An enzyme-linked (alkaline phosphatase) oligonucleotide probe for the detection of serum hepatitis B virus DNA Garcia FG, Quiros E, Bernal MC, Salmeron J, Maroto MC. An enzymelinked (alkaline phosphatase) oligonucleotide probe for the detection of serum hepatitis B virus DNA. Liver 1992: 12: 179-182. Abstract: Serum HBV-DNA detection represents a new and important diagnostic tool for the hepatologist, as well as a prognostic and therapeutic guide. Though most laboratories use genomic radioactive probes, the appearance of commercially available enzyme-linked oligonucleotide probes, which simplify the method, represents an advance in HBV-DNA detection. In order to verify the value of an enzyme-linked (alkaline phosphatase) oligonucleotide probe in HBV-DNA detection, we have first compared the results of the detection of serum HBV-DNA in 46 patients at different serological stages of HBV infection using this probe and with those obtained using a genomic P3’-labeled probe. Fisher’s test analysis of the results shows statistical significance; this indicates a high correlation between methods. Secondly, we have tested 296 sera of patients at different serological stages of HBV infection with the enzyme-linked probe; we have found HBV-DNA in 8 patients with acute hepatitis (61.54%), 61 HBeAg (70.12%), and 7 anti-HBe (12.07%) chronic hepatitis. These results show the value of this probe for routine survey of serum HBV-DNA using a simple method which avoids the use of radioactivity.
Serum HBV-DNA detection by molecular hybridization has modified the traditional concept by which the detection of serum HBeAg corresponded with HBV active replication, while anti-HBe detection corresponded to the integrated phase of infection (1, 2). Usually, patients chronically infected by the virus who are positive for HBeAg show high levels of HBV-DNA; when these patients seroconvert to anti-HBe, HBV-DNA becomes undetectable (3). However, some of these patients remain positive for HBV-DNA after seroconversion even though they are negative for HBeAg (4). Viral DNA represents a direct measure of the virion, while HBeAg detection depends on its concentration related to anti-HBe. There are also patients who are positive for HBeAg and negative for HBV-DNA (5). It seems, therefore, that HBV-DNA is a better marker of active replication and infectivity. Patients suffering from acute hepatitis B are generally positive for HBV-DNA, though it becomes undetectable before or when HBeAg disappears (6, 7). Serum HBV-DNA detection represents a new and important diagnostic tool for the hepatologist,
F. 6. Garcia, E. Quiros, M. C. Bernai, J. Salmeron’ and M. C. Maroto Department of Microbiology and ‘Unit of Hepatology, University Hospital, Granada, Spain
Key words: enzymatic probe - HBV-DNA radioactive probe - serum detection Federico Garcia Garcia, Departamento de Microbiologia, Facultad de Medicina. Av. Madrid-11, Granada, 18012, Spain Received 3 September 1991, accepted for publication 23 April 1992
