Abstract
This study evaluated the applicability of quantitative PCR (Q-PCR) and branched-chain DNA assays for detection of hepatitis B virus (HBV) DNA in sera. For 42 samples, the detection rates were 81 and 41%, respectively, with a correlation coefficient of 0.633. The Q-PCR is useful for early monitoring of HBV load in high-risk patients.
Hepatitis B virus (HBV) is an important etiologic agent of liver diseases. Reinfection of HBV in liver transplant patients often results in a poor prognosis (1), and reactivation of HBV is also seen with hepatocellular carcinoma (HCC) after surgical intervention (4). Laboratory surveillance on HBV activity in those high-risk patients is urgently needed. Viral load assay has been considered the most effective method for evaluating the effectiveness of anti-HBV drugs, as well as for monitoring the efficacy of prophylactic measures (10, 16, 17). The prevailing HBV DNA assays include probe hybridization-based and PCR-based viral DNA target detection. However, studies evaluating the performance of these assays in clinical settings are scanty, especially those involving patients with low serum HBV levels. The present study compared the efficacy of quantitative PCR (Q-PCR) (AcuGen HBV quantitative test; Biotronic Corp., Lowell, Mass.) (15, 23) with the branch-chained DNA (b-DNA) assay (Quantiplex HBV DNA; Chiron Corp., Emeryville, Calif.) (7, 8, 20–22) for detection of HBV in patients with liver diseases.
Forty-two serum samples from 17 patients (14 HCC and 3 liver transplant) admitted to the surgical ward of Queen Mary Hospital (Pokfulam, Hong Kong) were collected perioperatively using different time schedules and were stored in aliquots at −80°C until analysis. Four HBV serological markers (HBsAg, HBeAg, HBeAb, and HBcAb) were evaluated in each sample, using a monoclonal quantitative enzyme immunoassay (Auszyme; Abbott) and the IMX HBe 2, anti-HBe 2, and core assay kits (Abbott), respectively. The tests were performed by following the manufacturer's procedures for the microparticle enzyme immunoassay for qualitative determination of HBeAg, HBeAb, and HBcAb in human serum or plasma.
PCR was performed using 1 μl of a viral DNA template extracted from 200 μl of sera, 2.5 pmol of primers (5′-TTACAGGCGGGGTTTTTCTT-3′ and 5′-AAGATGTTGTACAGACTTGG-3′, which amplify a 585-bp fragment of the HBV surface antigen [S] gene), a 62.5 μM concentration of each deoxynucleoside triphosphate, 2 μM MgCl2, and 1.25 U of Taq DNA polymerase (GIBCO). The PCR was carried out in a thermal cycler (MJ Research Inc., Waltham, Mass.) in 40 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min. The amplification products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining.
The Quantiplex HBV DNA assay was employed according to the manufacturer's instructions. Briefly, samples were added to microplates precoated with two different sets of oligonucleotides derived from the conserved regions of the HBV genome: the 3′ regions of surface and core antigen genes. Viral DNA was captured by oligonucleotides used to coat the plate. After incubation with the HBV DNA target probes, the b-DNA amplifier hybridized to the target probes and was further amplified by an alkaline phosphatase-dioxetane substrate reaction. The chemiluminescent signal was read using a luminometer, and each reading represented the mean ± the standard deviation from duplicate determinations.
The AmpliSensor HBV assay (AcuGen) consists of two amplification steps: (i) an initial asymmetric amplification to overproduce one strand of the target sequence and (ii) a subsequent heminested amplification to monitor AmpliSensor (a fluorogenic primer duplex) as it is converted into the amplification product. The asymmetric primer set (sense, 5′-TGCTCGTGTTACAGGCGCCGT-3′; antisense, 5′-GAGGCATAGCAGCAGGATGAAGAG-3′) was designed to asymmetrically amplify a 241-bp HBV S DNA fragment (14). AmpliSensor primer 5′-TTATCGCTGGATGTGTCTGCGGCGT-3′ (sense) was used to further amplify and detect a 64-bp fragment from within the asymmetric amplification products. The profiles for amplification and detection were as follows: (i) 95°C for 25 s, 60°C for 25 s, and 72°C for 40 s and (ii) 95°C for 20 s, 60°C for 20 s, and 2°C for 30 s, respectively.
