and qPCR2 probe staining. Primer and probe sequences of the two qPCR methods are shown in Table 1. For both qPCR techniques, hydrolysis probes (also called TaqMan probes), were selected. qPCR1 used a primers/probe set targeting a conserved region within the HBV X gene, and qPCR2 a conserved region within the HBV S gene. Alignment of nucleotide sequences within the X and S genes of HBV showed that the primers and probe selected for qPCR1 generated much more frequent mismatches than those employed for qPCR2. Based on 12 published complete HBV genomic sequences from genotypes A to F (Stuyver et al., 2000) and using global gene HBX sequence alignments, we identified 10, 7 and 14 nucleotide mismatches for forward primer, reverse primer and probe of qPCR1, respectively. By contrast, only one mismatch was observed for each primer and probe of qPCR2 using global HBS gene sequence alignments and the HBV complete genomes cited above (see supplementary data). For each qPCR, 10 l of DNA extract was added to 40 l of master mixture incorporating 5 l of forward primer at 10 pmol/l, 5 l of reverse primer at 10 pmol/l, 1 l of dual-labelled fluorogenic probe at 10 pmol/l, 25 l of 2X TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA) and 4 l of RNase/DNase-free water. Each reaction consisted of 2 min at 50 ◦ C, 10 min at 95 ◦ C followed by 50 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C each. The two qPCR assays were performed using the LightCycler 480 II Real-Time PCR Instrument (Roche Diagnostics GmbH, Mannheim, Germany), and amplification data were analysed using the LightCycler 480 Software (Roche Diagnostics). 2.5. Standard and control samples The 2nd World Health Organization (WHO) International Standard for HBV DNA Nucleic Acid Amplification Techniques (NAT), product code 97/750, was obtained from the National Institute for Biological Standards and Control (NIBSC; Hertfordshire, United Kingdom) and used as an external standard for each qPCR assay. The lyophilised material of this standard was reconstituted in 0.5 ml of sterile nuclease-free water and the final concentration obtained was 1,000,000 (6 Log10 ) IU/ml (according to the manufacturer’s instructions). For each experiment, 200 l of the reconstituted standard were extracted together with the clinical samples and serially
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Table 1 Primer and hydrolysis probe sequences used for HBV in-house quantitative real-time PCR assays (qPCR1 and qPCR2). The primers and probes described below are adapted from previous published work (Daniel et al., 2009; Welzel et al., 2006). All the probes were labelled at the 5’ end with the reporter dye (R) FAM (6-carboxyfluorescein) and at the 3’ end with the quencher dye (Q) TAMRA (6-carboxytetramethylrhodamine).
Forward primer Reverse primer Probe
qPCR 1
qPCR 2
HBV-F3: 5’-GGC CAT CAG CGC ATG C-3’ HBV-R3M3: 5’-C [5-NitIdl] GCT GCG AGC AAA ACA-3’ HBV-P3: 5’–CTC TGC CGA TCC ATA CTG CGG AAC TC– 3’
HBV TAQ 1: 5’ -GTG TCT GCG GCG TTT TAT CA- 3’ HBV TAQ 2: 5’-GAC AAA CGG GCA ACA TAC CTT- 3’ HBV TAQ PR: 5’ - CCT CTT CAT CCT GCT GCT ATG CCT CAT C - 3’
diluted tenfold to obtain 5 different HBV DNA concentrations (1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 and 1 × 102 IU/ml). The standard curve obtained from these concentrations was used to estimate HBV DNA amount in clinical samples in each run of the qPCR assays. In addition, we prepared a low-positive control (LPC) by combining 4 clinical samples with known HBV DNA load, yielding an LPC with an estimated final concentration of approximately 2,500 IU/ml. Negative controls consisted of RNase/DNase-free water. By contrast to the qPCRs using a calibrated curve produced by an external control, the reference assay uses an internal control as HBV quantitation standard (QS). The QS was added to each specimen at a known copy number and was carried through all subsequent steps of specimen preparation, simultaneous PCR amplification and detection. The Cobas TaqMan Analyzer calculates the HBV DNA concentration in the test specimens by comparing the HBV signal to the HBV QS signal for each specimen and control. 