as well as a prognostic and therapeutic guide. Molecular biology offers the choice of several DNA probes for its detection, depending on their molecular weight, the detection system, and the hybridization format. Though most laboratories use genomic radioactive probes, the appearance of commercially available enzyme-linked oligonucleotide probes that simplify the method represents an advance in HBV-DNA detection. The aim of this study is to evaluate an enzymelinked oligonucleotide probe as a worthwhile alternative in HBV-DNA detection. Patients and methods
Our study was in two phases. First, we compared HBV-DNA detection by a genomic radioactive probe and an enzyme-linked oligonucleotide probe in serum samples from 46 individuals: 7 asymptomatic carriers, 29 chronic hepatitis, and 10 seronegative controls. Secondly, we tested sera from 296 individuals (66 asymptomatic carriers, 145 chronic hepatitis, 13 acute hepatitis, 19 resolved hepatitis, and 53 seronegative controls) with the oligonucleotide probe. 179
Garcia et al. Serological assays
Commercial enzyme immune assays (Abbott Laboratories) were used for screening HBsAg, antiHBs, anti-HBc, anti-HBc IgM, HBeAg, and antiHBe. HBV-DNA assays
1. Genomic radioactive probe. A spot hybridization technique for the detection of HBV-DNA was used; 250 pl of each sample and 600 pl of STENMBSA (Tris-HC1 0.1 M pH=7.4; C l N A 4 M; EDTA (Na), 0.1 M; BSA 10 mg/ml; Sucrose 0.3%) were spun for 4 h at 24500 g/4"C. After centrifugation, the pellet was resuspended in 160 p1 of Summer's buffer (Tris-HC1 5 mM, NaCl 75 mM, EDTA (Na), 5 mM) and incubated for 2 h with 28 pl SDS 25%, 32 pl Pronase 10 mg/ml, 4 pl t-RNA 10 mg/ml at 37"C, and DNA was extracted from the sample by phenol treatment. Samples were then incubated with 50 p1 I N NaOH for 10 rnin at room temperature, neutralized with 100 pl 1 M Tris-HCl+ 1.5 M NaCl p H = 7 and 100 pl0.5 M HCI+2.5 M NaCI, and transferred by means of a filtration apparatus onto a nylon filter soaked in 6 x SSC. Filters were air-dried after sample immobilization. Prehybridization was performed for 1 h at 70°C in sealed plastic bags containing 10 ml prehybridization solution (20 x Denhardt 0.1%, 20 x SSC 0.33%). Hybridization was performed using a P3' nick-translated cloned HBV-DNA pBR322 probe (specific activity = 10' cpm) in sealed plastic bags containing 10 ml hybridization solution (Deionized formamide 50%, 20 x SSC 33%, 100 mM Tris-HC1 pH = 7.95, salmon sperm DNA 2 mg/ml 0.008%, BSA 40 mg/ml 0.004%, SDS 0.08%) for 16-18 h at 37°C. Filters were washed five times in 2 x SSC + 0.1% SDS for 15 rnin and five times in 0.1 x SSC + 0.1% SDS for 15 rnin with agitation at 70°C. After drying at room temperature, filters were autoradiographed at - 70°C for 24 h.
2. Oligonucleotide enzyme-linked probe. A spot hybridization technique for the detection of HBVDNA was used. 25 p1 of each sample were incubated with 50 pl 1N NaOH and 25 pl 2 M NaCl for 10 rnin at room temperature and transferred by means of a filtration apparatus onto a nylon filter soaked in 5 x SSC. Neutralization was performed adding 100 pl 2 M ammonium acetate to each well. After sample immobilization, filters were washed several times in 5 x SSC and air-dried at room temperature. Prehybridization was performed for 15 min at 55°C in sealed plastic bags containing 10 ml prehy180
Table 1. Probe comparison results Method Radioactive
Enzymatic
Group
n
HEW-DNA+
%
HBV-DNA+
%
Seronegative Asymp. carriers HBeAg chronic hep. AntiHBe chronic hep.
