Abstract. The presence of hepatitis B virus (HBV) DNA in sera of 56 chronic carriers of hepatitis B surface antigen (HBsAg) was determined by three methods: the ...
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15. Plummer FA, Simonsen JN, Cameron DW, NdinyaAchola JO, KreissJK, GayinkaMN, Waiyaki P,Cheang M, Piot P, Ronald AR, Ngugi EN: Cofactors in malefemale sexualtransmissionof human immunodeficiency virus type I. Journal of Infectious Diseases 1991, 163: 233-239.
Detection of Hepatitis B Virus D N A in Chronic Carriers of Hepatitis B Surface Antigen in Southwestern Greece M. C h r i s t o f i d o u 1., A . A t h a n a s s i a d o u 2, A . S k o u t e l i s 3, E . D . A n a s t a s s i o u 1
The presence of hepatitis B virus (HBV) DNA in sera of 56 chronic carriers of hepatitis B surface antigen (HBsAg) was determined by three methods: the Abbott hybridization assay, the polymerase chain reaction (PCR) followed by gel electrophoresis and UV visualization (PCR-GE), and PCR followed by DNA enzyme immunoassay (PCR-DEIA). HBV DNAwas detected in four samples positive for hepatitis Be antigen (HBeAg) by all methods used. Both PCR-GE and PCR-DEIA detected viraemia in two anti-HBe, anti-HBc IgM positive samples. In the group of 50 anti-HBe positive samples the sensitivity of the three methods was 10 %, 24 % and 32 %, respectively. PCR-GE and PCR-DEIA results correlated well with the patients' clinical status; of 20 patients with elevated ALT levels, 12 (60 %) were found to be positive in the PCR-GE and another 2 were found to be positive in the PCRDEIA (70 %). These data indicate that PCR-DEIAis the most sensitive method for detection of HBV DNA. This method can be relatively easily applied in the clinical laboratory for monitoring the progression of disease and/or interferon therapy in patients with chronic hepatitis B.
Infection with hepatitis B virus (HBV) is endemic throughout the world and is responsible for a variety of liver diseases. Over 200 million people worldwide are chronic carriers of the virus while the number of fatalities is estimated to be ap1Department of Microbiology, ~Department of Biology and 3Department of Internal Medicine,School of Medicine, University of Patras, 26110 Patras, Greece.
Eur. J. Clin. Microbiol. Infect. Dis.
proximately two million individuals per year (1). The prevalence of HBV infection in the southwestern part of Greece, as determined with conventional serological markers, is relatively high (2). Although these markers have been shown to be sensitive and convenient for detecting acute, chronic or past infection, thus providing some prognostic information for the disease's outcome, they are not always good indicators of replicative activity of the virus (3) or precore HBV mutants which do not produce hepatitis Be antigen (HBeAg) and are responsible for severe hepatitis in Mediterranean patients positive for hepatitis B surface antigen (HBsAg) (4). Development of specific nucleic acid probes have facilitated hybridization, usually involving a radioactive signal, for the detection and quantitation of HBV DNA sequences in serum, thus providing an alternative, direct and reliable indicator of viral replication and infectivity (3, 5). HBV DNA determination for the assessment of the disease's progress and the effectiveness of antiviral therapy with greater accuracy has been established as a reference method for monitoring chronic hepatitis (6). Subsequently, polymerase chain reaction (PCR) has dramatically increased the sensitivity of HBV DNA detection (7-9). However, detection of the PCR product is based largely on visualization after gel electrophoresis or on dot-blot, methods which are not well suited to clinical laboratories. Several nonisotopic techniques for detecting DNA-DNA hybrids have been developed recently (7, 8); in particular, D N A enzyme immunoassay (DEIA) has been claimed to offer adequate sensitivity for the detection of amplified DNA, as well as being simple, rapid and easily applied in a clinical laboratory (10). In the present study we used three different methods to detect HBV D N A in the sera of 56 patients with chronic HBV infection, in order to determine the sensitivity of these methods in comparison with serological assays, and to assess their applicability in our hospital laboratory on a routine basis. If applicable, in the long run these methods will greatly facilitate monitoring the progression of disease and the a-interferon therapy in patients with chronic hepatitis B. Patients and Methods. Fifty-six HBsAg chronic carriers (55 men and 1 woman; mean age 65 years, range 35-75) from southwestern Greece were studied. These patients had had HBsAg in serum for at least one year and fell into one of the three following groups: Group A consisted of 50 subjects, 30 with normal liver function tests (healthy carriers) and 20 with chronic liver disease, with
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Notes
was replaced by distilled water were also included. Five ~1 of amplified material was electrophoresed on 1.2 % (w/v) agarose gels containing ethidium bromide (5 ~tg/ml). A • 174 x HaelII digest was used as DNA size marker. Commercially available reagents were used for DEIA (Gen-ETI-K HBV-core, Sorin Biomedica, Italy), based on single-stranded versus double-stranded DNA discrimination by an anti-DNA monoclonal antibody. In brief, 20 ~tl of denatured PCR product was hybridized at 50°C for 1 h with a single-stranded DNA, containing a 75-mer sequence included between nucleotides 2047 and 2121 of the HBV core gene region with a 5' biotinylation, and fixed to the wall of 48 microtiter plate wells with a streptavidin-biotin bond. The amplified DNA-probe hybrid was incubated for 1 h at room temperature with a monoclonal antibody specific for double-stranded DNA, washed and reincubated for I h at room temperature with an anti-mouse IgG antibody conjugated with horseradish peroxidase. A negative control containing Tris-HCl buffer, MgCI2 and BSA; a positive control containing a solution of singlestranded D N A complementary to the immobilized probe in solid phase, Tris-HC1, EDTA and BSA; and negative, positive and reagent controls of PCR were always used in DEIA. Detection and quantitation of HBV DNA was performed according to the manufacturer's instructions.
