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Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay
Journal of Virological Methods ELSEVIER
Journal of Virological Methods 49 (1994) 141-152
Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay Val&ie Barlet a1*,Marielle Cohard aTb,Marie Ange Thelu a~b, Marie Jeanne Chaix a, Christine Baccard a, Jean Pierre Zarski a2b, Jean Marie Seigneurin a aLaboratoire de Virologie Midicale Mole’culaire, Facultt? de Midecine/ CHU Grenoble, BP 217 X, 38043 Grenoble Cedex 09, France b Service d’Hkpato-Gastroenthologie, I-CHU Grenoble, France
Accepted 14 February 1994
Abstract A quantitative, non-radioactive hybrid capture HBV DNA assay (Digene Diagnostics), which uses an efficient solution hybridization procedure coupled to a sensitive chemiluminescent signal amplification system, was compared with the quantitative, radioactive solution hybridization assay (Genostics, Abbott Laboratories), in hepatitis B virus carriers, particularly in those undergoing antiviral therapy. The qualitative reproducibility of the chemiluminescent method, tested on 30 sera, was acceptable, with a reproducibility rate of 93.3%. A comparison of this hybrid capture HBV DNA assay with the radioactive test on 113 sera obtained from 48 patients (39 HBsAg-positive patients) gave a sensitivity of 87.2%, a specificity of 100% and an agreement between the two tests of 89.4% (101 sera including 82 HBV DNA positive and 19 negative samples). Changes in HBV DNA levels measured by the two assays showed a good correlation with each other during interferon therapy. However, the hybrid capture values were higher than the radioactive assay values, with the ratio of the two values being variable in the same patient during the course of treatment. The Genostics assay therefore seems to be a more accurate procedure for evaluating changes in viral replication, particularly at high HBV DNA levels. However, the hybrid capture method is faster and has the advantage of being a non-radioactive procedure. This chemilumines-
* Corresponding author. Fax: + 33 76.54.80.74. 0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00035-F
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cent assay is easy to perform as a routine diagnostic procedure and may be a useful alternative to the radioactive solution hybridization method. Keywords: Hepatitis B virus DNA; Quantitative chemiluminescent assay; Solution hybridization method; Antiviral therapy monitoring
1. Introduction The detection of serum hepatitis B virus (HBV) DNA by molecular hybridization represents the most informative method for determining viral replication activity and serum infectivity (Bonino et al., 1981; Weller et al., 1982; Scotto et al., 1983; Pontisso et al., 1989). Although the polymerase chain reaction (PCR) is the most sensitive method for HBV DNA detection in serum, it cannot be regarded as a routine test. Rapid detection methods have primarily used radiolabelled probes c3*P DNA probes) (Beminger et al., 1982; Scotto et al., 1983; Feinman et al., 1984; Fagan et al., 1985) but the interpretation of results is semi-quantitative and subjective. Recently, a quantitative and standardized solution hybridization assay (Genostics hepatitis B viral DNA, Abbott Laboratories) has been developed which uses a ‘251-labelled DNA probe (Kuhns et al., 1988). This assay has provided useful information for the clinical assessment of antiviral therapy (Kuhns et al., 1989; Zarski et al., 1989). Jalava et al. (1992) also developed a sandwich solution hybridization method for semi-quantitative detection of serum HBV DNA, which uses 35S-isotope as label and hybrid collection based on biotin-avidin affinity. However, these different radioactive assays have limited application in clinical laboratories due to the short half-life of the radioactive probe, as well as the special licensing requirements and safety considerations involved in handling. Recently, various tests using non-radioactive detection have been developed (Bronstein et al., 1989; Quibriac et al., 1989; Santantonio et al., 1990; Buti et al., 1991; Naoumov et al., 1991; Akar et al., 1992). However, some of these techniques appear to be slightly less sensitive than the methods requiring radioisotopes (Larzul et al., 1987; Saldanka et al., 1987; Manzin et al., 1989; Valentine-Thon et al., 1991). The hybrid capture HBV DNA assay (Digene Diagnostics) overcomes these problems by utilizing an efficient solution hybridization procedure coupled to a highly sensitive chemiluminescent signal amplification system based on LumiPhos@ 530. The purpose of the present study was to compare this new quantitative, non-radioactive hybridization assay with the quantitative radioactive method (Genostics, Abbott Laboratories), as regards its sensitivity, specificity and reproducibility, and its use for monitoring of antiviral therapy.