10 7 20 9
17 2
a5 22.2
-
-
15
75
-
-
bridization solution (20 x SSC 25%, BSA 0.5%, PVP 40000 0.5%, SDS 0.5%). Hybridization was performed using an enzyme-linked oligonucleotide probe from Dupont Lab. in sealed plastic bags containing 2.4 ml hybridization solution (same as prehybridization) for 15 rnin at 55°C. In this assay, the enzyme alkaline phosphatase is covalently linked to the C-5 position of a thymidine base through a spacer arm and the probe is a 2 5 4 0 bp long DNA fragment that anneals with the S region of HBV genome. Filters were washed twice in 1 x SSC+ 1% SDS for 5 rnin at 50"C, twice in 1 x SSC+ 1'%0 Triton X-100 for 5 rnin at 50"C, twice in 1 x SSC+ 1% Triton X-100 for 5 rnin with agitation at room temperature, and twice in 1 x SSC for 5 rnin with agitation at room temperature. Colour was developed by incubating the filter in sealed plastic bags with 7.5 ml substrate buffer, 50 pl NBT (Nitro Blue Tetrazolium) solution and 38 pl BCIP (5-bromo-4-Chloro-3-indolylphosphate)solution (Dupont Lab.) for 6 h at room temperature and 9 h at 37°C. The reaction was stopped by washing the filters with deionized water. For HBV-DNA assays all sera were tested twice; only concordant results were considered. Results Probe comparison
We detected HBV-DNA in samples from 17 HBeAg chronic hepatitis patients (85%) and 2 antiHBe chronic hepatitis patients (22.2%) by the genomic radioactive probe, while by the enzyme-linked probe we detected HBV-DNA in 15 HBeAg chronic hepatitis. Serum samples from asymptoTable 2. Probe comparison: Fisher's test Radioactive
Enzymatic
Positive Negative
Positive
Negative
15 4
27 Significant
Serum HBV-DNA detection by an enzymatic probe
Fig. I. Genomic radioactive probe. Spots IB to 4B correspond to serial dilutions of a HBV-DNA standard (lo', lo2, 10 and 1 pdml).
matic carriers and seronegative individuals were always negative for HBV-DNA (Table 1). These results are compared by Fisher's test in Table 2. Fig. 1 represents a filter hybridized with the genomic radioactive probe; this method was able to detect up to 1 pg/ml of an HBV-DNA standard. Spots 1 B to 4B correspond to serial dilutions of an HBV-DNA standard (lo3,lo2, 10 and 1 pg/ml). In Fig. 2, a filter hybridized with the enzyme-linked probe is shown; this probe could detect up to 10 pg/ml of the same HBV-DNA standard. Spots 2A to 2F correspond to serial dilutions of an HBV-DNA standard (lo6 to 10 pg/ml), while in positions 3A to 3F negative controls were tested. Enzyme-linked probe evaluation
We detected HBV-DNA in eight patients suffering from acute hepatitis (61.%YO).Sixty-one chronically infected patients were found positive for HBeAg (70.12'30)and seven were found positive for antiHBe ( I 2.07%). Asymptomatic carriers, resolved hepatitis, and seronegative individuals were always negative for HBV-DNA (Table 3). All sera were tested twice and we found that the test showed great reproducibility. Discussion
Molecular biology development and, more specifically, nucleic acid hybridization, cannot overlook HBV infection diagnosis (8). This is why different probes for the detection of serum HBV-DNA have recently appeared. The most recent ones are synthetic probes which are made of an oligonucleotide able to recognize a specific region of HBV genome. We have evaluated an enzyme-linked oligonuc-
Fig. 2. Enzyme-linked probe. Spots 2A to 2 F correspond to serial dilutions of a HBV-DNA standard (lo6 to 10 pg/ml), while in position 3A to 3 F negative controls were tested.
leotide probe to detect HBV-DNA in serum samples of patients at different stages of HBV infection, for its use at the clinical level as a worthwhile alternative to genomic radioactive probes. The method is much easier to perform and is available to a greater number of diagnostic laboratories (9). Although this is a strong reason for using this probe, its sensitivity and specificity must be evaluated, and that is why we first compared the results of HBV-DNA detection with this probe to those obtained with a genomic radioactive probe. Our study shows the detection of HBV-DNA in 85% of the HBeAg patients (n= 17) and 22.2% of the anti-HBe chronic hepatitis patients (n = 2) when detection was carried out with the radioactive probe; these results are similar to those previously reported (10, 1 I). Using the enzyme-linked probe, HBV-DNA was detected in 75% (n = 15) of HBeAg chronic hepatitis patients (Table 1). These results show that the enzymatic probe is able to detect similar positivity ranges to the radioactive one. The four serum samples positive using the radioactive probe, but negative by the enzymatic assay, showed low levels of HBV-DNA; the range of these levels was from 1-10 pg/ml, which is not detectable by the enzymatic method. For statistical comparison Table 3. Enzymatic probe evaluation HBV-DNA +
HBV-DNA-
Group
n
n
%
n
Seronegative Resolved hepatitis Asymp. carriers Acute hepatitis HBeAg chronic hep. Anti-HBe chronic hep.