elevated ALT (chronic hepatitis). All of them Were positive for HBsAg, antibodies to hepatitis B core antigen (anti-HBc) and antibodies to hepatitis Be antigen (anti-HBe). Group B consisted of 4 patients with chronic liver disease who were positive for HBsAg, anti-HBc and HBeAg. Group C consisted of 2 patients with chronic liver disease who were positive for HBsAg, anti-HBc, anti-HBe and anti-HBc IgM (Table 1). All Patients were HIV-negative, had no history of alcohol abuse and did not take any medications, and their tests for autoimmune diseases were negative. HBV serological markers were detected with commercial enzyme immunoassays (Abbott Laboratories, USA), according to the inStructions of the manufacturer. Serum samples from four healthy blood donors with no serological evidence of HBV infection were consistently Used as negative controls. HBV DNA was initially determined by a quantitative radiological hybridization assay (Abbott Laboratories, USA) according to the manufacturer's instructions. DNA amplification in PCRGE and PCR-DEIA was carried out by using Specific primers for the highly conserved region of HBV core gene sequences: C1, 1955-1974 5'T~GCCTTCTGACTrCTITCC3', and C2, 2401-2381 5'TCTG CGAGGCGAGGGACTTCT3' (Sorin Biomedica, Italy). DNA for PCR amplification was prepared by boiling 100 ~1 of Serum for 45 min and centrifugation at 12,000 x g for 20 min at 4°C (10). Reaction volume was 100 Ixl containing 5 ~tl of prepared testing serum, 50 pmoles of each primer, 20 ~tM of each dNTP (dATP, dCTP, dGTP, dTTP-Pharmacia, LKB BiOtechnology, Sweden), 10 mM Tris-HC1 (pH 8.3), 50 raM KC1, 1.5 mM MgC12, 100 Ixg/ml BSA and 2 U Taq polymerase (Minotech Molecular Biology Products, Greece). PCR was performed in a thermal cycler (MJ Research model PTC 100, USA) for 34 cycles at 93°C for 1 min, 48°C for 1 rain and 68°C for 2 min, preceded by a first cycle With a denaturation step of 2 min at 93°C. A Strongly positive HBeAg serum, a negative control serum and a reagent control in which serum
Results and Discussion. Fifty-six HBsAg chronic carriers were included in this study. Their complete serological status is shown in Table 1. In group A, 30 patients with normal levels of ALT were healthy carriers, whereas 20 patients had chronic hepatitis with elevated values of ALT (mean 151 U/l) (Table 2). In the subgroup of healthy carriers, 2 individuals were HBV DNApositive by PCR-DEIA only. In the subgroup of chronic hepatitis, 5 (25 %) had mean HBV DNA of 11.5 pg/ml with a range of 2.5-28.0 by hybridization; 12 (60 %) were positive for HBV DNA by PCR-GE and 14 (70 %) by PCR-DEIA. These results suggest that the absence of serum HBeAg does not exclude active HBV replication al-
Table 1: S e r o l o g i c a l s t a t u s of 56 H B s A g chronic carriers. Serological status
No. of
samples GroupA Group B Group C
50 4 2
465
HBsAg
HBeAg
Anti-HBc
Anti-HBe
Anti-HBc IgM
+ + +
+ -
+ + +
+ +
+
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T a b l e 2: D e t e c t i o n o f H B V D N A i n 56 chronic H B s A g c a r d e r s . No. of patients
Liver biopsy
HBVDNA
ALT (U/I)
Hybridization
PCR-GE
PCR-DEIA
(mean pg/ml)
Group A
28 1 I 6 1 4 5 2 2
-GZ h GZb 11.5 -
+ + + +d
+
CA H
normal" normal" normaP 69-115 78 55-515 55-713 53-55 78-120
Group B
CAH
85-140
77.0
+
+
Group C
CAH CAH
118 116
18.0 -
+ +
+ +
CAH CAH CAH a
+
+ + + + +
CAH: Chronic active hepatitis. = Normal values :~ 40 U/I. bValue in gray zone (1.0-1.4 pg/ml). c One of the five patients had chronic active hepatitis and cirrhosis. dQuadrupllcate amount of PCR product was electrophoresed in these two positive cases,
