438

Clinical and laboratory observations

The Journal of Pediatrics March 1992

Use of the polymerase chain reaction to demonstrate hepatitis B virus DNA in serum of children with chronic hepatitis B Stefan Wirth, MD, UIrike MOilers,* Klaus-Michael Keller, MD, a n d Andreas W i n t e r p a c h t , DiplBiol From Children's Hospital of the Johannes Gutenberg University, Mainz, Germany The polymerase chain reaction was used to investigate the presence of hepatitis B virus DNA in sera of 64 children with chronic hepatitis B and negative results on dot blot hybridization tests. Our results demonstrate that most chronic carriers of hepatitis B surface antigen in childhood have hepatitis B virus DNA d e t e c t a b l e by polymerase chain reaction in their serum and must be considered infectious. (J PEDIATR4992;120:438-40)

The development of modern molecular hybridization techniques has allowed the detection and identification of viral D N A in serum and liver tissue of patients with hepatitis B. In serum, the D N A of hepatitis virus B is conventionally determined by dot blot hybridization, but this technique has limited sensitivity,l, 2 For example, only 25% of patients who have antibodies reactive with hepatitis B e antigen can be shown to have HBV D N A by dot blot hybridization. 1The polymerase chain reaction can now be used to identify HBV D N A in serum from patients with negative results on dot blot hybridization. 3 This is of clinical significance because children whose serum contains HBV D N A must be considered infectious. We have used the PCR to investigate the presence of HBV D N A in sera of children with chronic hepatitis B and negative results on dot blot hybridization. The findings were related to other serologic markers and serum biochemical changes of chronic HBV infection. METHODS Sera from 190 children with chronic hepatitis B were tested for HBV D N A by dot blot hybridization; findings were negative in 61. In these 61 patients, aged 11/2 to 18 years (mean 14 years), PCR was performed. The diagnosis

Submitted for publication July 17, 1991; accepted Oct. 11, 1991. Reprint requests: Stefan Wirth, MD, Children's Hospital of the Johannes Gutenberg University, Langenbeckstr. 1, D-6500 Mainz, Germany. *Medical student. 9/22/34375

of hepatitis B was based on the determination of hepatitis B surface antigen and the HBeAg/anti-HBe status. Histologic diagnoses were chronic active hepatitis in 22, chronic persistent hepatitis in 20, and minimal hepatitis in 8 children. Five were otherwise healthy HBsAg carriers, and in 6 patients histologic data were not available. When the serum samples were collected, activities of aspartate and alanine aminotransferases were measured. We determined HBsAg, HBeAg, and anti-HBe antibody by commercial immunoassays (Abbott Laboratories DiagnosHBeAg HBsAg HBV PCR

Hepatitis B e antigen Hepatitis B surface antigen Hepatitis B virus Polymerase chain reaction

tic Division, Abbott Park, Ill.). Dot blot hybridization was performed by using a cloned HBV D N A probe labeled with phosphorus 32. PCR analysis. For the PCR assay, sequences specific for the HBV core region, as published by Kaneko and Miller, 4 were synthesized and used as primers: primer 1763, 5 ' G C T T T G G G G C A T G G A C A T T G A C C C G T A T A A - 3', and primer 2032 R, 5 ' - C T G A C T A C T A A T T C C C T G GATGCTGGGGTCT-3 '. Amplification was carried out in a final volume of 100 #1 containing 1 t~g purified D N A from 100 #l serum by phenol-chloroform extraction, 0.4 #mol/L of each primer, 200 gmol/L of each deoxyribonucleoside triphosphate, 50 mmol/L potassium chloride, 1.5 mmol/L magnesium chloride, 10 mmol/L TRIS-HC1 buffer (pH 8.3), 0.01% gelatin, and 2.5 units Taq polymerase (PerkinElmer Cetus, Norwalk, Conn.). For denaturation, samples

