AMERICAN JOURNAL OF PERINATOLOGY/VOLUME 9, NUMBER 1

January 1992

PRENATAL TRANSMISSION OF HEPATITIS B VIRUS TO NEONATES BORN TO SERUM HEPATITIS B VIRUS DNA-POSITIVE MOTHERS Chia C. Pao, Ph.D., Ding-Shyan Yao, B.S., Chieh-Yu Lin, B.S., and T'sang-T'ang Hsieh, M.D.

ABSTRACT

Infants born to women who are carriers of the hepatitis B surface antigen (HBsAg) are at high risk of acquiring hepatitis B virus (HBV) and developing chronic hepatitis and other liver diseases.1 This mother-to-infant transmission of HBV is closely related to maternal hepatitis B e antigen (HBeAg) and HBV DNA.2 It has been suggested that vertical transmission takes place during delivery or immediately after birth.3 However, in utero HBV infection of the fetus has been suggested as another possible route of transmission of HBV to the neonates.4-6 DNA hybridization has made it possible to detect HBV DNA in clinical specimens from both healthy individuals and patients with hepatitis.7-8 The advent of polymerase chain reaction (PCR) has provided a rapid and direct method that can detect extremely small quantities of DNA.9 PCR has been shown to be the most sensitive method currently available in detecting HBV DNA1011 and can detect HBV DNA in sera of patients who are negative for all HBV serologic markers12 or positive for antibodies directed against HBsAg.13 In the following text, we report the results of using PCR in the direct identification of HBV DNA sequences in sera of infants born to HBV DNApositive mothers at birth.

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Hepatitis B virus (HBV) DNA was detected by in vitro enzymatic DNA amplification techniques in 66.7% (six of nine) of hepatitis B virus surface antigen (HBsAg)-positive and in 21.1% (7 of 33) of HBsAg-negative pregnant women. Fiveof the HBV DNA and HBsAgpositive women and one HBV DNA-positive but HBsAg-negative woman gave birth to infants positive for serum HBV DNA at time of birth. These results suggest that HBsAgnegative pregnant women are potentially capable of transmitting HBV DNA to their infants.

MATERIALS AND METHODS Source of Serum Specimens

Serum specimens were obtained from 42 pregnant women receiving routine prenatal care at Chang Gung Memorial Hospital in Taipei, Taiwan. Blood specimens were also obtained by heel puncture from neonates born to these women on the day of their birth (see later). Informed consent was obtained from all pregnant women to obtain blood from them and their infants. Immunologic Assays

Serological HBV markers, including HBsAg, HBeAg, and antibodies directed against HBV core antigen (anti-HBc), were determined with commercial enzyme-linked immunosorbent assay and radioimmunoassay kits obtained from Behring (Marburg, Germany) and Abbott Laboratories (North Chicago, IL). Polymerase Chain Reaction

DNA was first extracted with phenol and chloroform from 5 (xl of serum and further purified by

Department of Biochemistry, Chang Gung Medical College, and Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei, Taiwan, Republic of China This study was supported by Medical Research grants CMRP-235 and CMRP-286 from Chang Gung Medical College and Memorial Hospital awarded to C.C.P. Reprint requests: Dr. Pao, Department of Biochemistry, Chang Gung Medical College, 259 WenHwa Road, KweiSan, TaoYuan, Taiwan, Republic of China Copyright © 1992 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.