2.6. Determination of the analytical performance of both qPCR assays The LDL (or threshold) for each assay was determined using 16 serial concentrations of the WHO International Standard (10; 20; 30; 40; 50; 60; 70; 80; 90; 100; 125; 200; 250; 400;500; 1,000 IU/ml) in at least 10 separate runs. The linearity of each qPCR technique was established by testing in 24 separate PCR-runs, five dilution series (1 × 106 , 1 × 105 , 1 × 104 , 1 × 103 and 1 × 102 IU/ml) of the WHO HBV DNA International Standard. Intra-assay variability of each qPCR assay was assessed by analyzing in a single PCR-run, nine replicates of the LPC at 2500 IU/ml, four replicates of four serial concentrations of the WHO International Standard (1x105 , 1x104 , 1x103 and 1x102 IU/ml) and a triplicate of six clinical samples quantified by means of the Roche Cobas test (503; 2,502; 5,500; 53,400; 564,000; and 6,220,000 IU/ml). The inter-assay variability of each qPCR technique was evaluated (i) by a same operator testing in 10 separate PCR-runs, the LPC and the five serial concentrations of the WHO International Standard; and furthermore, (ii) with two different operators determining five times on separate PCR-runs, the five serial concentrations of the WHO International Standard. qPCR assay specificity was assessed by testing the 59 HBVnegative serum samples including eight HCV-RNA positive samples, four HIV-RNA positive samples and one dual HIV RNA/HCV RNA-positive sample. 2.7. Statistical analysis Data analysis was performed using the SAS version 9.3 software package (SAS Institute, Cary, NC). HBV DNA concentrations obtained were accordingly converted to logarithm of ten (Log10 ) values before performing statistical analysis. The LDL of each qPCR assay was estimated using probit analysis. Coefficient of variation ([(SD/Means) × 100]; SD, the standard deviation) was calculated to estimate intra-assay and inter-assay variability. Continuous variables were described as median and inter-quartile range
(IQR) and were compared using the non-parametric Wilcoxon Mann–Whitney test due to the absence of normal distribution. Chi-square test was used to compare categorical variables. Statistical significance was set at a p value of < 0.05. The Spearman correlation coefficients were calculated to determine the relationship between the HBV DNA levels obtained with the commercial and qPCR assays. Deming regression analysis was used to compare quantitative results obtained between both qPCR and the reference assay. The Bland–Altman method (Bland and Altman, 1995) was used to measure agreement between assays. 3. Results 3.1. Analytical performance of the two qPCR assays Using serial concentrations of the WHO International Standard on repeated runs, the lowest HBV DNA concentration giving 100% detection signal was 125 IU/ml using the qPCR1 assay and 90 IU/ml using the qPCR2. Probit analysis predicts a 95% LDL of 104 IU/ml (95% confidence interval [CI]: 78–176) for qPCR1 and 91 IU/ml (95% CI: 73–292) for qPCR2. Linearity of the measures was observed from 100 to 1,000,000 IU/ml for both assays (Fig. 1). The inter-quartile ranges were increasingly smaller from low concentrations to high concentrations. The coefficient of determination was R2 = 0.998 for qPCR1 and R2 = 0.999 for qPCR2. The mean of within-run and between-run standard deviations for each qPCR assay and its coefficients of variation are represented in Table 2. All 59 samples that were found to be negative for HBV by the commercial assay, were also non-reactive by the two qPCR methods, conferring an analytical specificity of 100% for both assays. Of 122 samples with HBV DNA detected by commercial assay, 121 were confirmed positive using the qPCR1 and 122 using the qPCR2 assay, indicating a clinical sensitivity of 99.2% for qPCR1 and 100% for qPCR 2. The false negative HBV DNA result obtained using the qPCR1 (HBV DNA undetectable), exhibited a low HBV DNA viral load value of 2.87 Log10 IU/ml by reference assay, and 2.67 Log10 IU/ml by qPCR2. The negative result obtained by the qPCR1 was confirmed on a repeated assessment of this sample, classified as HBV genotype D. 3.2. Correlation and agreement of the two qPCR techniques with the Roche CAP/CTM HBV test v2.0 For 122 specimens the three assays yielded a median HBV DNA load of 3.45 Log10 IU/ml (IQR 25-75: 2.83–4.98) for the reference assay, 3.65 Log10 IU/ml (IQR 25-75: 2.87–4.84) for qPCR1 assay and 3.61 Log10 IU/ml (IQR 25-75: 3.05–4.95) for qPCR2 assay. Both qPCR assays were correlated with the reference test (r = 0.834, p < 0.0001 [qPCR1] and r = 0.870, p = 0.0001 [qPCR2]) (Fig. 2). No significant difference was observed between reference and qPCR tests (p = 0.720 and 0.301, respectively) but a visible large dispersion is observed in Fig. 2A, suggesting more discrepant values using the qPCR1 compared to those obtained by qPCR2. There was good agreement by Bland–Altman analysis between qPCR assays and
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Fig. 1. qPCR standard curves established using 5 serial dilutions of the WHO HBV DNA International Standard. The qPCR1 standard curve is represented by (A) and the qPCR2 standard by (B). The cycle threshold (Ct) is the number of cycles before the fluorescence passes a fixed limit (time to positivity). The solid line connects the median Ct values and the 25% and 75% inter-quartile ranges (box plot) for each dilution.