though seroconversion has occurred, as previously reported (7). Alternatively, the existence of precore mutants may explain these serological findings. Thus, patients positive for anti-HBe with elevated ALT levels should be considered highly infectious. These Jesuits also suggest that PCR dramatically increases the sensitivity of HBV D N A detection in HBsAg-positive persons. The HBsAg-positive, PCR-negative persons may have either viral replication below the detection limit of PCR or hepatocellular HBsAg production without HBV replication (8). The discrepancy between PCR-GE and PCR-DEIA is in agreement with a previous report (10) and is attributed to inadequate sensitivity of ethidium bromide visualization of nanogram amounts Of DNA. In group B, all subjects were positive for HBV DNA in all three methods used. The mean concentration of HBV DNA was estimated by hybridization to be 77.0 pg/ml (range 20.0-167.0 pg/ml). In group C, one subject was HBV DNApositive in all three assays and the respective hybridization result was 18.0 pg/ml. The other patient was HBV DNA-positive only by PCR-GE and PCR-DEIA (Table 2). Results obtained for group B show that HBeAg is a reliable traditional marker for viral replication (5, 11, 12). However, precore HBV mutants which do not produce HBeAg and are responsible for severe chronic hepatitis in Mediterranean countries have been described in Greece and elsewhere (4, 11, 13). In these cases, serum HBV D N A detection is used as a good marker of active viral replication.
Concerning electrophoresis of PCR-amplified products, HBV DNA-positive sera were revealed by the distinct and specific amplified 447 bp band. Representative positive samples obtained from all groups are presented in Figure 1. The smaller but more intensely stained DNA band in lane 6 may either be nonspeeifie or suggest deletions in the amplified sequence of HBV DNA. Deletions seem to be frequent in anti-HBe-positive chronic hepatitis B and are indicative of a long duration of the disease. This sample belongs to a 70-year-old man with chronic active hepatitis and cirrhosis. As mentioned earlier, two healthy chronic carriers from group A with normal ALT levels were HBV DNA-positive in PCR-DEIA. One of them exhibited results in the gray zone on hybridization (Table 2). PCR-GE did not confirm this resuit. This may suggest either a low level of HBV DNA or impairment of PCR amplification by DNA polymerase inhibitors to such an extent that only D E I A is efficient to detect the amplified DNA. To address the latter possibility, Vandenvelde et al. (14) proposed the use of a sodium octanoate thermoprotection method to suppress the inhibitory effect of serum proteins on the Taq polymerase in PCR. In the present study, the boiling method for DNA preparation (10) was used as an easy method for routine application, since the classical proteinase K/phenol-chloroform/ethanol precipitation resuits in a great loss of HBV D N A (15). Our results show that D E I A is feasible and may rescue a
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1
Notes
5
6
7
8
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ent areas of research such as virology, haematology, genetics and basic immunology (i0, 16). Although more data are required to correlate PCR-DEIA results with clinical significance, our findings suggest that the metlaod is very useful when used in conjugation with serological and histochemical findings, to evaluate the disease's progress and/or its response to interferon therapy. This method had the highest sensitivity of the three methods used; moreover, it is nonisotopic, can be performed in a day's shift, does not require particular technical skills and can be relatively easily applied in a routine clinical laboratory.
Acknowledgements !