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Establishment of HBV DNA Positivity

Southern blot hybridization was performed on all PCR products to enhance both the sensitivity and specificity of the amplification and to establish the HBV DNA positivity. For the Southern blot hybridization, 10 (xl amplified reaction mixture was fractionated by 2% agarose gel electrophoresis and transferred to nitrocellulose membrane filters. An internal oligonucleotide, TTGCTCACCTCACCATACTGCACTCAGGCAAGCCATTCTC, based on nucleotide sequences located between the two primers on the HBV genome, was synthesized and labeled at its 5'-end with [gamma-32P]-adenosine triphosphate by polynucleotide kinase and used as a probe. Procedures for Southern blot hybridization have been described previously by others,15 but we have made the following modifications. After hybridization, the filters were washed sequentially in 2 x SSC (1 X SSC is composed of 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.5% sodium dodecylsulfate twice for 10 minutes each at room temperature; and then in 0.1 X SSC and 0.5% sodium dodecylsulfate three times for 20 minutes each at 55°C. Restriction endonucleases were purchased from either Boehringer Mannheim (Mannheim, Germany) or BRL (Gaithersburg, MD) and used according to manufacturers' recommendations.

Precautions Against Contaminations and False-Positive Results in PCR

Because of the extraordinary amplification power of the PCR techniques, precautionary measures were taken to minimize any possible contaminations dur

ing sampling and subsequent processing. Gloves were always worn when the specimens were being handled and processed. Areas of skin were extensively cleaned before puncturing took place. Special steps were also taken to minimize sample-to-sample contamination and PCR-product carryover in order to avoid false-positive results. These steps include aliquoting of all reagents, physical separation of preand post-PCR products and reagents, and meticulous laboratory techniques. Furthermore, all reagents were irradiated with ultraviolet light to inactivate any possible contaminated double-stranded DNA already present in the reagents16 before sample DNA was added and the PCR started. Fifty nanograms of human DNA or 1 ng of Escherichia coli DNA was used as negative control, which always yielded negative results. Multiple reagent controls were also included in each PCR assay and gave negative results. Repeated DNA amplification assays of the same specimens at different times produced the same results.

RESULTS

The presence of HBV DNA in the serum is indicated by the 433 base pair DNA after amplification (Fig. 1, lane B). The lower limit of detection by our method (that is, amplification by PCR followed with Southern blot hybridization) is approximately 10~5 pg or 3 HBV viral genome equivalents. This figure was estimated by amplifying serial dilutions of a purified cloned HBV DNA solution of known concentration (data not shown). The level of detection is not adversely affected by the presence of as much as 50 ng of human or 20 ng of bacterial E. coli DNA in the amplification reaction mixture (data not shown). DNA prepared from Chlamydia trachomatis, Mycoplasmahominis, human papillomaviruses, EpsteinBarr virus, human cytomegalovirus, and herpes simplex viruses types I and II do not cross-amplify with the HBV primers used in this study (data not shown). The specificity of the amplification and the authenticity of the 433 base pair DNA were evaluated and confirmed by two independent methods. First, the actual restriction endonuclease digestion patterns of the amplified DNA were compared with and found to agree exactly with those predicted from known DNA sequences and locations of the restriction sites. When the amplified 433 base pair HBV DNA was treated with restriction endonuclease Taql or Bglll, the original 433 base pair DNA disappeared and was replaced with new DNA of 309 and 124, and 339 and 94 base pairs, respectively (Fig. 1, lanes D and E). Second, the results of Southern blot hybridization showed that the 433 base pair DNA produced by amplifying either cloned HBV DNA or DNA prepared from serum of a patient with HBV DNA-positive chronic active hepatitis hybridized with the oligonucleotide probe specific for HBV DNA sequences (Fig. 2, lanes A and B).

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ethanol precipitation before used in PCR. The nucleotide sequences of the two primers are GCTTTGGGGCATGGACATTGACCC and TGATAAGATAGGGGCATTTGGTGG, which cover a 433 base portion of HBV gene codes for the core antigen. The procedures used for the PCR were those we have described earlier14 with the following modifications. Briefly, the 100 (xl DNA amplification reaction mixture contains 10 mM Tris hydrochloric acid buffer, pH 8.3; 50 mM potassium chloride; 1.5 mM magnesium chloride; 0.01% gelatin; 20 pmol each of the two primers; 2.5 nmol each of the four deoxyribonucleoside triphosphates; 1 unit of Tag DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT), and DNA from either specimen, positive, or negative control (see later). The temperature of the reaction mixture was first raised to 94°C for 20 seconds to denature the DNA, then cooled to 63°C for another 20 seconds to allow annealing of primers to putative target DNA sequences. The reaction mixture was then raised to 72°C for 1 minute to extend the DNA chain. This whole process was repeated 32 times with a 10minute incubation at 72°C at the end.