2. Materials and methods 2.1. Patients and serum samples One hundred and twenty-three serum samples from 58 patients (47 HBsAg-positive carriers, 10 HBsAg-negative carriers and one patient with unknown HBV serological
V. Barlet et al. /Journal
of Virological Methods 49 (1994) 141-I 52
143
status) were studied. Sera were chosen at random among serum samples sent to the laboratory for quantitative HBV DNA analysis. All serum samples were kept at - 20°C until use. The HBV serological status of the patients was unknown during the study. Comparative analysis between the non-radioactive, hybrid capture assay and the radioactive, solution hybridization method was performed on 113 serum samples from 48 patients (39 HBsAg-positive carriers, eight HBsAg-negative carriers and one patient with unknown HBV serological status). Twenty out of the 21 sera with low Genostics values (between 1.5 and 10 pg/ml) were also tested by genomic amplification (PCR). The reproducibility of the chemiluminescent molecular hybridization procedure was evaluated on 30 sera from 27 patients. Finally, a comparative study was performed for the HBV DNA level detected by Digene hybrid capture and Genostics assays in eight patients undergoing antiviral therapy with interferon. 2.2. Serological
HBV markers
HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe EIA kits (Abbott Labs, North Chicago, IL). 2.3. Quantitative
were assayed with commercial
serum HBV DNA detection
2.3.1. Radioactive solution hybridization assay (Genostics test) The radioactive solution hybridization assay was carried out according to the procedure described by Kuhns et al. (1988). This technique used an ‘251-labelled DNA probe. Results were expressed in pg HBV DNA per ml. Samples with a value equal to or greater than 1.5 pg/ml HBV DNA were considered positive. 2.3.2. Non-radioactive chemiluminescent molecular hybridization assay (Digene hybrid capture system) The chemiluminescent molecular hybridization assay was carried out according to the procedure described by the manufacturers (Digene Diagnostics, Silver Spring, MD). 50 ~1 serum were diluted and prepared by the addition of reagents 1 and 2. After incubation at 65 + 2°C during 20 & 5 min, a denaturation step was carried out with 50 ml of reagent 3. Hybridization of the target DNA was carried out for 60 f 5 min at 65 + 2°C after the addition of 50 ml of specific full length genomic HBV RNA probe. The resultant RNA-DNA hybrid was captured onto the surface of a tube coated with an anti RNA-DNA hybrid antibody. The immobilized hybrid was then incubated with a second anti-hybrid antibody conjugated to alkaline phosphatase and detected with a chemiluminescent substrate (LumiPhos @ 530). As the substrate was cleaved by the bound alkaline phosphatase, light was emitted which was measured as relative light units (RLUs) on a luminometer. The intensity of the light emitted was proportional to the amount of target DNA in the specimen. A calibration curve of the standards was plotted and the RLU value of each specimen was compared to the RLU values of the standards (one negative control and three positive controls containing 10 pg/ml, 200 pg/ml and 2 ng/ml of HBV DNA in specimens, respectively). Specimens with RLU values equal to or greater than the HBV cutoff value were considered positive. Results were
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expressed in pg HBV DNA/ml pg/ml to 2 ng/ml of serum.
of Virological Methods 49 (1994) 141-152
of serum and the assay was linear in the range of 10
2.4. HBV DNA detection by PCR HBV DNA was extracted from 500 ml of serum sample by the standard phenol/ chloroform procedure (Maniatis et al., 1989). Two conserved sequences of the HBV genome, located in the pre-S and pre-C regions, were amplified. The primers MD 19 (nt 2816-2833) and MD 16 (nt 187-170) (Gerken et al., 1991) were used to amplify the pre-S sequence. The pre-C region was amplified with the primer VB3 (nt 1735-1755) defined in our laboratory and the primer BCl (nt 1974-1955) (Carman et al., 1989). Thirty-five cycles of amplification were carried out with the pre-C primer pair, and 40 cycles with the pre-S primer pair. The specificity of the amplification was confirmed by Southern blot analysis with 32P end-labelled probes. Negative controls included sera from HBV-seronegative patients and the substitution of water for the DNA template. P-Globin-specific primers were used as positive PCR controls. 2.5. Statistical
analysis
Statistical analysis was carried out using the c2 test with Yates correction. The reproducibility of the chemiluminescent assay, concerning the qualitative detection of HBV DNA in sera, was evaluated by the Kappa test. The quantitative reproducibility of the test in HBV DNA-positive samples was expressed by plotting pairs of HBV DNA measurements, according to the method of Bland et al. (1986).
3. Results 3.1. Reproducibility
of the chemiluminescent
assay
The chemiluminescent assay was tested for reproducibility with 30 sera: two independent experiments were carried out on 24 sera (7 with two differents kits, 17 with the same kit) and three experiments were carried out on the other six sera (all with the same kit). Only two samples out of the 30 sera tested (6.7%) gave discordant results in two separate hybrid capture measurements (patients 29 and 30, Table 1). These divergent determinations were obtained in successive experiments with two different kits. The overall reproducibility rate in the tests was 93.3% (P < O.OOl>,with a Kappa test value of 0.86. Except for the two sera giving discordant results, 12 serum samples were positive with the chemiluminescent assay (2 10 pg/ml). Fig. 1 shows that the difference between the results of two determinations increased as the HBV DNA level increased. There is therefore a lack of agreement between the two measurements, especially at high HBV DNA values. The mean (absolute value) of the differences between two HBV DNA hybrid capture determinations was equal to 132 pg/ml in the group of sera with low HBV DNA levels ( < 1200 pg/ml) (patients 1, 2, 3, 24 and 25). For the seven sera
V. Barlet et al./Journal Table 1 Reproducibility Serum number (II = 30)
of the chemiluminescent
41 169 861
145
hybrid capture assay in 30 sera Difference
HBV DNA value (pg/ml) No. of determinations 1
1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16
of Virological Methods 49 (1994) 141-1.52
(pg/ml)
2
3
14 565 1130 _
11 464 1070 -
_
-
3 420 2930 3930 2810 2 270 3170 2000
4420 3 420 5 660 2250 2 740 3 980 2160 _
in HBV DNA (l-2)
36 - 396 - 269
-1000 - 490 -1730 560 - 470 -810 - 160
_
II
18 19 20 21 22 23 24 25 26
-
_ _
81 23 -
102 34 _
27 28 29 30
-21 -11
_ 94 34
HBV DNA determinations 1 and 2 were performed in two independent experiments (patients 1 to 23 with the same kit, patients 24 to 30 with 2 different kits). Patients 20 and 30 gave discordant results in two separate Hybrid Capture measurements; - , negative result.
with HBV DNA equal to or higher than 2000 pg/ml (patients (absolute value) of the differences was equivalent to 586 pg/ml.
