Vol. 28, No. 1

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 39-42 0095-1137/90/010039-04$02.00/0 Copyright © 1990, American Society for Microbiology

Evaluation of the Commercially Available HepProbe Kit for Detection of Hepatitis B Virus DNA in Serum ELIZABETH VALENTINE-THON,1* JOCHEN STEINMANN,l AND WOLFGANG ARNOLD2 Abteilung Virologie, Staatliches Hygiene-Institut, St.-Jurgen-Strasse,' and Klinikum fur Innere Medizin, Zentralkrankenhaus St.-Jurgen-Strasse,2 D-2800 Bremen 1, Federal Republic of Germany Received 9 June 1989/Accepted 26 September 1989

The commercially available HepProbe kit involving the use of a 32P-labeled RNA probe was evaluated for its

sensitivity, specificity, and reproducibility in detecting hepatitis B virus (HBV) DNA in patient serum by dot blot hybridization. The level of detection was 0.3 pg, corresponding to 3 x 104 genomes in 50 ,ul of serum. A total of 181 serum samples were tested; 53 (82%) of 65 patients positive for both hepatitis B surface antigen and hepatitis e antigen were positive for HBV DNA compared with only 12 of 74 (16%) hepatitis B surface antigen-positive but hepatitis e antigen-negative individuals. In addition, among all patients positive for HBV DNA, there was a statistically significant correlation between the concentration of HBV DNA in serum and the presence or absence of hepatitis e antigen. None of the 42 hepatitis B surface antigen- and hepatitis e antigen-negative patients tested was positive for HBV DNA. Reproducibility was 87%, with all discordant results representing borderline positives. The results indicate that HepProbe can be employed as a sensitive and reliable assay for HBV DNA in patient serum. Since no clinical evaluation of HepProbe has been published to date, we carried out the present study to determine the applicability of this commercially available kit for assaying HBV DNA in patient serum in routine clinical hepatitis laboratories.

The detection of hepatitis B virus (HBV) DNA in patient by using molecular hybridization techniques, in particular dot blot hybridization, has been shown to be a more direct and sensitive assay of viremia and consequent infectivity than conventional serological tests for viral products such as hepatitis B surface antigen (HBsAg) or e antigen (HBeAg) or for HBV-specific DNA polymerase (1-4, 11, 17). As such, screening for HBV DNA can be used clinically to distinguish viremic from nonviremic HBsAg carriers (4, 6, 20), to monitor response to antiviral therapy (7, 12), or to screen for HBV DNA in apparently healthy blood donors lacking all other markers of an HBV infection (18). In 1988 the HepProbe HBV DNA detection kit for the detection of human HBV in serum became commercially available in Europe and Asia. Produced by Life Technologies, Inc., Gaithersburg, Md., and employed in the United States since 1986, this kit is distributed in the Federal Republic of Germany by GIBCO-BRL GmbH, Eggenstein. Although HepProbe involves the use of a radiolabeled probe, several aspects and consequent advantages of this assay system could render it particularly suitable for routine clinical laboratories. First, a 32P-labeled RNA probe complementary to the long or negative strand of the HBV genome, prepared by in vitro transcription of a recombinant plasmid containing the entire HBV genome, is included in the kit. This obviates the necessity to radiolabel one's own HBV nucleic acid probe. Second, the probe in each kit has been labeled to an activity of less than 10 ,uCi of 32p, thereby rendering the contents exempt from Nuclear Regulatory Commission or Agreement State licensing requirements. Third, the specificity of hybridization is enhanced by the use of an RNA probe followed by digestion with RNase to remove unhybridized, nonspecifically bound probe, thereby reducing background. Finally, all reagents (including denaturing solution, prehybridization and hybridization solutions, probe, RNase, and washing buffers) required for assaying 96 serum samples are included in a single kit for improved standardization. serum

*

MATERIALS AND METHODS

Patient sera. Sera from 181 patients with various HBV serological marker constellations were collected from the routine Hepatitis Laboratory of the Institute of Hygiene, Bremen, Federal Republic of Germany. All patients had been tested for HBsAg, HBeAg, and anti-HBe by using enzyme immunoassay kits obtained commercially from Abbott Laboratories (Wiesbaden-Delkenheim, Federal Republic of Germany). All sera had been stored at -20°C before use.