53 19 66 13 87 58
-
-
8 61 7
61.54 70.12 12.07
53 19 66 5 26 51
%
100 100 100 38.46 29.88 87.93
181
Garcia et a]. we used Fisher’s test: data analysis (Table 2) showed statistical significance; therefore, there is a strong correlation between methods, and there is a high probability that similar results would be obtained by both methods. Specificity is the same for both probes: we could not detect HBV-DNA in serum samples of seronegative and asymptomatic carriers by any method. Having found that both methods showed similar results, we evaluated the enzymatic probe in a greater number of samples in order to prove its usefulness at the clinical level. Specificity was again found to be very high: no positivity for HBV-DNA was detected in seronegative, resolved hepatitis or asymptomatic carriers of HBV (Table 3). For acute hepatitis we detected HBV-DNA in 8 cases (61.54%): these results are very similar to those previously reported for acute hepatitis by Krogsgaard et al. (12), Govindarajan et al. (13) and Genesca et al. (14), though they are higher than those presented by Pontisso et al. (1 5) and Fong et al. (16). For chronic hepatitis we detected HBVDNA in 61 HBeAg (70.12%) and 7 anti-HBe (12.07%) patients; these results are very similar to those previously reported for radioactive probes (1 1, 12). The test’s reproducibility was also found to be very high: samples were always tested twice and no discordance was observed. HBV-DNA detection represents a therapeutic guide for the hepatologist. When monitoring antiviral therapy, a quantification of HBV-DNA levels is necessary. Dot-blot techniques allow a semiquantification of the serum HBV-DNA level by comparison of the spot signal (radioactive or enzymatic) of the patient’s serum to serial dilutions of a positive HBV-DNA standard. For this purpose, spots must be easy to evaluate; though the enzymatic methodology spots could appear to be more difficult to evaluate, and the experienced observer could easily discriminate a positive from a negative result. However, to monitor antiviral therapy and to quantify HBV-DNA serum levels, the best option is to use an ELISA test, such as the doublestranded enzyme immunoassay described by Mantero et al. (17), to evaluate the hybridization. There are methodological advantages in the use of this enzymatic probe: for example, it avoids the use of radioactivity, prehybridization and hybridization times are shorter so results can be obtained in 24 h, and no special equipment unknown to a virology laboratory and no specialized personnel are required. These advantages and our results lead us to propose this probe as a worthwhile alternative at the clinical level for the routine survey of
182
HBV-DNA in hospitals. This is a useful method for screening HBV-DNA in serum samples before applying PCR technology which will help us to save time and money in diagnosis of HBV infection. References 1. BRECHOT C, HADCHOUEL M. SCOTTOJ et al. Detection of
hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Luncet 1981: ii: 765-768. 0. Analysis of DNA 2. IMAZEKIF, OMATAM, YOKOSUKA polymerase reaction products for detecting hepatitis B virus in serum. Comparison with spot hybridization technique. Hepatology 1985: 5: 783-788. 3. MATSUYAMA 0 et al. Discordance Y, OMATA M, YOKOSUKA of hepatitis B e antigen/antibody and hepatitis B virus DNA in serum. Gastroenterology 1985: 89: 11041 108. 4. BONINOF, CRIVELLI 0, RlZZETTo M. Hepatitis B virus infection. In: Bonino F, Crivelli 0, Rizzetto M eds. Progress in hepatitis research: hepatitis B virus (HBV) and hepatitis delta virus (HDV). Saluggia: Sorin Biomedica, 1988: 7-25. 