7 to 13), the mean
3.2. Comparison between the two methods The radioactive and the chemiluminescent methods were tested in parallel on 113 sera (Table 2). The sera were divided into four groups according to their HBV DNA values with the Genostics assay (< 1.5 pg/ml, 1.5 to 10 pg/ml, 10 to 100 pg/ml and 2 100 pg/ml). In 101 (89.4%) cases, the results of the radioactive and chemiluminescent assays were similar. All sera with Genostics values less than 1.5 pg/ml (n = 19) gave negative results with the chemiluminescent test, except for one (patient 30, Table
V. Barlet et al./Journal
146
2 C
of Virological Methods 49 (1994) 141-152
.___________________--______
1000,
Mea”+pSD
soo-
0
SF n.22 g cm .cm
.s
0
0.80 _,__O-___
I
-5oo.m
~_~-~~~~~~~~~
Mean 0
c
0
0 g .E 9
-lOOO.-
:
-1500-:______________________________________________
0
Mean-2SD
E m
g
0
-2ooor
I 0
Average
1000
2000
HBV DNA between
3000
4000
the two Digene independent
5000
determinations
(pg/ml)
Fig. 1. Difference between the absolute values from two independent HBV DNA hybrid capture determinations plotted against the mean, for each serum sample. SD, standard deviation.
1) which was found to be positive in a second chemiluminescent test. The two hybrid capture values obtained for this serum sample were 3 pg/ml and 34 pg/ml. Three further serum samples from this patient gave negative results in both the radioactive and non-radioactive assays. In patients with a Genostics value between 1.5 and 10 pg/ml (21 sera from 19 patients), the results observed by chemiluminescent and radioactive methods were discordant. Indeed, only nine serum samples (from seven patients) gave positive results with the hybrid capture assay (one of these presented the following two Digene values: 94 and 4.5 pg/ml) (patient 29, Table 1). The mean HBV DNA value of these patients was 54 pg/ml(19 to 121 pg/ml) with the chemiluminescent assay and 7 pg/ml with the radioactive test. All patients were HBsAg-positive carriers and 5/7 were HBeAg-positive. On the other hand, eight sera among the 12 remaining serum samples negative for the hybrid capture assay were HBsAg-positive and only one was HBeAg-positive. The mean Genostics value of these 12 patients was 3.5 pg/ml. PCR amplification was carried out on 20 of the sera with low Genostics values for HBV DNA (1.5 to 10 pg/ml). The two amplified HBV regions were found to be present in 17/20 of the serum samples tested. The three patients which gave negative results after
Table 2 Comparison of the hybrid capture HBV DNA assay to the radioactive sera (48 patients) Genostics (pg/ml) (radioactive assay) Digene (pg/ml) (chemiluminescent + (210) - ( < 10)
solution hybridization
method in 113
< 1.5 (?I = 19)
1.5-10 (it = 21)
10-100 (n = 30)
2100 (n = 43)
9b 12
30
19 a
43 -
assay)
a One patient presented two discordant b One patient presented two discordant
values with hybrid capture assay (3 and 34 pg/ml). values with hybrid capture assay (94 and 4.5 pg/ml).
V. Barlet et al. /Journal
of Virological Methods 49 (1994) 141-152
147
HBV DNA pwnl
0
1
2
3
4
5
Months after start of therapy
Fig. 2. Comparison of the HBV DNA chemiluminescent assay (Digene) and the radioactive method (Genostics) in serum from patient A during antiviral interferon therapy. This patient presented an anti-HBe (+ 1, chronic active hepatitis. IFN, interferon.
genomic amplification were HBV DNA-negative with the non-radioactive assay. Two were HBsAg-negative and the third was an HBsAg-positive and anti-HBe-positive carrier. All sera with a Genostics value greater than 10 pg/ ml (n = 73) were positive by hybrid capture assay. The mean Genostics and hybrid capture values were 39 pg/ml and 426 pg/ ml, respectively, for sera with Genostics value between 10 and 100 pg/ ml. For serum samples in which the quantitative, radioactive method gave high HBV DNA values (2 100 pg/ml), the mean Genostics and Digene values were 566 and 2263 pg/ ml, respectively. Finally, a comparative analysis of the chemiluminescent assay with the radioactive solution hybridization method gave a sensitivity of 87.2% and a specificity of 100%.
3500 3000 2500 HBV DNA
2000
(pg/mU 1500 1000 500 0 0
0.5
1
2
3
4
6
16
Fig. 3. Serum HBV DNA detected by the chemiluminescent assay (Digene) and the radioactive method (Genostics) in another patient (patient B) undergoing interferon treatment. This patient presented a HBeAg ( + 1, chronic active hepatitis. IFN, interferon.
148
K Barlet et al. /Journal
of Virological Methods 49 (1994) 141-152
Only 82 out of the 94 serum samples positive with the radioactive assay (HBV DNA 2 1.5 pg/ml) were found to be positive with the chemiluminescent test. In the 82 sera found to be positive by both methods, the Digene values were, on average, U-times higher than the Genostics values. On the other hand, all sera from HBsAg-negative patients (n = 10) gave negative results with the hybrid capture HBV DNA assay. 3.3. HBV DNA levels in patients
undergoing
antiviral treatment
Comparison of relative changes in HBV DNA levels with radioactive and hybrid capture assays in eight patients treated with interferon and monitored monthly during the course of treatment showed that HBV DNA values determined by the two methods followed the same general pattern. However, estimates of the HBV DNA levels differed noticeably between the two methods, particularly at high HBV DNA levels (Digene values > Genostics values). Figs. 2 and 3 show representative curves for two patients undergoing antiviral therapy. The ratio between the Digene and the Genostics values varied from 1.3 to 6.4 in patient A (an anti-HBe-positive chronic hepatitis B carrier) and from 3.5 to 23.8 in patient B (an HBeAg-positive carrier).