HBV DNA standards. For semiquantitation of HBV DNA results, serial dilutions of two standards of known concentration were included in each test: (i) an HBV DNA-positive reference serum kindly donated by the National Reference Laboratory for Hepatitis B Virus in Gottingen, Federal Republic of Germany, and (ii) a cloned HBV DNA (Du Pont de Nemours GmbH, Bad Homburg, Federal Republic of Germany). Positive controls. Positive controls included sera from patients known to be HBV DNA positive from independent testing with other assays as well as the positive control serum included in each kit. Negative controls. Negative controls included sera from two patients known to be HBV DNA negative from independent testing with other assays, a normal human serum lacking all markers of an HBV infection (the diluent used to titrate the reference serum), distilled water (the diluent used to titrate the cloned HBV DNA), and the negative control serum included in each kit. HepProbe detection protocol. The protocol followed was essentially that recommended by the manufacturer. Briefly, sera were thawed, clarified by centrifugation for 10 min at 14,926 x g to facilitate filtration, and in 50-il samples deposited into single or duplicate wells of a microdilution

Corresponding author. 39

40

J. CLIN. MICROBIOL.

VALENTINE-THON ET AL.

,1 i,

A

i .

B

.' .

c

(J.IJ)NwL:)

1113V

DNA^

l), _2 T:.'

FIG. 1. Autoradiograms of serial dilutions of two HBV standards: an HBV reference serum (A) and a cloned HBV DNA (B). Filters were hybridized with the 32P-labeled HBV RNA probe included in the HepProbe hepatitis B virus DNA detection kit from GIBCO-BRL. Films were developed after 48 h of autoradiographic exposure. Negative controls are as shown in Fig. 2.

plate followed by the addition of 50 ,ul of denaturing reagent (4 N NaOH) and incubation for 5 min in a 65°C water bath. Each sample was then transferred to the wells of a Minifold I filtration apparatus (Schleicher & Schuell, Dassel, Federal Republic of Germany) and vacuum filtered onto the nylon membrane with underlay included in the kit. The filter was removed under gentle vacuum, blotted, air dried (30 min), and baked for 1 h at 80°C. Filters can be used immediately or stored at room temperature for several weeks. In these experiments filters were stored overnight at room temperature. Filters were prehybridized in a heat-sealed bag with 4 ml of prehybridization reagent (10% formamide, blocking reagent, stabilizers, and kinetic enhancer in a buffered solution) for 15 min in a 65°C water bath. After transfer to a new sealed bag, the filters were hybridized with 4 ml of hybridization reagent (prehybridization reagent containing

,

FIG. 2. Dot blot hybridization results obtained after hybridization of patient serum with the 32P-labeled HBV RNA probe in the HepProbe kit and autoradiographed after 48 h. Sera from 21 patients tested in duplicate are represented in dots A1/A2 through H1/H2, A3/A4 through H3/H4, and A5/A6 through E5/E6. Dots F5/F6 and H5/H6 represent negative controls in duplicate, and dot G5 is the positive control serum from the HepProbe kit.

Typical hybridization results obtained with patient sera are depicted in Fig. 2. Of the 21 patient sera represented here, 18 were HBV DNA positive, 3 were negative (dots A3/A4, G3/G4, and H3/H4. The two negative controls (dots F5/F6 and H5/H6) were negative, and the positive control serum from the HepProbe kit (single dot G5) was clearly positive. Table 1 summarizes the results obtained from testing 181 sera for HBV DNA in correlation with serological markers. Of 65 patients positive for HBsAg and HBeAg, 53 (82%) were HBV DNA positive compared with only 12 (16%) of 74 patients positive for HBsAg but negative for HBeAg. From the latter 74 patients, the majority (53 [72%]) had anti-HBe antibodies, whereas 21 (28%) lacked both HBeAg and antiHBe. Only 4 of these 21 patients were HBV DNA positive (data not shown). None of the 42 HBsAg- and HBeAgnegative individuals was positive for HBV DNA. TABLE 1. Correlation between HBV DNA and serological markers in 181 serum samples No. (%) with the indicated reaction: HBV DNAa

HBsAg+ HBeAg+

HBsAg+ HBeAg-

HBsAg- HBeAg-

=

=

=

(n

Positive' Negative a

b

65)

53 (82) 12 (18)

(n

74)

12 (16) 62 (84)

Hepatitis B DNA detected with HepProbe kit. .0.3 pg (3 x 104 genomes).