5. GOVINDARAJAN S. FONGTL, VALINLUCK B et al. Markers of viral replication in patients with chronic hepatitis B virus infection. Am J Clin Pathol 1988: 89: 233-237. 6. LOKASF, KARAYIANNIS P, JOWETTT P et al. Studies of HBV replication during acute hepatitis followed by recovery and acute hepatitis progressing to chronic disease. J Hepatol 1985: 1: 671-679. HF, CZAJAAJ et al. Pattern and dur7. WOODJR, TASWELL ation of HBV DNA seropositivity in acute hepatitis B. Dig Dis Sci 1988: 33: 477480. FC. Diagnostic deoxyribonucleic acid probes for 8. T’ENOVER infectious diseases. Clin Microbiol Rev 1988: 1: 82-101. 9. MANZINA, PAURI P, BAGNARELLI P et al. Detection Of hepatitis B virus DNA in serum using synthetic non-radioactive oligonucleotides. J Clin Pathol 1989: 42: 1206-1210. 10. THIERSV, BOUCHERDEAU AM et al. Hepatitis F, COUROUCE B virus DNA as a marker of viral multiplication: comparison with HBe antigen and anti-HBe antibody. Presse Med 1986: 15: 1219-1222. 1 1 CHUANG WL, CHANGWY. CHANGSF et al. Factor analysis for the presence of serum HBV-DNA in chronic hepatitis B virus infection. Taiwan I Hsueh Hui Tsa Chi 1989: 88: 874-878. 12 KROGSCAARD K, KRIGERP, ALDERSHVIL I et al. Hepatitis B virus DNA in serum from patients with acute hepatitis B. Hepatology 1985: 1: 10-13. 13 GOVINDARAJAN S, DE COCKKM, VALI’NLUCK B et al. Serum hepatitis B virus DNA in acute hepatitis B. Am J Clin Path01 1986: 86: 352-354. 14 GENESCA J, JARDIR, BUTIM et al. Hepatitis B virus replication in acute hepatitis B, acute hepatitis B virus-hepatitis delta virus coinfection and acute hepatitis delta superinfection. Hepatology 1987: 3: 569-572. F, SCHIAVON E et al. Serum hepa15 PONTISSO P, BORTOLOTTI titis B virus DNA in acute hepatitis type B. Digestion 1986: 34: 46-50. A. AKRIVIADIS M D et al. Serum 16 FONCTL, EVANGELOS hepatitis B viral DNA in acute viral hepatitis B. Ann Intern Med 1989: 110: 936-937. G, ZONARO A, ALBERTIN1 A et al. DNA enzyme 17 MANTERO immunoassay: general method for detecting products of polymerase chain reaction. Clin Chem 1991: 37: 422429.
Serum HBV-DNA detection by molecular hybridization has modified the traditional concept by which the detection of serum HBeAg corresponded with HBV active replication, while anti-HBe detection corresponded to the integrated phase of infection (1, 2). Usually, patients chronically infected by the virus who are positive for HBeAg show high levels of HBV-DNA; when these patients seroconvert to anti-HBe, HBV-DNA becomes undetectable (3). However, some of these patients remain positive for HBV-DNA after seroconversion even though they are negative for HBeAg (4). Viral DNA represents a direct measure of the virion, while HBeAg detection depends on its concentration related to anti-HBe. There are also patients who are positive for HBeAg and negative for HBV-DNA (5). It seems, therefore, that HBV-DNA is a better marker of active replication and infectivity. Patients suffering from acute hepatitis B are generally positive for HBV-DNA, though it becomes undetectable before or when HBeAg disappears (6, 7). Serum HBV-DNA detection represents a new and important diagnostic tool for the hepatologist,
F. 6. Garcia, E. Quiros, M. C. Bernai, J. Salmeron’ and M. C. Maroto Department of Microbiology and ‘Unit of Hepatology, University Hospital, Granada, Spain
Key words: enzymatic probe - HBV-DNA radioactive probe - serum detection Federico Garcia Garcia, Departamento de Microbiologia, Facultad de Medicina. Av. Madrid-11, Granada, 18012, Spain Received 3 September 1991, accepted for publication 23 April 1992