PRENATAL HBV TRANSMISSION/Pao, et al.

B

C

D

Figure 1. Agarose gel electrophoresis of polymerase chain reaction (PCR) amplified hepatitis B virus (HBV) DNA. DNA was extracted from 5 |xl of serum and was amplified with procedures described in "Materials and Methods." Lanes A and F are restriction endonuclease Hael 11-digested Phi-X 174 phage DNA used as size standards and the sizes of the DNA bands are (from top to bottom) 1353, 1078, 872, 603, 310, 281, 271, 234,194,118, and 72 base pairs. Lane B shows a representative of a HBV DNA positive serum with the 433 base pair band indicating HBV DNA positivity. Lane C is a negative control in which 50 ng of human DNA was amplified. When the amplified HBV DNA shown in lane B was treated with restriction endonuclease Taq\ or BglW, the original 433 base pair DNA disappeared and was replaced with two new DNA of 309 and 124 base pairs after TaqI (lane D) or of 339 and 94 base pairs after BglW treatment (lane E). The band of the 94 base pair DNA in lane E is too short to be visible.

Table 1 summarizes the prevalence of serum HBV DNA in mothers and their newborns with respect to other HBV serologic markers. HBsAg was detected in 21.4% or 9 of 42 otherwise healthy pregnant women, and this HBsAg carrier rate agrees with that of the general population of Chinese living in Taiwan.i? Sera from 7 of 33 (21.2%) of the HBsAg-negative and six of nine (66.7%) of the HBsAg-positive pregnant women were found to be positive for HBV DNA. All 13 HBV DNA-positive individuals (seven HBsAg negative and six HBsAg positive) were also positive for anti-HBc, indicating their prior exposure to HBV. A total of six neonates were found to have serum HBV DNA at birth; five were born to HBsAg- and HBV DNA-positive mothers and one to a HBsAg-negative but HBV DNA-positive mother. The HBsAg negative but HBV DNApositive mother who gave birth to an HBV DNA-positive infant was positive only for anti-HBc. Southern blot hybridization analysis of amplified HBV DNA from two mothers and their newborns is shown in Figure 2.

Figure 2. Southern blot DNA hybridization of amplified hepatitis B virus (HBV) DNA with an internal oligonucleotide probe. Lanes A and B are amplification of cloned HBV DNA and DNA prepared from serum of a HBV DNA positive chronic active hepatitis patient, respectively. Lanes C and D are amplification of DNA prepared from sera of a HBsAg and HBV DNA-positive mother and her newborn daughter, respectively. Lanes E and F are amplification of DNA prepared from sera of the only HBsAg-negative but HBV DNA-positive mother (lane E) who gave birth to a HBV DNA-positive son (lane F). Lane G is negative control with DNA prepared from serum of a HBV DNA-negative mother. Lanes H and I are primers and Taq polymerase negative controls. The arrow indicates the position of DNA of 433 base pairs.

DISCUSSION

Prenatal transmission of HBV to neonates born to HBsAg-positive carrier mothers has been reported by a number of investigators.4"6 Results presented in this study seem to suggest that the presence of HBV DNA at birth in infants born to HBsAg and HBV DNA-positive mothers is a rather frequent event (five of six) and that HBV DNA can also be found at birth in an infant born to an HBV DNApositive mother even if she is seronegative for HBsAg. PCR is an extremely sensitive method for detecting target DNA sequences.9 The lower limit of detection of approximately 3 HBV viral genomes equivalent in this study confirms reports of others that PCR is the most sensitive method currently