(n

42)

0 42 (100)

DETECTION OF HBV DNA WITH HEPPROBE

VOL. 28, 1990

TABLE 2. Comparison of HBV DNA concentration in patients with or without HBeAga HBeAg

HBV DNA concn (pg/50

kd)

Presence

n

Mean

SD

Range

Positive Negative

53 12

98b 2

165.2 2.3

0.5-2:500 0.5-8

a All patient sera were HBsAg positive. b Student's t test; P < 0.01.

Among all HBsAg-positive patients positive for HBV DNA (n = 65), a comparison of the concentration of HBV DNA in serum was made in relation to the presence or absence of HBeAg. The 53 HBeAg-positive serum samples had a significantly higher concentration of HBV DNA on the average (98 pg, corresponding to 12 x 106 genomes) than the 12 HBeAg-negative serum samples (2 pg, corresponding to 2

105) (Table 2). Of the 181 serum specimens tested here with HepProbe, 13 were assayed as well with an HBV DNA probe consisting of the complete HBV genome inserted in M13 (gift from the National Reference Laboratory for Hepatitis B Virus, Gottingen) and labeled in our laboratory with [a-32P]dCTP to 108 cpm4,Lg of DNA. Two serum samples were negative with both tests, 10 serum samples were positive with both tests, and 1 serum sample was positive only with HepProbe, presumably due to the lower level of detection of HepProbe (0.3 pg) compared with that achieved with our own assay (2 to 4 pg) (data not shown). The reproducibility of the HepProbe assay was evaluated by testing 53 sera on two separate occasions with two different HepProbe kits; 87% of the results were concordant, but 13% were discordant (r = 0.73). Seven HBsAg-positive serum samples were HBV DNA positive in the first test but negative upon retesting. AHl seven, however, were borderline positive (less than 2 pg of HBV DNA) in the first test, five of seven were HBeAg negative, and two of the last five were also anti-HBe negative. One serum sample gave a weak positive signal in the second test after 7 days of autoradiographic exposure. x

DISCUSSION The HepProbe kit evaluated in this study provided a sensitive and reliable assay of HBV DNA in patient serum. The level of detection achieved, 0.3 pg of HBV DNA or 3 x 104 HBV genomes in 50 ktl of serum after 48 h of autoradiographic exposure, compares favorably with detection levels reported by other groups using radiolabeled probes but in a variety of assays with various exposure times, i.e., 100 pg/50 ,ul after 72 h (16), 10 pg/20 ,ul after 48 h (14), or 0.3 pg/ml after 3 to 5 days (20). Extending the exposure time beyond 48 h would lower the detection level of HepProbe but would simultaneously prolong the assay. The ability to HepProbe to detect HBV DNA was supported by the high qualitative and quantitative correlation seen between HBV DNA and another recognized marker of viral replication and infectivity, HBeAg: 82% of our 65 HBsAg- and HBeAg-positive patients were HBV DNA positive. All reports published to date confirm the high but not absolute association between these two markers. Morace et al. (14), for example, reported HBV DNA positivity in 67% of 45 HBsAg- and HBeAg-positive patients, whereas Zyzik et al. (20) found 93% HBV DNA positivity in 294 such patients. The latter figure (93%) probably reflects the higher