4. Discussion Quantitative detection of HBV DNA in the sera of chronic HBV carriers represents the most accurate marker of viral replication and is useful for monitoring patient response to antiviral therapy (Kuhns et al., 1989). Recently, several nonisotopic hybridization methods have been developed to overcome the disadvantages of radioisotopes (Santantonio et al., 1990; Buti et al., 1991; Naoumov et al., 1991; Valentine-Thon et al., 1991; Akar et al., 1992). In this study, we investigated the sensitivity, specificity and use for antiviral therapy management, of a non-radioactive assay (Digene), in comparison with a radioactive method (Genostics) for the quantitation of HBV DNA in human serum samples. The Digene hybrid capture system is a rapid sandwich capture molecular hybridization assay which utilizes chemiluminescent detection. The time required for this test is approximately 4-5 hours. The data show an agreement between the two methods of 89.4%, which demonstrates an almost similar sensitivity and specificity. However, the two methods are not comparable in terms of HBV DNA quantification, particularly for high values. Indeed, HBV DNA titers, although both expressed in pg/ml, were very different in the two tests. The ratio between the Digene and the Genostics values was, on average, equal to 8.5 (7.6 for Genostics values between 1.5 to 10 pg/ml and 8.6 for Genostics values > 10 pg/ml) but varied from one patient to another, and in the same patient during the follow-up. A similar comparison of the hybrid capture HBV DNA assay to the Genostics method in sera from 30 HBsAg-positive patients (Garcia et al., 1992) gave sensitivity, specificity and agreement values of lOO%, 71% and 93%, respectively. In our work, samples with Abbott values equal to or greater than 1.5 pg/ ml were considered reactive for HBV DNA. However, the positive limit level of the radioactive solution hybridization assay has been re-evaluated by several investigators to around 10 pg/ml of serum
V. Barlet et al. /Journal
of Virological Methods 49 (1994) 141-152
149
HBV DNA. This could explain the differences observed between our data and the study of Garcia et al. (1992) concerning the sensitivity and specificity of the chemiluminescent test compared to the radioactive assay. In this study, the reproducibility of the hybrid capture assay was also evaluated. There was good qualitative agreement between the values from two or three Digene determinations in 28 out of 30 samples (12 positive and 16 negative). In the patients which gave divergent results in two successive determinations, the positive Digene values for HBV DNA were low ( < 100 pg/ ml). Moreover, the Genostics test gave negative results for one patient and values between 1.5 to 10 pg/ ml for the other. It also appears that the qualitative reproducibility defect of the chemiluminescent test occurred in patients with a low level of viral replication. Patients with high HBV DNA values ( 2 2000 pg/ml) showed a mean variation of around 590 pg/ml in Digene values from same sample. Therefore, variations in HBV DNA levels between two tests which are lower than this value should not be considered significant. The hybrid capture assay is a quantitative method, with a linear range of HBV DNA detection from 10 to 2000 pg/ml. For HBV DNA levels higher than 2000 pg/ml, quantitative variations between two determinations on the same serum sample were probably due to the analysis procedure (luminometer). However, in our study, we also observed large differences between separate Digene determinations in sera with low HBV DNA values. Such variation in the measurements of HBV DNA level should be taken into account when monitoring viral replication in patients during antiviral treatment. In a previous study, we showed that it was not possible to draw a firm conclusion as to the presence or absence of HBV DNA in patients with low HBV DNA values (between 1.5 and 10 pg/ml) in the Genostics test. In these cases, the results have to be interpreted according to the clinical and serological status of the patients. Genomic amplification by PCR is also useful to confirm the presence of viral particles in these sera (Barlet et al., 1991). Our data show discrepancies between the radioactive and chemiluminescent techniques for this group of patients with low Genostics values for HBV DNA. In these patients, the hybrid capture results showed good agreement (84%) with the presence of the HBeAg serological marker. The number of HBeAg-positive and Digene-positive patients was significantly higher than the number of HBeAg-positive carriers and hybrid capture-negative patients (P < 0.02). The chemiluminescent method may therefore be a useful tool for clinical diagnosis in patients with low amount of HBV DNA. However, 9 out of the 12 hybrid capture-negative patients gave positive results after genomic amplification, indicating a persistent viral replication. The quantitation of serum HBV DNA in patients with chronic hepatitis treated with interferon is a valuable means of evaluating the efficacy of antiviral therapy (Kuhns et al., 1989) and is considered as an important factor predicting response to interferon (Kuhns et al., 1989; Thomas et al., 1991). Response to antiviral treatment was defined as the loss of detectable HBV DNA and HBeAg, either during or after therapy. Previous studies (Kuhns et al., 1989; Zarski et al., 1989) have shown that the radioactive solution hybridization test represents a standardized and quantitative method for monitoring viral replication before, during and after antiviral therapy. In the present work, changes in HBV DNA levels measured by the Digene and Genostics assays showed a good correlation in patients undergoing interferon treatment. However, the Digene values
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of Vimlogical Methods 49 (1994) 141-152
were higher than the Genostics values, with a variable ratio in the same patient during the course of therapy, particularly at high HBV DNA levels. Therefore, changes in high viral replication levels seem better resolved by the Genostics assay. In conclusion, we believe that this non-radioactive assay is useful for the detection of HBV DNA in serum and that it may be a good alternative to the ‘251-labelled probe-based assay. This test is rapid, sensitive, and easy to use, and therefore suitable for routine testing. The performance characteristics of the Digene hybrid capture assay are similar to the solution hybridization assay with a radioactive probe. The only disadvantage of the chemiluminescent method is its low reproducibility at high HBV DNA values, which could hamper the monitoring of viral replication during therapy in patients with particularly high HBV DNA levels.
Acknowledgement We are grateful to Prof. M. Suh for critical reading of the manuscript.