Table 1. Prevalence of Hepatitis B Virus (HBV) DNA in Maternal Serum and the Presence of Serum HBV DNA in Newborns Maternal Markers* HBsAg (-) HBsAg (+) HBeAg (+) HBeAg (-)

No. Persons 33 (78.6) 9 (21.4) 4 5

No. HBV No. HBV DNA (+) DNA (+) Persons (%) Newborns 7(21.2) 6 (66.7) 4 (100.0) 2 (40.0)

*HBsAg: hepatitis B surface antigens; HBeAg: hepatitis B e antigen; (+): positive; (-): negative. 63

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shown that HBV DNA can be found in most HBsAg positive as well as in a substantial proportion of HBsAg seronegative pregnant women. The results also suggest HBV DNA can be frequently found in sera of neonates born to HBsAg and HBV DNApositive mothers at birth and even in infant born to a HBV DNA-positive mother who is seronegative for HBsAg.

REFERENCES

1. Steven CE, Beasley RP, Tsui J, Lee WC: Vertical transmission of hepatitis B antigen in Taiwan. N Engl J Med 292: 771-774, 1975 2. Lee SD, Lo KJ, Tsai YT, et al: Maternal hepatitis B virus DNA in mother-infant transmission. Lancet 1:719, 1989 3. Beasley RP, Hwang LY: Postnatal infectivity of hepatitis B surface antigen-carrier mothers. J Infect Dis 147:185190,1983 4. Alexander GJM, Eddleston ALWF: Does maternal antibody to core antigen prevent recognition of transplacental transmission of hepatitis-B-virus infection? Lancet 1: 296-297, 1986 5. Mitsuda T, Yokota S, Mori T, et al: Demonstration of mother-to-infant transmission of hepatitis B virus by means of polymerase chain reaction. Lancet 2:886—888, 1989 6. Li L, Sheng MH, Tong SP, Chen HZ, Wen YM: Transplacental transmission of hepatitis B virus. Lancet 2:872, 1986 7. Scotto J, Hadchouel M, Hery C, Yvart J, Tiollais P, Brechot C: Detection of hepatitis B virus DNA in serum by simple spot hybridization technique: Comparison with results for other viral markers. Hepatology 3:279-284, 1983 8. Liebermann HM, LaBrecque DR, Kew MC, Hadziyannis SJ, Shafritz 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 carriers. Hepatology 3:285-291, 1983 9. Saiki RK, Gelfand DH, Stoffel S, et al: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491, 1988 10. Ulrich PP, Bhat RA, Seto B, Mack D, Sninsky J, Vyas GN: Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity testing in chimpanzees. J Infect Dis 160:37-43, 1989 11. Larzul D, Guigue F, Sninsky JJ, Mack DH, Brechot C, Guesdon JL: Detection of hepatitis B virus sequences in serum by using in vitro enzyme amplification. J Virol Methods 20:227-237, 1988 12. Thiers V, Nakajima E, Kremsdorf D, et al: Transmission of hepatitis B from hepatitis-B-seronegative subjects. Lancet 2:1273-1276, 1988 13. Kaneko S, Miller RH, Feinstone SM, et al: Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Natl Acad Sci USA 86:312-316, 1989 14. Pao CC, Lin CY, Maa JS, Lai CH, Wu SY, Soong YK: Detection of human papillomavirus in cervicovaginal cells by polymerase chain reaction. J Infect Dis 161:113— 115, 1990 15. Southern E: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503-517, 1975 16. Sarkar G, Sommer SS: Shedding light on PCR contamination. Nature 343:27, 1990 17. Wu JS, Chen CH, Chiang YH, et al: Hepatitis B virus infection in Taiwan: With reference to anti-HBc versus HBsAg and anti-HBs. J Formosan Med Assoc 79:760767,1980 18. Kaneko S, Feinstone SM, Miller RH: Rapid and sensitive method for the detection of serum hepatitis B virus DNA