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were heated to 94 ~ C for 5 minutes in the first cycle, cooled to 55 ~ C for 2 minutes, and incubated at 72 ~ C for 3 minutes. In the following 40 cycles the denaturation time was 2 minutes. After the last cycle the mixture was incubated at 72 ~ C for 10 minutes. Great care was taken to prevent any contamination. Reagents were divided and stored in new containers. Different places were used for DNA extraction, preparation of the reaction mixture, and PCR. The work was carried out under a hood to prevent aerosol contamination. Negative and defined positive control sampies, including reaction mixture and water without DNA, were analyzed in each experiment. After amplification, 10 #1 aliquots of each sample were fractlonated by 2% agarose gel electrophoresis. The DNA was visualized by ethidium bromide staining and subsequently transferred to a nylon membrane (Gene Screen Plus, NEN Research Products, Boston, Mass.) for Southern blot hybridization. Molecular hybridization was performed by using a complete, denatured, single-stranded HBV DNA probe labeled with [32P]cytidine 5'-triphosphate by nick translation to a specific activity of 2 to 5 • 108 cpm/#g DNA. Autoradiography was done at a temperature of - 7 0 ~ C with an intensifying screen (Hyperfilm-MT; Amersham International Ltd., Little Chalfont, United Kingdom). RESULTS Fourteen children were seropositive for HBeAg and 44 for anti-HBe antibodies; three had neither HBeAg nor anti-HBe antibodies in the serum. Elevated serum aminotransferase values were found in nine patients with HBeAg (range 24 to 313 U/L) and in five with anti-HBe antibodies (range 22 to 58 U/L). Detection of serum HBV DNA by PCR was characterized by a signal at the 259 base-pair position in the ethidium bromide-stained gel. The results in the subsequently performed Southern blot tests were essentially the same. The detection limit of HBV DNA was equivalent to about 30 virions. All control sera showed no signal. The Table summarizes the PCR results from the sera of the 61 children, related to the HBeAg/anti-HBe status and serum aminotransferase values. Viral sequences were detected in 47 (77%) of the investigated sera. Of the 47 patients with HBV DNA, 14 had HBeAg and 30 had anti-HBe antibodies in their serum; 3 had neither. All 14 chronic HBsAg carriers with undetectable HBV DNA had anti-HBe antibodies. Thus, in 68.2% of the serum samples containing anti-HBe antibodies, HBV DNA could be demonstrated by PCR. All patients with elevated serum aminotransferase values, regardless of their HBeAg/anti-HBe status, had HBV DNA as determined by PCR.

Clinical and laboratory observations

439

Table. PCR-detectable HBV DNA in sera of 61 children with chronic hepatitis B and negative results on dot blot hybridization tests, related to HBV markers and liver enzymes HBV-DNA Marker

Positive

Negative

H BeAg-positive Anti-HBe antibody-positive HBeAg/anti HBe antibody-negative AST and ALT levels Elevated Normal

14 30 3

0 14 0

14 47

0 14

AST, Aspartateaminotransferase;ALT, alanineaminotransferase.

DISCUSSION Previous studies have shown that PCR represents a more sensitive test for determination of HBV DNA than do other, conventional hybridization assays. However, PCR, because of its high degree of sensitivity, will detect even minor contamination; thus several technical precautions are required.3, 5 No contaminated control samples could be found in our studies, suggesting that no false-positive results were obtained. Chronic hepatitis B in childhood is characterized by two phases. During the first phase there is biochemical or pathologic evidence of disease activity; the sera of these patients contain HBeAg. During the course of the disease the second phase can be recognized by seroconversion from HBeAg to anti-HBe antibody, usually indicating a decrease of inflammatory activity in the liver and normalization of liver enzyme values.6 The clearance of HBV DNA, as determined by conventional hybridization methods, has suggested that replication may have ceased in most of these patients. Previous studies have demonstrated that HBV DNA is detectable by dot blot hybridization in more than 80% of adults with HBeAg, but patients with anti-HBe antibodies whose liver enzyme values are normal usually have no HBV DNA. 1 The PCR studies in adults, however, revealed that almost all HBsAg carriers with HBeAg and up to 80% of the patients with anti-HBe antibodies have HBV DNA in their serum. 7, 8 Reports of comparable studies of children with chronic HBV infection have not been available. Our data now demonstrate that HBV DNA is detectable by PCR in the serum of all HBsAg carriers with HBeAg. Furthermore, about two thirds of the patients with anti-HBe antibodies have viral sequences in the serum. When the data are related to serum aminotransferase activities, it becomes obvious that all children with elevated