Table 2 Intra-run and inter-run variability. For intra-run variability, WHO HBV DNA International Standard serial dilutions, low-positive control (LPC) and selected clinical samples were tested at least 3 times in the same run using each in-house qPCR assay. For inter-run variability, LPC and WHO International Standard serial dilutions were tested at least 5 times in different runs using each in-house qPCR assay and performed by two different operators. In-house qPCR1
Intra-run Sample 1 (2.70 Log10 IU/ml) Sample 2 (3.74 Log10 IU/ml) Sample 3 (4.73 Log10 IU/ml) Sample 4 (5.75 Log10 IU/ml) Sample 5 (6.79 Log10 IU/ml) LPC (3.40 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (4.11 Log10 IU/ml) Inter-run Operator 1 LPC (3.40 Log10 IU/ml) STD 1 (6.00 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (3.90 Log10 IU/ml) Operator 2 STD 1 (6.00 Log10 IU/ml) STD 2 (5.00 Log10 IU/ml) STD 3 (4.00 Log10 IU/ml) STD 4 (3.00 Log10 IU/ml) STD 5 (2.00 Log10 IU/ml) Average (4.00 Log10 IU/ml)
In-house qPCR2
Mean (Log10 IU/ml)
SD (Log10 IU/ml)
%CV
Mean (Log10 IU/ml)
SD (Log10 IU/ml)
%CV
2.43 2.44 4.47 5.82 6.64 3.05 4.99 3.98 3.07 1.96 3.89
0.07 0.15 1.01 0.01 0.00 0.11 0.04 0.02 0.07 0.05 0.05
2.80 6.18 0.31 0.19 0.06 3.66 0.80 0.46 2.40 2.36 1.38
2.31 3.64 4.50 5.79 6.52 3.24 5.00 4.00 2.99 2.00 4.00
0.24 0.06 0.02 0.01 0.01 0.05 0.00 0.01 0.02 0.01 0.04
10.18 1.58 0.34 0.10 0.22 1.51 0.08 0.21 0.71 0.66 1.06
3.54 5.98 5.00 4.00 3.02 1.95 3.92
0.14 0.04 0.03 0.04 0.05 0.13 0.07
3.96 0.60 0.59 0.92 1.70 6.75 1.81
3.58 5.99 5.01 3.99 3.03 1.98 3.93
0.15 0.04 0.04 0.03 0.07 0.05 0.06
4.14 0.61 0.84 0.79 2.34 2.41 1.60
5.99 5.01 4.00 2.99 2.25 4.05
0.02 0.01 0.02 0.05 0.47 0.11
0.26 0.23 0.56 1.58 20.81 2.79
5.92 5.06 4.02 3.00 1.97 4.00
0.19 0.18 0.07 0.07 0.03 0.11
3.15 3.60 1.81 2.18 1.64 2.70
LPC, low-positive control; STD, standard; SD, standard deviation; CV, coefficient of variation; IU, international unit; ml, milliliter; Log10, logarithm of ten; qPCR, quantitative real-time polymerase chain reaction.
reference assay (Fig. 3). The mean difference (bias ± SD) between the qPCR and reference tests was + 0.07 ± 0.77 Log IU/ml for qPCR1, and + 0.13 ± 0.52 Log IU/ml for qPCR2. Out of the 122 HBV positive samples, 113 (92.6%) of the qPCR1 results, and 114 (93.4%) of the qPCR2 results, were within 95% limit of agreement. As shown in Fig. 3A, a proportional bias was observed using qPCR1 in which HBV DNA concentrations tended to be underestimated in samples exhibiting a low viral load level, and overestimated when the values
were high. qPCR1 gave HBV DNA measurements equivalent (i.e., difference in HBV DNA < 0.5 Log10 IU/ml) to the reference assay results in 73 out of 122 (59.8%) samples. In contrast, equivalent HBV DNA results were obtained in 87 out of 122 (71.3%) samples using the qPCR2. A trend for a higher concordance between the reference assay and the qPCR2 assay was observed when compared to qPCR1 (p = 0.059). Among the discrepant results with ≥ 0.5 Log10 IU/ml difference, qPCR values were lower than reference values in
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D. Kania et al. / Journal of Virological Methods 201 (2014) 24–30
Fig. 2. Correlation between Cobas TaqMan v2.0 test and the in-house qPCR assays for HBV DNA quantitation: (A) Cobas TaqMan v2.0 test versus qPCR1; and (B) Cobas TaqMan v2.0 test versus qPCR2. The fitted regressions are represented with solid lines.