as well as a prognostic and therapeutic guide. Molecular biology offers the choice of several DNA probes for its detection, depending on their molecular weight, the detection system, and the hybridization format. Though most laboratories use genomic radioactive probes, the appearance of commercially available enzyme-linked oligonucleotide probes that simplify the method represents an advance in HBV-DNA detection. The aim of this study is to evaluate an enzymelinked oligonucleotide probe as a worthwhile alternative in HBV-DNA detection. Patients and methods
Our study was in two phases. First, we compared HBV-DNA detection by a genomic radioactive probe and an enzyme-linked oligonucleotide probe in serum samples from 46 individuals: 7 asymptomatic carriers, 29 chronic hepatitis, and 10 seronegative controls. Secondly, we tested sera from 296 individuals (66 asymptomatic carriers, 145 chronic hepatitis, 13 acute hepatitis, 19 resolved hepatitis, and 53 seronegative controls) with the oligonucleotide probe. 179
Garcia et al. Serological assays
Commercial enzyme immune assays (Abbott Laboratories) were used for screening HBsAg, antiHBs, anti-HBc, anti-HBc IgM, HBeAg, and antiHBe. HBV-DNA assays
1. Genomic radioactive probe. A spot hybridization technique for the detection of HBV-DNA was used; 250 pl of each sample and 600 pl of STENMBSA (Tris-HC1 0.1 M pH=7.4; C l N A 4 M; EDTA (Na), 0.1 M; BSA 10 mg/ml; Sucrose 0.3%) were spun for 4 h at 24500 g/4"C. After centrifugation, the pellet was resuspended in 160 p1 of Summer's buffer (Tris-HC1 5 mM, NaCl 75 mM, EDTA (Na), 5 mM) and incubated for 2 h with 28 pl SDS 25%, 32 pl Pronase 10 mg/ml, 4 pl t-RNA 10 mg/ml at 37"C, and DNA was extracted from the sample by phenol treatment. Samples were then incubated with 50 p1 I N NaOH for 10 rnin at room temperature, neutralized with 100 pl 1 M Tris-HCl+ 1.5 M NaCl p H = 7 and 100 pl0.5 M HCI+2.5 M NaCI, and transferred by means of a filtration apparatus onto a nylon filter soaked in 6 x SSC. Filters were air-dried after sample immobilization. Prehybridization was performed for 1 h at 70°C in sealed plastic bags containing 10 ml prehybridization solution (20 x Denhardt 0.1%, 20 x SSC 0.33%). Hybridization was performed using a P3' nick-translated cloned HBV-DNA pBR322 probe (specific activity = 10' cpm) in sealed plastic bags containing 10 ml hybridization solution (Deionized formamide 50%, 20 x SSC 33%, 100 mM Tris-HC1 pH = 7.95, salmon sperm DNA 2 mg/ml 0.008%, BSA 40 mg/ml 0.004%, SDS 0.08%) for 16-18 h at 37°C. Filters were washed five times in 2 x SSC + 0.1% SDS for 15 rnin and five times in 0.1 x SSC + 0.1% SDS for 15 rnin with agitation at 70°C. After drying at room temperature, filters were autoradiographed at - 70°C for 24 h.
2. Oligonucleotide enzyme-linked probe. A spot hybridization technique for the detection of HBVDNA was used. 25 p1 of each sample were incubated with 50 pl 1N NaOH and 25 pl 2 M NaCl for 10 rnin at room temperature and transferred by means of a filtration apparatus onto a nylon filter soaked in 5 x SSC. Neutralization was performed adding 100 pl 2 M ammonium acetate to each well. After sample immobilization, filters were washed several times in 5 x SSC and air-dried at room temperature. Prehybridization was performed for 15 min at 55°C in sealed plastic bags containing 10 ml prehy180
Table 1. Probe comparison results Method Radioactive
Enzymatic
Group
n
HEW-DNA+
%
HBV-DNA+
%
Seronegative Asymp. carriers HBeAg chronic hep. AntiHBe chronic hep.