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Journal of Virological Methods 49 (1994) 141-152
Quantitative detection of hepatitis B virus DNA in serum using chemiluminescence: comparison with radioactive solution hybridization assay Val&ie Barlet a1*,Marielle Cohard aTb,Marie Ange Thelu a~b, Marie Jeanne Chaix a, Christine Baccard a, Jean Pierre Zarski a2b, Jean Marie Seigneurin a aLaboratoire de Virologie Midicale Mole’culaire, Facultt? de Midecine/ CHU Grenoble, BP 217 X, 38043 Grenoble Cedex 09, France b Service d’Hkpato-Gastroenthologie, I-CHU Grenoble, France
Accepted 14 February 1994
Abstract A quantitative, non-radioactive hybrid capture HBV DNA assay (Digene Diagnostics), which uses an efficient solution hybridization procedure coupled to a sensitive chemiluminescent signal amplification system, was compared with the quantitative, radioactive solution hybridization assay (Genostics, Abbott Laboratories), in hepatitis B virus carriers, particularly in those undergoing antiviral therapy. The qualitative reproducibility of the chemiluminescent method, tested on 30 sera, was acceptable, with a reproducibility rate of 93.3%. A comparison of this hybrid capture HBV DNA assay with the radioactive test on 113 sera obtained from 48 patients (39 HBsAg-positive patients) gave a sensitivity of 87.2%, a specificity of 100% and an agreement between the two tests of 89.4% (101 sera including 82 HBV DNA positive and 19 negative samples). Changes in HBV DNA levels measured by the two assays showed a good correlation with each other during interferon therapy. However, the hybrid capture values were higher than the radioactive assay values, with the ratio of the two values being variable in the same patient during the course of treatment. The Genostics assay therefore seems to be a more accurate procedure for evaluating changes in viral replication, particularly at high HBV DNA levels. However, the hybrid capture method is faster and has the advantage of being a non-radioactive procedure. This chemilumines-
* Corresponding author. Fax: + 33 76.54.80.74. 0166-0934/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00035-F
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cent assay is easy to perform as a routine diagnostic procedure and may be a useful alternative to the radioactive solution hybridization method. Keywords: Hepatitis B virus DNA; Quantitative chemiluminescent assay; Solution hybridization method; Antiviral therapy monitoring
1. Introduction The detection of serum hepatitis B virus (HBV) DNA by molecular hybridization represents the most informative method for determining viral replication activity and serum infectivity (Bonino et al., 1981; Weller et al., 1982; Scotto et al., 1983; Pontisso et al., 1989). Although the polymerase chain reaction (PCR) is the most sensitive method for HBV DNA detection in serum, it cannot be regarded as a routine test. Rapid detection methods have primarily used radiolabelled probes c3*P DNA probes) (Beminger et al., 1982; Scotto et al., 1983; Feinman et al., 1984; Fagan et al., 1985) but the interpretation of results is semi-quantitative and subjective. Recently, a quantitative and standardized solution hybridization assay (Genostics hepatitis B viral DNA, Abbott Laboratories) has been developed which uses a ‘251-labelled DNA probe (Kuhns et al., 1988). This assay has provided useful information for the clinical assessment of antiviral therapy (Kuhns et al., 1989; Zarski et al., 1989). Jalava et al. (1992) also developed a sandwich solution hybridization method for semi-quantitative detection of serum HBV DNA, which uses 35S-isotope as label and hybrid collection based on biotin-avidin affinity. However, these different radioactive assays have limited application in clinical laboratories due to the short half-life of the radioactive probe, as well as the special licensing requirements and safety considerations involved in handling. Recently, various tests using non-radioactive detection have been developed (Bronstein et al., 1989; Quibriac et al., 1989; Santantonio et al., 1990; Buti et al., 1991; Naoumov et al., 1991; Akar et al., 1992). However, some of these techniques appear to be slightly less sensitive than the methods requiring radioisotopes (Larzul et al., 1987; Saldanka et al., 1987; Manzin et al., 1989; Valentine-Thon et al., 1991). The hybrid capture HBV DNA assay (Digene Diagnostics) overcomes these problems by utilizing an efficient solution hybridization procedure coupled to a highly sensitive chemiluminescent signal amplification system based on LumiPhos@ 530. The purpose of the present study was to compare this new quantitative, non-radioactive hybridization assay with the quantitative radioactive method (Genostics, Abbott Laboratories), as regards its sensitivity, specificity and reproducibility, and its use for monitoring of antiviral therapy.