Fig. 3. Bland–Altman representations for agreement between Cobas TaqMan v2.0 test and in-house qPCR assays: (A) Cobas test versus qPCR1; and (B) Cobas test versus qPCR2. The mean differences and the 95% limits of agreement are represented by horizontal solid lines. For symbols, white circles represent HBV genotype A (n = 22), black circles genotype B (n = 3), black triangles genotype C (n = 2), white triangles genotype D (n = 29), white squares genotype E (n = 11), black squares genotype G (n = 1) and asterisks non-defined genotypes (n = 54). Discontinuous oblique line in Fig. 3A indicates the proportional bias observed between the qPCR1 and the Cobas TaqMan.
21 out of 49 cases (42.9%) for qPCR1, and 10 out of 35 cases (28.6%) for qPCR2.
The main characteristics of the three assays (Roche CAP/CTM, qPCR1 and qPCR2) were described in the Table 3.
3.3. Impact of HBV genotypes on DNA quantification by the Roche CAP/CTM HBV test v2.0 and qPCR assays
4. Discussion
Available HBV genotype (n = 68), was classified into 6 genotypes as follows: A (22), B (3), C (2), D (29), E (11) and G (1). The impact of HBV diversity was explored in the three most frequent genotypes (A, D and E). Similar HBV DNA levels were obtained by reference and qPCR assays for genotypes A (p = 0.782), D (p = 0.923) and E (p = 0.795). Discrepant results (difference in HBV DNA ≥ 0.5 Log10 IU/ml), were found in 7/29 (D), 4/11 (E) and 2/22 (A), genotyped samples by qPCR1, whereas, qPCR2 yielded discrepant results in 4/29 (D), and 1/11 (E) genotyped samples. Frequent discrepant results between the commercial and qPCR assays were observed for genotype B (2/3) and G (1/1), with lower HBV DNA values found in 2 cases for qPCRs (Fig. 3). However, due to the low number of those genotypes this observation did not permit a clear conclusion.
In this study, two qPCR techniques were compared to a commercial assay using a large number of clinical samples with different HBV genotypes. The two qPCR techniques showed high analytical performance for HBV DNA quantitation in serum. A notable linearity was observed between qPCR and reference assays over a broad quantitative range with the ability to detect DNA values over 107 Log10 IU/ml. The intra-assay and inter-assay variations were also very good and close to those reported for other commercial molecular assays (Laperche et al., 2006; Ismail et al., 2011). The coefficients of variation were excellent over a 3 Log10 IU/ml range with variability below 5%. As expected, technique variability was greatest near the LDL of each assay (20.8% for qPCR1 and 10.2% for qPCR2).
D. Kania et al. / Journal of Virological Methods 201 (2014) 24–30
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Table 3 Main characteristics of the two in-house real-time PCR assays (qPCR1 and qPCR2) compared to the Roche Cobas AmpliPrep/Cobas TaqMan test, version 2.0 for HBV DNA measurement. Characteristics
CAP/CTM
qPCR1
qPCR2
Target region Probe technology Extraction method Amplification system Amplification software Input volume for extraction (l) Lower detection limit (IU/ml) Upper detection limit (IU/ml) Linearity (R2 ) Intra-run %CV Inter-run %CV
pre-Core/Core gene TaqMan Cobas AmpliPrep Cobas TaqMan Amplilink v3.3.5 500a 19 [11-107]a 170000000a 0.9987a 14.50 [10.00-27.00]a 14.83 [10.00-28.00]a
X-gene TaqMan Qiagen manual LightCycler 480 LC 480 software 200 104 [78-176] 100000000 0.9979 1.38 [0.06-6.18] 2.30 [0.23-20.81]
S-gene TaqMan Qiagen manual LightCycler 480 LC 480 software 200 91 [73-292] 100000000 0.9995 1.06 [0.08-10.18] 2.15 [0.61-4.14]
a Data obtained from the document of procedure instructions of the Roche Cobas® AmpliPrep/Cobas TaqMan HBV kit (package insert informations); qPCR1, in-house real-time PCR assay 1; qPCR2, in-house real-time PCR assay 2; R2 , Coefficient of determination; %CV, coefficient of variation.