10 7 20 9
17 2
a5 22.2
-
-
15
75
-
-
bridization solution (20 x SSC 25%, BSA 0.5%, PVP 40000 0.5%, SDS 0.5%). Hybridization was performed using an enzyme-linked oligonucleotide probe from Dupont Lab. in sealed plastic bags containing 2.4 ml hybridization solution (same as prehybridization) for 15 rnin at 55°C. In this assay, the enzyme alkaline phosphatase is covalently linked to the C-5 position of a thymidine base through a spacer arm and the probe is a 2 5 4 0 bp long DNA fragment that anneals with the S region of HBV genome. Filters were washed twice in 1 x SSC+ 1% SDS for 5 rnin at 50"C, twice in 1 x SSC+ 1'%0 Triton X-100 for 5 rnin at 50"C, twice in 1 x SSC+ 1% Triton X-100 for 5 rnin with agitation at room temperature, and twice in 1 x SSC for 5 rnin with agitation at room temperature. Colour was developed by incubating the filter in sealed plastic bags with 7.5 ml substrate buffer, 50 pl NBT (Nitro Blue Tetrazolium) solution and 38 pl BCIP (5-bromo-4-Chloro-3-indolylphosphate)solution (Dupont Lab.) for 6 h at room temperature and 9 h at 37°C. The reaction was stopped by washing the filters with deionized water. For HBV-DNA assays all sera were tested twice; only concordant results were considered. Results Probe comparison
We detected HBV-DNA in samples from 17 HBeAg chronic hepatitis patients (85%) and 2 antiHBe chronic hepatitis patients (22.2%) by the genomic radioactive probe, while by the enzyme-linked probe we detected HBV-DNA in 15 HBeAg chronic hepatitis. Serum samples from asymptoTable 2. Probe comparison: Fisher's test Radioactive
Enzymatic
Positive Negative
Positive
Negative
15 4
27 Significant
Serum HBV-DNA detection by an enzymatic probe
Fig. I. Genomic radioactive probe. Spots IB to 4B correspond to serial dilutions of a HBV-DNA standard (lo', lo2, 10 and 1 pdml).
matic carriers and seronegative individuals were always negative for HBV-DNA (Table 1). These results are compared by Fisher's test in Table 2. Fig. 1 represents a filter hybridized with the genomic radioactive probe; this method was able to detect up to 1 pg/ml of an HBV-DNA standard. Spots 1 B to 4B correspond to serial dilutions of an HBV-DNA standard (lo3,lo2, 10 and 1 pg/ml). In Fig. 2, a filter hybridized with the enzyme-linked probe is shown; this probe could detect up to 10 pg/ml of the same HBV-DNA standard. Spots 2A to 2F correspond to serial dilutions of an HBV-DNA standard (lo6 to 10 pg/ml), while in positions 3A to 3F negative controls were tested. Enzyme-linked probe evaluation
We detected HBV-DNA in eight patients suffering from acute hepatitis (61.%YO).Sixty-one chronically infected patients were found positive for HBeAg (70.12'30)and seven were found positive for antiHBe ( I 2.07%). Asymptomatic carriers, resolved hepatitis, and seronegative individuals were always negative for HBV-DNA (Table 3). All sera were tested twice and we found that the test showed great reproducibility. Discussion
Molecular biology development and, more specifically, nucleic acid hybridization, cannot overlook HBV infection diagnosis (8). This is why different probes for the detection of serum HBV-DNA have recently appeared. The most recent ones are synthetic probes which are made of an oligonucleotide able to recognize a specific region of HBV genome. We have evaluated an enzyme-linked oligonuc-
Fig. 2. Enzyme-linked probe. Spots 2A to 2 F correspond to serial dilutions of a HBV-DNA standard (lo6 to 10 pg/ml), while in position 3A to 3 F negative controls were tested.