2. Materials and methods 2.1. Patients and serum samples One hundred and twenty-three serum samples from 58 patients (47 HBsAg-positive carriers, 10 HBsAg-negative carriers and one patient with unknown HBV serological
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143
status) were studied. Sera were chosen at random among serum samples sent to the laboratory for quantitative HBV DNA analysis. All serum samples were kept at - 20°C until use. The HBV serological status of the patients was unknown during the study. Comparative analysis between the non-radioactive, hybrid capture assay and the radioactive, solution hybridization method was performed on 113 serum samples from 48 patients (39 HBsAg-positive carriers, eight HBsAg-negative carriers and one patient with unknown HBV serological status). Twenty out of the 21 sera with low Genostics values (between 1.5 and 10 pg/ml) were also tested by genomic amplification (PCR). The reproducibility of the chemiluminescent molecular hybridization procedure was evaluated on 30 sera from 27 patients. Finally, a comparative study was performed for the HBV DNA level detected by Digene hybrid capture and Genostics assays in eight patients undergoing antiviral therapy with interferon. 2.2. Serological
HBV markers
HBsAg, anti-HBs, anti-HBc, HBeAg and anti-HBe EIA kits (Abbott Labs, North Chicago, IL). 2.3. Quantitative
were assayed with commercial
serum HBV DNA detection
2.3.1. Radioactive solution hybridization assay (Genostics test) The radioactive solution hybridization assay was carried out according to the procedure described by Kuhns et al. (1988). This technique used an ‘251-labelled DNA probe. Results were expressed in pg HBV DNA per ml. Samples with a value equal to or greater than 1.5 pg/ml HBV DNA were considered positive. 2.3.2. Non-radioactive chemiluminescent molecular hybridization assay (Digene hybrid capture system) The chemiluminescent molecular hybridization assay was carried out according to the procedure described by the manufacturers (Digene Diagnostics, Silver Spring, MD). 50 ~1 serum were diluted and prepared by the addition of reagents 1 and 2. After incubation at 65 + 2°C during 20 & 5 min, a denaturation step was carried out with 50 ml of reagent 3. Hybridization of the target DNA was carried out for 60 f 5 min at 65 + 2°C after the addition of 50 ml of specific full length genomic HBV RNA probe. The resultant RNA-DNA hybrid was captured onto the surface of a tube coated with an anti RNA-DNA hybrid antibody. The immobilized hybrid was then incubated with a second anti-hybrid antibody conjugated to alkaline phosphatase and detected with a chemiluminescent substrate (LumiPhos @ 530). As the substrate was cleaved by the bound alkaline phosphatase, light was emitted which was measured as relative light units (RLUs) on a luminometer. The intensity of the light emitted was proportional to the amount of target DNA in the specimen. A calibration curve of the standards was plotted and the RLU value of each specimen was compared to the RLU values of the standards (one negative control and three positive controls containing 10 pg/ml, 200 pg/ml and 2 ng/ml of HBV DNA in specimens, respectively). Specimens with RLU values equal to or greater than the HBV cutoff value were considered positive. Results were
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expressed in pg HBV DNA/ml pg/ml to 2 ng/ml of serum.
of Virological Methods 49 (1994) 141-152
of serum and the assay was linear in the range of 10
2.4. HBV DNA detection by PCR HBV DNA was extracted from 500 ml of serum sample by the standard phenol/ chloroform procedure (Maniatis et al., 1989). Two conserved sequences of the HBV genome, located in the pre-S and pre-C regions, were amplified. The primers MD 19 (nt 2816-2833) and MD 16 (nt 187-170) (Gerken et al., 1991) were used to amplify the pre-S sequence. The pre-C region was amplified with the primer VB3 (nt 1735-1755) defined in our laboratory and the primer BCl (nt 1974-1955) (Carman et al., 1989). Thirty-five cycles of amplification were carried out with the pre-C primer pair, and 40 cycles with the pre-S primer pair. The specificity of the amplification was confirmed by Southern blot analysis with 32P end-labelled probes. Negative controls included sera from HBV-seronegative patients and the substitution of water for the DNA template. P-Globin-specific primers were used as positive PCR controls. 2.5. Statistical
analysis
Statistical analysis was carried out using the c2 test with Yates correction. The reproducibility of the chemiluminescent assay, concerning the qualitative detection of HBV DNA in sera, was evaluated by the Kappa test. The quantitative reproducibility of the test in HBV DNA-positive samples was expressed by plotting pairs of HBV DNA measurements, according to the method of Bland et al. (1986).
3. Results 3.1. Reproducibility
of the chemiluminescent
assay
The chemiluminescent assay was tested for reproducibility with 30 sera: two independent experiments were carried out on 24 sera (7 with two differents kits, 17 with the same kit) and three experiments were carried out on the other six sera (all with the same kit). Only two samples out of the 30 sera tested (6.7%) gave discordant results in two separate hybrid capture measurements (patients 29 and 30, Table 1). These divergent determinations were obtained in successive experiments with two different kits. The overall reproducibility rate in the tests was 93.3% (P < O.OOl>,with a Kappa test value of 0.86. Except for the two sera giving discordant results, 12 serum samples were positive with the chemiluminescent assay (2 10 pg/ml). Fig. 1 shows that the difference between the results of two determinations increased as the HBV DNA level increased. There is therefore a lack of agreement between the two measurements, especially at high HBV DNA values. The mean (absolute value) of the differences between two HBV DNA hybrid capture determinations was equal to 132 pg/ml in the group of sera with low HBV DNA levels ( < 1200 pg/ml) (patients 1, 2, 3, 24 and 25). For the seven sera
V. Barlet et al./Journal Table 1 Reproducibility Serum number (II = 30)
of the chemiluminescent
41 169 861
145
hybrid capture assay in 30 sera Difference
HBV DNA value (pg/ml) No. of determinations 1
1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16
of Virological Methods 49 (1994) 141-1.52
(pg/ml)
2
3
14 565 1130 _
11 464 1070 -
_
-
3 420 2930 3930 2810 2 270 3170 2000
4420 3 420 5 660 2250 2 740 3 980 2160 _
in HBV DNA (l-2)
36 - 396 - 269
-1000 - 490 -1730 560 - 470 -810 - 160
_
II
18 19 20 21 22 23 24 25 26
-
_ _
81 23 -
102 34 _
27 28 29 30
-21 -11
_ 94 34
HBV DNA determinations 1 and 2 were performed in two independent experiments (patients 1 to 23 with the same kit, patients 24 to 30 with 2 different kits). Patients 20 and 30 gave discordant results in two separate Hybrid Capture measurements; - , negative result.
with HBV DNA equal to or higher than 2000 pg/ml (patients (absolute value) of the differences was equivalent to 586 pg/ml.