The two generic tests presented a LDL of almost 100 IU/ml which was higher when compared to the Roche assay (19 IU/ml in serum). However, this cut-off value is better than the LDL of the commercial signal amplification techniques from Bayer (347 IU/ml for the bDNA v3.0 VERSANT assay) (Yang et al., 2009). These levels of detection of HBV DNA load obtained by our qPCR assays would enable routine clinical management and are sufficient for detection of active HBV replication in patients with DNA levels more than 2,000 IU/ml (Shi and Shi, 2009; Chakravarty, 2012; European, 2012). Such displayed sensitivity could also be adequate for routine monitoring of response to antiviral therapy, although ultra-sensitive methods are more desirable to detect suboptimal suppression of viral replication in patients receiving nucleotide/nucleoside analogues. Likewise, the two qPCR assays may fail to detect serum HBV DNA in case of occult HBV infection, but this situation is infrequent (Zheng et al., 2011). Theoretically, it remains possible to improve the LDL of qPCR assays by increasing the quantity of serum used for the DNA extraction phase. In our study, a 2.5 fold less serum volume was used in the qPCR assays by comparison to the reference assay (200 l for qPCRs vs. 500 l for Roche). In addition, we only used 20% of total available eluted nucleic acid per sample in our qPCR reaction. Therefore, by increasing the amount of eluted nucleic acids, the LDL of the qPCR methods could potentially be set as low as 40 IU/ml when using an input volume of serum similar to those used in the commercial assay. Our study, based on a large number of samples with different genotypes, confirms the potential value of these assays. For each qPCR assay, good agreement and correlation were obtained with the reference test. Welzel et al. reported a good correlation using the primers and probe of the qPCR1 in comparison to the Bayer VERSANT HBV DNA 3.0 on HBV DNA-positive clinical specimens, however, discrepant results (differences of HBV DNA ≥ 0.5 Log10 IU/ml) were frequent (Welzel et al., 2006). Daniel et al. also reported a good correlation between a PCR using the primers and probe of the qPCR2 and the Artus HBV RG PCR assay (Daniel et al., 2009). Genotypes were taken into account in the first study, but not in the second one, and both explored a small number of samples, 29 and 30 HBV DNA positive samples, respectively. The mean nucleotide divergence is estimated at 8% between the eight distinct HBV genotypes denoted from A to H. (Norder et al., 1993; Stuyver et al., 2000). Geographic distribution of HBV variants differs in populations around the globe. Genotypes E, D and A are the most prevalent in sub-Saharan Africa (Vray et al., 2006; Hubschen et al., 2008; Mansour et al., 2012), whereas, genotypes A, D and G are overrepresented in European countries (Huy and Abe, 2004; Pujol et al., 2009). Polymorphisms within the primers/probe and the three different HBV regions targeted by the assays (C, X and S-genes) may affect accuracy of viral load measurements differently. Although we confirmed the good performance obtained with the qPCR1, we also
observed a lower agreement with the commercial assay, a proportional bias, and one HBV genotype D sample with a low DNA viral load that was not detected by this assay. These results suggest that the ability of the qPCR2 technique may be superior to the qPCR1 test for HBV DNA detection and quantitation, irrespective of genotype. In conclusion, this study evaluating two HBV DNA qPCR assays on an open real-time PCR system showed that both assays were reliable for accurate HBV detection and quantitation. The approximately 10-fold cost-saving implication of these assays, could assist in the implementation of national programmes in low- to middleincome countries for diagnosing and monitoring HBV infection, and encourage provision of access to anti-HBV therapy. Further development should validate automated extraction methods using small and robust open apparatus in order to standardize, facilitate and improve the performance of these assays. Lastly, validation of such assays on dried blood spots would render accessible HBV DNA quantitation for those where access to venous sample collection is not freely available, as found in most developing countries (WHO, 2010). Competing interests None declared. Acknowledgements We would like to thank the Inserm Unit 1058 and the Virology Laboratory of Lapeyronie University Hospital in Montpellier, France for their financial and technical supported. We would also like to thank the Virology Laboratory and the Purpan Federative Biology Institute of Toulouse University Hospital, France for supplying the HBV genotype. We acknowledge the Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS) site and the Service de Coopération et d’Action Culturelle of French Embassy in Burkina Faso for their constant support. Thanks to Cara-Chan Manville for her assistance with the English proofreading. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jviromet. 2014.01.015. References Bland, J.M., Altman, D.J., 1995. Comparing methods of measurement: why plotting difference against standard method is misleading. Lancet 346, 1085–1087.
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