Data were analyzed by SPSS statistical software (SPSS Inc., Chicago, Ill.). The Mann-Whitney or Wilcoxon rank test was used to compare continuous variables, while chi-square test or Fisher's exact test was used for discrete variables. Pearson correlation was to analyze data from two quantitative assays. Assay reproducibility was assessed by coefficient of variation. A two-tailed hypothesis was tested in all cases, and a P value of <0.05 was considered significant.
All patients selected were positive for one or more HBV serological markers (Table 1). In general, the HBV DNA titer obtained from the b-DNA assay correlated well with that from the AmpliSensor Q-PCR (correlation coefficient = 0.633, P < 0.001). The reproducibilities of both the AmpliSensor and b-DNA assays were good, with coefficients of variation of 6.4 and 6.5%, respectively. Table 2 summarizes the results of the conventional PCR, AmpliSensor Q-PCR, and b-DNA assays. The AmpliSensor Q-PCR was shown to be highly sensitive compared to the b-DNA assay and conventional PCR. Samples with low HBV titers (0.002 to 0.7 Meq/ml, i.e., below the cutoff of the b-DNA assay) were detectable by the AmpliSensor Q-PCR. By contrast, the performance of the b-DNA test was particularly good for samples with high titers of HBV DNA. Furthermore, the conventional PCR was found to be useful for confirmation of results for samples with titers of 1.23 Meq/ml or higher. Seventy-nine percent and 67% of the results from the b-DNA and AmpliSensor tests, respectively, were validated by PCR. Thus, a combination of the AmpliSensor or b-DNA test with PCR can enhance the accuracy of HBV detection. Each b-DNA assay, which costs $80 per test, takes about 24 h in order to report a definitive result, whereas the AcuGen Q-PCR costs around $5 per test and is completed in about 8 to 10 h.
TABLE 1.
Analysis of HBV DNA by conventional PCR, AuGen Q-PCR, and Chiron b-DNA assays in serum samples from HBV-seropositive patientsa
| Patient group and no. | Sample no. | Sexb/age (yr) | Presence of serological marker
|
HBV DNA result
|
|||||
|---|---|---|---|---|---|---|---|---|---|
| HBsAg | HBeAg | HBeAb | HBcAb | PCR | Q-PCR (Meq/ml) | b-DNA assay (Meq/ml) | |||
| HCC patients | |||||||||
| 1 | 379 | M/64 | + | + | − | + | + | 2.710 | <0.700 |
| 2 | 206 | M/57 | + | − | + | 0.015 | <0.700 | ||
| 275 | − | 0.015 | <0.700 | ||||||
| 368 | − | <0.002 | <0.700 | ||||||
| 3 | 354 | F/65 | − | − | − | + | − | <0.002 | <0.700 |
| 438 | − | 0.014 | <0.700 | ||||||
| 539 | − | 0.014 | <0.700 | ||||||
| 4 | 504 | M/72 | + | − | + | + | − | 0.029 | <0.700 |
| 569 | − | 0.023 | <0.700 | ||||||
| 5 | 610 | F/75 | + | − | + | + | + | 1.230 | <0.700 |
| 626 | + | 1.790 | <0.700 | ||||||
| 6 | 468 | M/74 | − | − | + | + | − | 0.019 | <0.700 |
| 577 | − | <0.002 | <0.700 | ||||||
| 7 | 488 | F/63 | + | − | + | + | + | <0.002 | <0.700 |
| 542 | − | 0.130 | <0.700 | ||||||
| 598 | − | 0.018 | <0.700 | ||||||
| 8 | 350 | M/45 | + | + | − | + | + | 12.800 | <0.700 |
| 512 | + | 153.000 | 8.931 | ||||||
| 9 | 541 | M/44 | + | − | + | + | + | 60.400 | 20.970 |
| 549 | + | 108.000 | 4.156 | ||||||
| 579 | + | 126.000 | 19.800 | ||||||
| 10 | 556 | M/42 | + | − | + | + | − | 2.140 | <0.700 |
| 11 | 573 | M/69 | − | − | + | + | − | <0.002 | 0.907 |
| 605 | − | 0.007 | <0.700 | ||||||
| 627 | − | 0.059 | <0.700 | ||||||
| 12 | 473 | M/58 | + | + | − | + | + | 18.300 | 2.719 |
| 513 | + | 4.380 | <0.700 | ||||||
| 567 | + | 15.800 | 0.737 | ||||||
| 13 | 263 | M/63 | − | − | + | + | − | 0.012 | <0.700 |
| 293 | − | <0.002 | <0.700 | ||||||
| 320 | − | <0.002 | 0.852 | ||||||
| 14 | 454 | M/52 | + | − | + | + | + | 4.240 | 1.224 |
| 495 | + | 89.800 | 1.773 | ||||||
| 516 | + | 5.780 | 9.129 | ||||||
| Liver transplant patients | |||||||||