7 to 13), the mean
3.2. Comparison between the two methods The radioactive and the chemiluminescent methods were tested in parallel on 113 sera (Table 2). The sera were divided into four groups according to their HBV DNA values with the Genostics assay (< 1.5 pg/ml, 1.5 to 10 pg/ml, 10 to 100 pg/ml and 2 100 pg/ml). In 101 (89.4%) cases, the results of the radioactive and chemiluminescent assays were similar. All sera with Genostics values less than 1.5 pg/ml (n = 19) gave negative results with the chemiluminescent test, except for one (patient 30, Table
V. Barlet et al./Journal
146
2 C
of Virological Methods 49 (1994) 141-152
.___________________--______
1000,
Mea”+pSD
soo-
0
SF n.22 g cm .cm
.s
0
0.80 _,__O-___
I
-5oo.m
~_~-~~~~~~~~~
Mean 0
c
0
0 g .E 9
-lOOO.-
:
-1500-:______________________________________________
0
Mean-2SD
E m
g
0
-2ooor
I 0
Average
1000
2000
HBV DNA between
3000
4000
the two Digene independent
5000
determinations
(pg/ml)
Fig. 1. Difference between the absolute values from two independent HBV DNA hybrid capture determinations plotted against the mean, for each serum sample. SD, standard deviation.
1) which was found to be positive in a second chemiluminescent test. The two hybrid capture values obtained for this serum sample were 3 pg/ml and 34 pg/ml. Three further serum samples from this patient gave negative results in both the radioactive and non-radioactive assays. In patients with a Genostics value between 1.5 and 10 pg/ml (21 sera from 19 patients), the results observed by chemiluminescent and radioactive methods were discordant. Indeed, only nine serum samples (from seven patients) gave positive results with the hybrid capture assay (one of these presented the following two Digene values: 94 and 4.5 pg/ml) (patient 29, Table 1). The mean HBV DNA value of these patients was 54 pg/ml(19 to 121 pg/ml) with the chemiluminescent assay and 7 pg/ml with the radioactive test. All patients were HBsAg-positive carriers and 5/7 were HBeAg-positive. On the other hand, eight sera among the 12 remaining serum samples negative for the hybrid capture assay were HBsAg-positive and only one was HBeAg-positive. The mean Genostics value of these 12 patients was 3.5 pg/ml. PCR amplification was carried out on 20 of the sera with low Genostics values for HBV DNA (1.5 to 10 pg/ml). The two amplified HBV regions were found to be present in 17/20 of the serum samples tested. The three patients which gave negative results after
Table 2 Comparison of the hybrid capture HBV DNA assay to the radioactive sera (48 patients) Genostics (pg/ml) (radioactive assay) Digene (pg/ml) (chemiluminescent + (210) - ( < 10)
solution hybridization
method in 113
< 1.5 (?I = 19)
1.5-10 (it = 21)
10-100 (n = 30)
2100 (n = 43)
9b 12
30
19 a
43 -
assay)
a One patient presented two discordant b One patient presented two discordant
values with hybrid capture assay (3 and 34 pg/ml). values with hybrid capture assay (94 and 4.5 pg/ml).
V. Barlet et al. /Journal
of Virological Methods 49 (1994) 141-152
147
HBV DNA pwnl
0
1
2
3
4
5
Months after start of therapy
Fig. 2. Comparison of the HBV DNA chemiluminescent assay (Digene) and the radioactive method (Genostics) in serum from patient A during antiviral interferon therapy. This patient presented an anti-HBe (+ 1, chronic active hepatitis. IFN, interferon.
genomic amplification were HBV DNA-negative with the non-radioactive assay. Two were HBsAg-negative and the third was an HBsAg-positive and anti-HBe-positive carrier. All sera with a Genostics value greater than 10 pg/ ml (n = 73) were positive by hybrid capture assay. The mean Genostics and hybrid capture values were 39 pg/ml and 426 pg/ ml, respectively, for sera with Genostics value between 10 and 100 pg/ ml. For serum samples in which the quantitative, radioactive method gave high HBV DNA values (2 100 pg/ml), the mean Genostics and Digene values were 566 and 2263 pg/ ml, respectively. Finally, a comparative analysis of the chemiluminescent assay with the radioactive solution hybridization method gave a sensitivity of 87.2% and a specificity of 100%.
3500 3000 2500 HBV DNA
2000
(pg/mU 1500 1000 500 0 0
0.5
1
2
3
4
6
16
Fig. 3. Serum HBV DNA detected by the chemiluminescent assay (Digene) and the radioactive method (Genostics) in another patient (patient B) undergoing interferon treatment. This patient presented a HBeAg ( + 1, chronic active hepatitis. IFN, interferon.
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of Virological Methods 49 (1994) 141-152
Only 82 out of the 94 serum samples positive with the radioactive assay (HBV DNA 2 1.5 pg/ml) were found to be positive with the chemiluminescent test. In the 82 sera found to be positive by both methods, the Digene values were, on average, U-times higher than the Genostics values. On the other hand, all sera from HBsAg-negative patients (n = 10) gave negative results with the hybrid capture HBV DNA assay. 3.3. HBV DNA levels in patients
undergoing
antiviral treatment
Comparison of relative changes in HBV DNA levels with radioactive and hybrid capture assays in eight patients treated with interferon and monitored monthly during the course of treatment showed that HBV DNA values determined by the two methods followed the same general pattern. However, estimates of the HBV DNA levels differed noticeably between the two methods, particularly at high HBV DNA levels (Digene values > Genostics values). Figs. 2 and 3 show representative curves for two patients undergoing antiviral therapy. The ratio between the Digene and the Genostics values varied from 1.3 to 6.4 in patient A (an anti-HBe-positive chronic hepatitis B carrier) and from 3.5 to 23.8 in patient B (an HBeAg-positive carrier).