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available in detecting HBV DNA.iO-H.i8.i9 Although the significance and meanings of the extremely low levels of HBV DNA are not clear at the present time, the exquisite sensitivity of the detection method is the most likely reason that all four of HBsAg- and HBeAg-positive pregnant women and their neonates were found to be positive for HBV DNA. High sensitivity of PCR is also probably responsible for the fact that one fifth of HBsAg seronegative pregnant women in this study are positive for serum HBV DNA. This finding of ours is in general accordance with a report of Paterlini et al20 that HBV DNA sequences were found in eight of the ten liver cancer patients with HBV antibodies and 6 of 13 without any HBV serologic markers, and with the report of Shih et al21 that over 80% of HBeAg-negative carriers could be infectious. Large amount of data in the literature indicate that HBsAg-negative patients may not be infectious. However, with more sensitive method of PCR, HBV DNA has been detected in HBsAg-negative individuals.i2,2O(22 The concentrations of serum HBV DNA in HBsAg-negative individuals are frequently too low to be detected by DNA blotting alone and are almost invariably much lower than those found in HBsAg-positive individuals. The fact that such HBsAg-negative and HBV DNA-positive individuals can be infectious was first demonstrated by Thiers et al,12 who reported that serum from HBsAg-negative but HBV DNA-positive (detectable only by PCR) person can induce acute hepatitis when inoculated into chimpanzees. Our identification of a HBV DNA-positive infant born to a HBsAg-negative but HBV DNA-positive mother underscores again the possibility that a HBsAg-negative person can be positive for HBV DNA and capable of transmitting HBV to others. Skin contamination from mother's blood on the newborn's heel is unlikely, since skin wipes of this and other newborns born to HBV DNA-positive mothers (both HBsAg positive and negative) were free of HBV DNA (data not shown). Although the data suggest that one fifth of HBsAgnegative mothers are potentially capable of transmitting HBV DNA to their infants, it is also possible that most of them contain serum HBV DNA at a concentration too low to cause actual transmission. What is evident from this study is that the clinical significance of these findings needs further investigations, especially follow-up studies on the infants born to women who are HBsAg negative but HBV DNA positive by the PCR method. It was not clear whether the HBV DNA found in the serum of the neonates was actually produced in the hepatocytes of the neonates or simply represented HBV passed through the placenta from the mothers during delivery. Analysis of HBV DNA and HBV-specific RNA in fetal liver or other tissues obtained from induced abortion may offer information on whether there was an indication of established HBV replication in utero. In summary, using PCR technique, we have

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viral DNA by polymerase chain reaction in patients with hepatitis B surface antigen. J Med Virol 30:159-162, 1990 22. Marcellin P, Martinot-Peignoux M, Loriot MA, et al: Persistence of hepatitis B virus DNA demonstrated by polymerase chain reaction in serum and liver after loss of HBsAg induced by antiviral therapy. Ann Intern Med 112:227-228,1990

Authors are indebted to the encouragement and support of Dr. Delon Wu and Dr. Chau-Hsiung Chang.

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using the polymerase chain reaction technique. J Clin Microbiol 27:1930-1933, 1989 19. Ulrich PP, Bhat RA, Seto B, Mack D, Sninsky J, Vyas GN: Enzymatic amplification of hepatitis B virus DNA in serum compared with infectivity.testing in chimpanzees. J Infect Dis 160:37-43, 1989 20. Paterlini P, Gerken E, Nakajima E, et al: Polymerase chain reaction to detect hepatitis B virus DNA and RNA sequences in primary liver cancers from patients negative for hepatitis B surface antigen. N Engl J Med 323:80— 85, 1990 21. Shih LN, Sheu JC, Wang JT, et al: Detection of hepatitis B

Prenatal transmission of hepatitis B virus to neonates born to serum hepatitis B virus DNA-positive mothers.

Hepatitis B virus (HBV) DNA was detected by in vitro enzymatic DNA amplification techniques in 66.7% (six of nine) of hepatitis B virus surface antige...
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