41

sensitivity of the assay employed by that group. In the present study HBV DNA was found as well in 12 (16%) of 74 HBsAg-positive, HBeAg-negative patients, most of whom had anti-HBe antibodies. The frequency of HBV DNA in such patients is extremely variable, ranging from 5.5% (8) to 63% (5), reportedly related to the geographic origin of the patients as well as to the severity of liver disease (8), in particular among asymptomatic HBsAg carriers (2). The patients in our study were not further classified as to geographic origin or clinical status. It is of interest to note that the concentration of HBV DNA in the 12 HBeAgnegative patients described here was significantly lower than that found in HBeAg-positive patients. Similar results were reported by Sun et al. (18) in a group of HBsAg-positive healthy blood donors. The high specificity of HepProbe was indicated by the lack of HBV DNA in the 42 HBsAg- and HBeAg-negative subjects presumably suffering from non-HBV-related hepatic diseases. Occasionally HBV DNA has been reported in such patients (8, 15) as well as in preportedly healthy individuals (18). Such studies emphasize the value of molecular hybridization techniques as an adjunct to serology in clinical hepatitis laboratories. With few exceptions, the hybridization results obtained with HepProbe could be reproduced. The seven discrepant cases were all initially borderline positive results and were not detected upon retesting under the same conditions. Presumably some or all of these cases represented sera with very low concentrations of HBV DNA, since one sample gave a weak positive signal in the second test after extended autoradiographic exposure (7 days). To minimize the frequency of false results, it is advisable when using HepProbe to perform all tests in duplicate, to repeat tests giving borderline positive results at least once, and to expose film for 7 days as well as for 48 h. The inclusion in the HepProbe kit of a 32P-prelabeled HBV RNA probe of low radioactivity offers several advantages for the user. First, it obviates the necessity to radiolabel one's own nucleic acid probe, with all the associated storage, handling, and disposal problems. Second, it allows the kit to be used in those clinical laboratories lacking a license for radioisotopes. The safety recommendations for handling radioactive or infectious materials, however, as outlined in the Instruction manual accompanying the kit, should still be followed. Third, the single-stranded RNA probe does not have to be denatured before use in hybridization. Finally, the use of an RNA probe permits highly sensitive detection and low backgrounds by allowing nonspecifically bound probe to be subsequently digested with RNase. The one disadvantage accompanying the use of the 32P-labeled probe is the requirement to perform the HBV DNA detection assay within 2 or 3 weeks after receipt of the kit. To avoid the use of radioactivity altogether, several attempts have been made to detect HBV DNA in serum nonradioactively by using probes labeled with biotin (13, 16) or 2-acetylaminofluorene (10) or sulfonated with sodium bisulfite (Valentine-Thon, manuscript in preparation). To date, however, none of these methods has proven sufficiently sensitive, specific, and reproducible to replace the use of radioisotopes. Such methods may eventually provide a feasible alternative to radioactivity when the HBV DNA in patient serum can be sufficiently amplified in routine clinical laboratories, i.e., by the polymerase chain reaction, as recently reported by Larzul et al. (9) and Thiers et al. (19).

42

VALENTINE-THON ET AL.