| 15 | 1153 | M/20 | +/− | − | − | 0.043 | <0.700 | ||
| 1227 | − | <0.002 | <0.700 | ||||||
| 16 | 1211 | F/46 | + | + | + | 247.000 | 55.250 | ||
| 17 | 1221 | M/37 | + | − | + | + | 294.000 | 819.000 | |
| 1255 | + | 9.500 | 306.400 | ||||||
| 1258 | + | 123.000 | 133.400 | ||||||
| 1265 | + | 37.200 | 25.770 | ||||||
| 1275 | + | 50.200 | 8.927 | ||||||
A total of 42 serum samples were tested from 17 patients (14 HCC and 3 liver transplant). Cutoff values for Q-PCR and the b-DNA assay were <0.002 and <0.7 Meq/ml, respectively, per the manufacturers' recommendations. The conventional PCR had a detection limit of approximately 0.0625 Meq/ml. The coefficients of variation for Q-PCR and the b-DNA assay were 6.4 and 6.5%, respectively; the correlation coefficient was 0.633 (P < 0.001).
M, male; F, female.
TABLE 2.
Qualitative relation among AmpliSensor Q-PCR, b-DNA assay, and conventional PCR results
| Status | No. (%) of samples |
|---|---|
| AmpliSensor +, b-DNA +, PCR + | 15 (36) |
| AmpliSensor +, b-DNA −, PCR − | 13 (31) |
| AmpliSensor +, b-DNA −, PCR + | 6 (14) |
| AmpliSensor −, b-DNA −, PCR − | 5 (12) |
| AmpliSensor −, b-DNA +, PCR − | 2 (5) |
| AmpliSensor −, b-DNA −, PCR + | 1 (2) |
| Total | 42 (100) |
The Quantiplex b-DNA assay is the most frequently used method for HBV DNA detection (7, 8, 20–22), with reported high accuracy and good reproducibility. However, its low sensitivity compared with that of the PCR assay is a major limitation. A handful of quantitative PCR methods, such as the Amplicor and AmpliSensor assays, were recently developed to enhance the sensitivity for viral DNA (6, 18, 24). The Amplicor assay for quantification of hepatitis C virus RNA was recently evaluated (5), and it also demonstrated better sensitivity than the Digene hybridization assay for HBV DNA detection in samples from patients with chronic HBV (9). Furthermore, the AmpliSensor HBV quantitative test, a format similar to the Amplicor assay, has also been applied with good success for screening HBV DNA in blood intended for transfusion (3).
HBV viral load has become a useful index for predicting disease progress and for evaluating the efficacy of antiviral treatment of patients with chronic HBV infection (10, 11, 16, 17, 19). For transplant patients, reinfection with HBV or emergence of virus mutants definitely influences the prognosis of graft survival. Thus, a slight change in viral load may have important clinical implications (13). HBV replication often happens at low titers in most HBV-infected HCC patients (2, 12), and unfortunately, surgical treatment for HCC and/or postoperative adjuvant chemotherapy may result in reactivation of viral activity, further destroying the liver parenchyma and inducing cirrhosis. Therefore, early surveillance of the changes in the viral load may yield a better prognosis for these patients.
Although the b-DNA assay provides accurate results, it may not be suitable for patients with low HBV titers. The AmpliSensor Q-PCR appears to provide a relatively cheap, fast, and sensitive approach, particularly useful in HBV surveillance schemes for early prediction of slight changes in viral DNA level in transplant patients. Because of the small number of samples used in this study, a broader prospective evaluation is being undertaken to validate the present data; the evaluation includes patients with pre- or postantiviral therapy, serial specimens from uncomplicated patients, and other patients whose sera contain a wide range of HBV DNA levels.
Acknowledgments
This work is supported by the Development Fund for Area of Excellence (University of Hong Kong) and the Hong Kong Research Grants Council (HKU7281/98M) to J.M.L.
We gratefully acknowledged Ms. Maggie Ho for preparing the manuscript.
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