leotide probe to detect HBV-DNA in serum samples of patients at different stages of HBV infection, for its use at the clinical level as a worthwhile alternative to genomic radioactive probes. The method is much easier to perform and is available to a greater number of diagnostic laboratories (9). Although this is a strong reason for using this probe, its sensitivity and specificity must be evaluated, and that is why we first compared the results of HBV-DNA detection with this probe to those obtained with a genomic radioactive probe. Our study shows the detection of HBV-DNA in 85% of the HBeAg patients (n= 17) and 22.2% of the anti-HBe chronic hepatitis patients (n = 2) when detection was carried out with the radioactive probe; these results are similar to those previously reported (10, 1 I). Using the enzyme-linked probe, HBV-DNA was detected in 75% (n = 15) of HBeAg chronic hepatitis patients (Table 1). These results show that the enzymatic probe is able to detect similar positivity ranges to the radioactive one. The four serum samples positive using the radioactive probe, but negative by the enzymatic assay, showed low levels of HBV-DNA; the range of these levels was from 1-10 pg/ml, which is not detectable by the enzymatic method. For statistical comparison Table 3. Enzymatic probe evaluation HBV-DNA +
HBV-DNA-
Group
n
n
%
n
Seronegative Resolved hepatitis Asymp. carriers Acute hepatitis HBeAg chronic hep. Anti-HBe chronic hep.
53 19 66 13 87 58
-
-
8 61 7
61.54 70.12 12.07
53 19 66 5 26 51
%
100 100 100 38.46 29.88 87.93
181
Garcia et a]. we used Fisher’s test: data analysis (Table 2) showed statistical significance; therefore, there is a strong correlation between methods, and there is a high probability that similar results would be obtained by both methods. Specificity is the same for both probes: we could not detect HBV-DNA in serum samples of seronegative and asymptomatic carriers by any method. Having found that both methods showed similar results, we evaluated the enzymatic probe in a greater number of samples in order to prove its usefulness at the clinical level. Specificity was again found to be very high: no positivity for HBV-DNA was detected in seronegative, resolved hepatitis or asymptomatic carriers of HBV (Table 3). For acute hepatitis we detected HBV-DNA in 8 cases (61.54%): these results are very similar to those previously reported for acute hepatitis by Krogsgaard et al. (12), Govindarajan et al. (13) and Genesca et al. (14), though they are higher than those presented by Pontisso et al. (1 5) and Fong et al. (16). For chronic hepatitis we detected HBVDNA in 61 HBeAg (70.12%) and 7 anti-HBe (12.07%) patients; these results are very similar to those previously reported for radioactive probes (1 1, 12). The test’s reproducibility was also found to be very high: samples were always tested twice and no discordance was observed. HBV-DNA detection represents a therapeutic guide for the hepatologist. When monitoring antiviral therapy, a quantification of HBV-DNA levels is necessary. Dot-blot techniques allow a semiquantification of the serum HBV-DNA level by comparison of the spot signal (radioactive or enzymatic) of the patient’s serum to serial dilutions of a positive HBV-DNA standard. For this purpose, spots must be easy to evaluate; though the enzymatic methodology spots could appear to be more difficult to evaluate, and the experienced observer could easily discriminate a positive from a negative result. However, to monitor antiviral therapy and to quantify HBV-DNA serum levels, the best option is to use an ELISA test, such as the doublestranded enzyme immunoassay described by Mantero et al. (17), to evaluate the hybridization. There are methodological advantages in the use of this enzymatic probe: for example, it avoids the use of radioactivity, prehybridization and hybridization times are shorter so results can be obtained in 24 h, and no special equipment unknown to a virology laboratory and no specialized personnel are required. These advantages and our results lead us to propose this probe as a worthwhile alternative at the clinical level for the routine survey of
182
HBV-DNA in hospitals. This is a useful method for screening HBV-DNA in serum samples before applying PCR technology which will help us to save time and money in diagnosis of HBV infection. References 1. BRECHOT C, HADCHOUEL M. SCOTTOJ et al. Detection of
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