The authors thank Miss Nicki Vouldis for secretaria! assistance. This work was supported in part by a grant from the Department of Health and Social Services.
t,c Figure 1" Detection of HBV DNA in HBsAg-positive sere by PCR-GE. Detection of 447 bp band indicates positive HBV DNA serum. Lane M: 4> 174 x Hae digest containing 1,353, 1,078, 872, 603, 310, 281,271,234, 194, 118 and 72 bp. Lanes 1 to 7: representative positive samples of group A. Lane 8: positive serum of group C. Lanes 9 and 10: positive sera of group B. Lanes 11 and 12: control sere negative for HBsAg. Lanes 13 and 14: two samples of group A where a fourfold amount of PCR-amplified Product was needed to detect the 447 bp band. Lane RC: reagent control in which the serum was replaced by water.
SUboptimal PCR. To test whether or not the preParation step of boiling the D N A is necessary to resolve all PCR-DEIA positive samples, an experiment was carried out omitting this step. This resulted in a 12-25 % loss in sensitivity, which is certainly undesirable. Furthermore, these results zndicate that naked D N A may be present in HBsAg positive sera and/or that the boiling time for DNA preparation may be shortened. Liver biopsy was performed in 18 patients; all of them had elevated ALT levels, were H B V DNAPositive by PCR-GE and P C R - D E I A and were fOUnd to have chronic active hepatitis (Table 2). One o f them had cirrhosis. The 18 patients with histological findings began therapy with c-interferon according to standard protocols. Recently, P C R - D E I A has been used successfully zn detecting specific nucleic acid hybrids in differ-
References 1. Szmuness W: Hepatocellular caminoma and the hepatitis B virus: evidence for a causal association. Progress in Medical Virology 1978, 24: 40-49. 2. Chdstofidou M, Dlmltracopoulos G, Anastassiou ED: Prevalence of hepatitis B markers. Acta Microbiologica Hellenica 1989, 35: 144.-149. 3. Scotto J, Hadchouel M, Hery C, Yvart J, "riollals P, Brschot C: Detection of hepatitis B virus DNA in serum by a simple spot hybridization technique: comparison with results for other viral markers. Hepatology 1983, 3: 279-284. 4. Carman WF, Fagan EA, Hadztyannis SJ, Karayiannis P, Tassopoulos NC, Williams R, Thomas HC: Association of a precore genomlc variant of hepatitis B virus with fulmlnant hepatitis. Hepatology 1991, 14: 219-222.' 5. Morace G, vonder Helm K, Jilg W, Deinhardt F: Detection of hepatitis B virus DNA In serum by a rapid filtrationhybridization assay. Journal of Virological Methods 1985, 12: 235-242. 6. Ueberman HM, Labrecque DR, Kew MC, Hadziyannis SJ, Shafrflz DA: Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carders. Hepatology 1983, 3: 285-291. 7. Kaneko S, Feinstone SM, Miller RH: Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. Joumal of Medical Microbiology 1989, 27: 1939-1934. 8. Zaaller HL, ter Borg F, Cuypers HTM, Hermus MCAH, Lelle PN: Comparison of methods for detection of hepatitis B virus DNA. Journal of Clinical Microbiology 1994, 32: 2088-2091. 9. Keller GH, Huang DP, Shih JWK, Manak MM: Detection of hepatitis B virus DNA in serum by polymerase chain reaction amplification and microtiter sandwich hybridization. Journal of Clinical Microbiology 1990, 28: 14111416.
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10. Mantero G, Zonaro A, Albertini A, Bertolo P, Pdmi D: DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction. Clinical Chemistry 1991, 37: 422-429. 11. Bonino F, Hoyer B, Nelson J, Engle R, Verme G, Gertn J: Hepatitis B virus DNA In the sera of HBsAg carders: a marker of active hepatitis B replication in the liver. Hepatology 1981, 1: 386-391. 12. Baker BL, DiBisceglie AM, Kaneko S, Miller R, Feinstone SM, Waggoner JG, Hoofnagle JG: Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology 1991, 13: 632-636. 13. Hadziyannis SJ, Lieberman HM, Karvountzis GG, Shafritz DA: Analysis of liver disease nuclear HBcAg, viral replication and hepatitis B virus DNA in liver and serum of HBeAg vs anti-HBe positive carriers of hepatitis B virus. Hepatology 1983, 3: 656-662.
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14. Vandenvelde C, Scheen R, Defoor M, Duys M, Duman J, Van Beers D: Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate: application to routine clinical detection of hepatitis B virus at its infectivity threshold .in serum. Journal of Virological Methods 1993, 42: 251263. 15. Maniatis T, Fritsch El=, Sambrook J: Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989. 16. Bettinardi A, Imberti L, Sottini A, Primi D: Analysis of amplified T cell receptor V beta transcripts by a nonisotopic immunoassay. Journal of Immunological Methods 1992, 146: 71-82.