4. Discussion Quantitative detection of HBV DNA in the sera of chronic HBV carriers represents the most accurate marker of viral replication and is useful for monitoring patient response to antiviral therapy (Kuhns et al., 1989). Recently, several nonisotopic hybridization methods have been developed to overcome the disadvantages of radioisotopes (Santantonio et al., 1990; Buti et al., 1991; Naoumov et al., 1991; Valentine-Thon et al., 1991; Akar et al., 1992). In this study, we investigated the sensitivity, specificity and use for antiviral therapy management, of a non-radioactive assay (Digene), in comparison with a radioactive method (Genostics) for the quantitation of HBV DNA in human serum samples. The Digene hybrid capture system is a rapid sandwich capture molecular hybridization assay which utilizes chemiluminescent detection. The time required for this test is approximately 4-5 hours. The data show an agreement between the two methods of 89.4%, which demonstrates an almost similar sensitivity and specificity. However, the two methods are not comparable in terms of HBV DNA quantification, particularly for high values. Indeed, HBV DNA titers, although both expressed in pg/ml, were very different in the two tests. The ratio between the Digene and the Genostics values was, on average, equal to 8.5 (7.6 for Genostics values between 1.5 to 10 pg/ml and 8.6 for Genostics values > 10 pg/ml) but varied from one patient to another, and in the same patient during the follow-up. A similar comparison of the hybrid capture HBV DNA assay to the Genostics method in sera from 30 HBsAg-positive patients (Garcia et al., 1992) gave sensitivity, specificity and agreement values of lOO%, 71% and 93%, respectively. In our work, samples with Abbott values equal to or greater than 1.5 pg/ ml were considered reactive for HBV DNA. However, the positive limit level of the radioactive solution hybridization assay has been re-evaluated by several investigators to around 10 pg/ml of serum
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of Virological Methods 49 (1994) 141-152
149
HBV DNA. This could explain the differences observed between our data and the study of Garcia et al. (1992) concerning the sensitivity and specificity of the chemiluminescent test compared to the radioactive assay. In this study, the reproducibility of the hybrid capture assay was also evaluated. There was good qualitative agreement between the values from two or three Digene determinations in 28 out of 30 samples (12 positive and 16 negative). In the patients which gave divergent results in two successive determinations, the positive Digene values for HBV DNA were low ( < 100 pg/ ml). Moreover, the Genostics test gave negative results for one patient and values between 1.5 to 10 pg/ ml for the other. It also appears that the qualitative reproducibility defect of the chemiluminescent test occurred in patients with a low level of viral replication. Patients with high HBV DNA values ( 2 2000 pg/ml) showed a mean variation of around 590 pg/ml in Digene values from same sample. Therefore, variations in HBV DNA levels between two tests which are lower than this value should not be considered significant. The hybrid capture assay is a quantitative method, with a linear range of HBV DNA detection from 10 to 2000 pg/ml. For HBV DNA levels higher than 2000 pg/ml, quantitative variations between two determinations on the same serum sample were probably due to the analysis procedure (luminometer). However, in our study, we also observed large differences between separate Digene determinations in sera with low HBV DNA values. Such variation in the measurements of HBV DNA level should be taken into account when monitoring viral replication in patients during antiviral treatment. In a previous study, we showed that it was not possible to draw a firm conclusion as to the presence or absence of HBV DNA in patients with low HBV DNA values (between 1.5 and 10 pg/ml) in the Genostics test. In these cases, the results have to be interpreted according to the clinical and serological status of the patients. Genomic amplification by PCR is also useful to confirm the presence of viral particles in these sera (Barlet et al., 1991). Our data show discrepancies between the radioactive and chemiluminescent techniques for this group of patients with low Genostics values for HBV DNA. In these patients, the hybrid capture results showed good agreement (84%) with the presence of the HBeAg serological marker. The number of HBeAg-positive and Digene-positive patients was significantly higher than the number of HBeAg-positive carriers and hybrid capture-negative patients (P < 0.02). The chemiluminescent method may therefore be a useful tool for clinical diagnosis in patients with low amount of HBV DNA. However, 9 out of the 12 hybrid capture-negative patients gave positive results after genomic amplification, indicating a persistent viral replication. The quantitation of serum HBV DNA in patients with chronic hepatitis treated with interferon is a valuable means of evaluating the efficacy of antiviral therapy (Kuhns et al., 1989) and is considered as an important factor predicting response to interferon (Kuhns et al., 1989; Thomas et al., 1991). Response to antiviral treatment was defined as the loss of detectable HBV DNA and HBeAg, either during or after therapy. Previous studies (Kuhns et al., 1989; Zarski et al., 1989) have shown that the radioactive solution hybridization test represents a standardized and quantitative method for monitoring viral replication before, during and after antiviral therapy. In the present work, changes in HBV DNA levels measured by the Digene and Genostics assays showed a good correlation in patients undergoing interferon treatment. However, the Digene values
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were higher than the Genostics values, with a variable ratio in the same patient during the course of therapy, particularly at high HBV DNA levels. Therefore, changes in high viral replication levels seem better resolved by the Genostics assay. In conclusion, we believe that this non-radioactive assay is useful for the detection of HBV DNA in serum and that it may be a good alternative to the ‘251-labelled probe-based assay. This test is rapid, sensitive, and easy to use, and therefore suitable for routine testing. The performance characteristics of the Digene hybrid capture assay are similar to the solution hybridization assay with a radioactive probe. The only disadvantage of the chemiluminescent method is its low reproducibility at high HBV DNA values, which could hamper the monitoring of viral replication during therapy in patients with particularly high HBV DNA levels.
Acknowledgement We are grateful to Prof. M. Suh for critical reading of the manuscript.
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