ACKNOWLEDGMENTS supported by the Tonjes-Vagt Stiftung, Bremen, This work was Federal Republic of Germany. We thank W. Gerlich and A. Uy of the National Reference Laboratory for Hepatitis B Virus, Gottingen, for providing the HBV reference serum and the HBV DNA probe cloned in M13. LITERATURE CITED 1. Berninger, M., M. Hammer, B. Hoyer, and J. L. Gerin. 1982. An assay for the detection of the DNA genome of hepatitis B virus in serum. J. Med. Virol. 9:57-68. 2. Berris, B., R. E. Sampliner, and R. Sooknanan, and S. V. Feinman. 1987. Hepatitis B virus DNA in asymptomatic HBsAg carriers: comparison with HBeAg/anti-HBe status. J. Med. Virol. 23:233-239. 3. Bonino, F., B. Hoyer, J. Nelson, R. Engle, G. Verme, and J. Gerin. 1981. Hepatitis B virus DNA in the sera of HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology 1:386-391. 4. Brechot, C., M. Hadchouel, J. Scotto, F. Degos, P. Charnay, C. Trepo, and P. Tiollais. 1981. Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet ii:765-768. 5. Chu, C.-M., P. Karayiannis, M. J. F. Fowler, M. Monjardino, Y. F. Liaw, and H. C. Thomas. 1985. Natural history of chronic hepatitis B virus infection in Taiwan: studies of hepatitis B virus DNA in serum. Hepatology 5:431-434. 6. Hadziyannis, S. J., H. M. Lieberman, G. G. Karvountzis, D. A. Shafritz. 1983. Analysis of liver disease, nuclear HBeAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 3:656-662. 7. Heîjtink, R. A., P. Small, F. J. ten Kate, J. Kruining, and S. W. Schalm. 1987. Detection of HBV-DNA in liver biopsy and serum: its significance in the selection of hepatitis B patients for antiviral therapy. Antiviral Res. 7:329-340. 8. Karayiannis, P., M. J. F. Fowler, A. S. F. Lok, C. Greenfield, J. Monjardino, and H. C. Thomas. 1985. Detection of HBV DNA by molecular hybridization: correlation with HBeAg/anti-HBe status, racial origin and liver histology. J. Hepatol. 1:99-106. 9. Larzul, D., F. Guigue, J. J. Sninsky, D. H. Mack, C. Brechot, and J.-L. Guesdon. 1988. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J.

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Virol. Methods 20:227-237. 10. Larzul, D., V. Thiers, A. M. Courouce, C. Brechot, and J. L. Guesdon. 1987. Nonradioactive hepatitis B virus DNA probe for detection of HBV-DNA in serum. J. Hepatol. 5:199-204. 11. Lieberman, H. M., D. R. LaBrecque, M. C. Kew, S. J. Hadziyannis, and D. A. Shafritz. 1983. Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers. Hepatology 3:285-291. 12. Lok, A. S., P.-C. Wu, G.-L. Lai, and E. K. Y. Leung. 1988. Long-term follow-up in a randomised controlled trial of recombinant c2-interferon in Chinese patients with chronic hepatitis B infection. Lancet i:298-302. 13. Molden, D. P., R. M. Nakamura, H. Suzuki, S. Greer, R. G. Pergolizzi, and C. L. Brakel. 1985. Comparison of radio-labeled DNA probe with a nonisotopic probe for assay of serum hepatitis B virus DNA. Clin. Physiol. Biochem. 3:174-183. 14. Morace, G., K. von der Helm, W. Jilg, and F. Deinhardt. 1985. Detection of hepatitis B virus DNA in serum by a rapid filtration-hybridization assay. J. Virol. Methods 12:235-242. 15. Nalpas, B., P. Berthelot, V. Thiers, G. Duhamel, A. M. Courouce, P. Tiollais, and C. Brechot. 1985. Hepatitis B virus multiplication in the absence of usual serological markers: a study of 146 chronic alcoholics. J. Hepatol. 1:89-97. 16. Quibriac, M., J. Petitjean, M. Novel, and F. Freymuth. 1987. Non-radioactive spot hybridization test for detection of hepatitis B virus DNA in serum. Ann. Inst. Pasteur Virol. 138: 377-384. 17. Scotto, J., M. Hadchouel, C. Hery, J. Yvart, P. Tiollais, and C. Brechot. 1983. Detection of hepatitis B virus DNA in serum by a single spot hybridization technique: comparison with results for other viral markers. Hepatology 3:279-282. 18. Sun, C.-F., C. C. Pao, S.-Y. Wu, and Y.-F. Liaw. 1988. Screening for hepatitis B virus in healthy blood donors by molecular DNA hybridization analysis. J. Clin. Microbiol. 26: 1848-1852. 19. Thiers, V., D. Kremsdorf, H. Schellekens, A. Goudeau, J. Sninsky, E. Nakajima, D. Mack, F. Driss, J. Wands, P. Tiollais, and C. Brechot. 1988. Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet ii:1273-1276. 20. Zyzik, E., W. H. Gerlich, A. Uy, H. Kochel, and R. Thomssen. 1986. Analysis of hepatitis B virus genome titers in sera of infected subjects. Eur. J. Clin. Microbiol. 5:330-335.