440

Clinical and laboratory observations

liver enzyme values have PCR-detectable HBV D N A , regardless of their H B e A g / a n t i - H B e status. This is in accordance with the findings in adult chronic H B s A g carriers, as recently noted by Baker et al. 9 Our results confirm that P C R is the best technique to detect serum H B V D N A in children with negative dot blot results. Most chronic H B s A g carriers in childhood have low-level virus replication and have PCR-detectable H B V D N A in their serum. The clinical significance of these observations is supported by the demonstration that sera containing extremely small amounts of H B V D N A , detectable only by P C R , can cause acute hepatitis when inoculated into chimpanzeesJ ~ Thus children with ongoing viral replication, as detected by P C R , must be considered infectious. Our data underline the necessity for vaccination of close relatives of these patients.

REFERENCES 1. Kroogsgard K. Hepatitis B virus DNA in serum: applied molecular biology in the evaluation of hepatitis B infection. Liver 1988;8:257-83. 2. Bonino F. The importance of hepatitis B viral DNA in serum and liver. J Hepatol 1986;3:136-41.

The Journal of Pediatrics March 1992

3. Brechot C. Polymerase chain reaction: a new tool for the study of viral infections in hepatology. J Hepatol 1990; 11:124-9. 4. Kaneko S, Miller R. Characterization of primers for optimal amplification of hepatitis B virus DNA in the polymerase chain reaction assay. J Virol Methods 1990;29:225-30. 5. Malter JS, Gerber MA. The polymerase chain reaction for hepatitis B virus DNA. Hepatology 1991;13:188-9. 6. Bortolotti F, Cadrobbi P, Crivellaro C, et al. Long-term outcome of chronic type B hepatitis in patients who acquire hepatitis B virus infection in childhood. Gastroenterology 1990; 99:805-10. 7. Kaneko S, Miller RH, Di Biseglie AM, Feinstone SM, Hoofnagle JH, Purcell RH. Detection of hepatitis B virus DNA in serum by polymerase chain reaction. Gastroenterology 1990;99:799-804. 8. Pao CC, Yao DS, Lin CY, et al. Serum hepatitis B virus DNA in hepatitis B virus seropositive and seronegative patients with normal liver function. Am J Clin Pathol 1991;95:591-6. 9. Baker BL, Di Bisceglie, Kaneko S, et al. Determination of hepatitis B virus DNA in serum using the polymerase chain reaction: clinical significance and correlation with serological and biochemical markers. Hepatology 1991;13:632-6. 10. Thiers V, Nakajima E, Kremsdorf D, et al. Transmission of hepatitis B from hepatitis B seronegative subjects. Lancet 1988;2:1273-6.

Complete recovery from hemorrhagic shock and encephalopathy J a m e s R. Bonham, PhD, A n d r e w Meeks, MRCPI, M i c h a e l Levin, FRCP, Alan Gibson, MRCP, Kay Hawkins, MRCP, a n d M a r y Gerrard, MRCP From the Departments of Chemical Pathology and Paediatrics, Children's Hospital, Sheffield, the Department of Paediatrics, St. Mary's Hospital Medical School, London, and the Department of Paediatrics, County Hospital, Lincoln, United Kingdom

We describe three infants (aged 9 weeks to 4 months) with the classic features of hemorrhagic shock and encephalopathy syndrome, including sudden onset of shock, neurologic disturbance, bleeding, disseminated intravascular coagulation, and impaired hepatic and renal function. Unlike the cases previously described, all three children recovered rapidly without evidence of long-term neurologic damage. These findings may modify the perception that this disorder has a uniformly bad outcome. (J PEDIATR1992;120:440-3)

Submitted for publication May 30, 1991; accepted Oct. 21, 1991. Reprint requests: James R Bonham, PhD, Department of Chemical Pathology, Children's Hospital, Sheffield SI0 2TH, United Kingdom. 9/22/34488

The common features of hemorrhagic shock and encephalopathy in children include the sudden onset of shock, coma and convulsions, bleeding, disseminated intravascular coagulation, watery diarrhea, and impaired hepatic and renal function. 14 The disorder was included in the United King-

Use of the polymerase chain reaction to demonstrate hepatitis B virus DNA in serum of children with chronic hepatitis B.

The polymerase chain reaction was used to investigate the presence of hepatitis B virus DNA in sera of 61 children with chronic hepatitis B and negati...
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