FEMS MicrobiologyLetters 83 (1991)35-38 © 1991 Federation of European MicrobiologicalSocieties 0378-1097/91/$03.50 Published by Elsevier ADONIS 0378109791004077

35

FEMSLE 04602

Detection of hepatitis B virus DNA by polymerase chain reaction in HBsAg negative Senegalese patients suffering from cirrhosis or primary liver cancer P i e r r e C o u r s a g e t 1, P i e r r e L e C a n n 1, D i d i e r L e b o u l l e u x 2 M a r i e - T h 6 r ~ s e , D i o p 3, O m a r Bao 3 and A w a - M a r i e Col! 2 1 Institut de Virologie de Tours, Tours, France, 2 Sert.,ice des Maladies Infectieuses, HSpital de Fann, Dakar, and "~SerL,ice de Mddecine Interne, HSpital Le Dantec, Dakar, Senegal

Received 7 June 1991 Accepted 21 June 1991 Key words: Hepatitis B; Cirrhosis; Liver cancer; Polymerase chain reaction

l. S U M M A R Y The polymerase chain reaction was used to search for hepatitis B virus (HBV)-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer. Amplified H B V - D N A sequences were detected by hybridization with a digoxigenin-labelled HBV-DNA probe, HBV-DNA was detected in 17% of HBsAg negative Senegalese subjects from the general population and in 44% and 58% of the patients suffering from cirrhosis or primary hepatocellular carcinoma (PHCC) respectively. In the control group, amplified HBV-DNA was detected in 25% of the subjects without HBs a g and anti-HBs antibodies, and in 6% of subjects positive for anti-HBs antibodies. This study

P. Coursaget, Institut de Virologie de Tours, 2 bis boulevard Tonnell~, 37042 Tours Cedex, France. Correspondence to."

confirmed the hypothesis that there is an etiologic link between HBV and PHCC in HBsAgnegative patients.

2. I N T R O D U C T I O N It is now clear that chronic hepatitis B virus (HBV) infection causes not only chronic active hepatitis and cirrhosis but also hepatocellular carcinoma (HCC) [1] although the precise role of HBV in the development of liver cancer remains unclear. However, in tropical Africa, two groups of patients suffering from primary hepatocellular carcinoma (PHCC) can be distinguished. The first comprises young adults in which the relationship between HBV and primary liver cancer (PLC) seems to be well established. In contrast, the frequency of HBV replication markers in older patients is not different from that observed in the general population [2] and thus the role of HBV in HBsAg-negative PLC patients has been de-

36 bated. For this group of patients another etiology for PHCC could be put forward since the development of cirrhosis and primary liver cancer were also reported in patients who had previously contracted non-A, non-B hepatitis after blood transfusion [3,4]. However, several investigators have reported the presence of HBV-DNA sequences in the liver and in the serum of HBsAg-negative patients, suggesting that some HBsAg-negative/HBV-DNA-positive liver disease may be related to an HBV infection [5-9] since such patients have to be considered as HBV chronic carriers. The recently developed polymerase chain reaction (PCR) has offered a very sensitive tool for investigation of the presence of HBV-DNA [1012], and is particularly helpful for detecting low HBV-DNA quantities as observed in HBsAgnegative anti-HBc-positive subjects [11,13-15]. Recent data using PCR amplification of HBVDNA suggests that HBV may be involved in the development of HBsAg-negative hepatocellular carcinoma [13]. We have used the polymerase chain reaction to search for HBV-DNA sequences in the sera of HBsAg-negative Senegalese patients suffering from liver cirrhosis or liver cancer.

3. M A T E R I A L S AND M E T H O D S A total of 115 Senegalese adults were included in this study: 32 HBsAg carriers, 42 control subjects from the general population without clinical evidence of liver disease and negative for HBsAg, and 40 HBsAg-negative patients suffering from cirrhosis a n d / o r PHCC. Serum was analysed for the presence of HBsAg and anti-HBs using commercial radioimmunoassays (Abbott Laboratories, IL) or ELISA (Behring). HBV-DNA was purified from serum according to the procedure described by Thiers et al. [8]. Two oligonucleotide primers [8] selected from the S gene region of HBV were synthesised using a DNA synthesiser apparatus (Gene Assembler Plus, Pharmacia). The primers 5 ' - C A T C T T C T T G T T G G T T C T T C (position 427-450) and 5 ' - T T A G G G T T T A -

A A T G T A T A C C (position 824-844) are located in the S gene region. Amplification of HBV-DNA was carried out by the polymerase chain reaction according to a modification of the procedure described by Saiki et al. [16] using a commercially available reagent kit (Gene Amp DNA amplification kit, Perkin Elmer, Uberlingen, F.R.G.). The target sequences were amplified in a total volume of 50 #1 containing an equivalent to 25 #1 of sera. The samples were subjected to 30 cycles of amplification using a DNA Thermal cycler (Perkin-ElmerCetus, Norwalk, U.S.A.). For each cycle, denaturation was at 94 ° C for 30 s, annealing of primers at 5 0 ° C for 30 s, and elongation at 7 5 ° C for 1 min. The temperature was maintained at 75 °C for 3 min after the completion of the last cycle. Amplified HBV-DNA was visualized by slot blot hybridization onto a nitrocellulose membrane (Hybond C, Amersham International, Amersham, U.K.) using a digoxigenin-labelled DNA probe. For the generation of this probe we used a 417-base pairs-long fragment which was obtained by PCR of an HBsAg-positive, HBeAgpositive serum sample, chosen as positive reference. This sample was obtained from a healthy chronic carrier. The probe is obtained by random primed incorporation of digoxigenin-labelled deoxyuridine-triphosphate (dUTP) during the amplification. After hybridization of this probe with target amplified HBV-DNA portions, the hybrids are detected by enzyme-linked immunoassay using an anti-digoxigenin alkaline phosphatase conjugate and subsequent enzyme-catalysed colour reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium salt (DNA labelling and detection kit, Boehringer Mannheim, F.R.G.). PCR assay, electrophoresis and dot blot analysis were r e p r o d u c e d at least twice.

4. RESULTS PCR-amplified HBV-DNA sequences were detected in 23 out of 32 (70%) Senegalese HBsAg carriers (Table 1). It should be noted that HBVDNA was found in all (100%) HBeAg-positive

37 Table 1 Detection of HBV-DNA by PCR in HBsAg negative Senegalese patients suffering from cirrhosis or primary liver cancer No. Tested

HBsAg chronic carriers HBsAg-negative controls HBsAg-negative cirrhosis HBsAg-negative PHCC

HBV-DNA detected by slot blot hybridization No.

%

32

23

(70%)

42

7

(17%)

9

4

(44%)

31

18

(58%)

chronic carriers as opposed to only 13 out of 22 (59%) in HBeAg-negative HBsAg chronic carriers. Among the 42 HBsAg-negative controls HBV-DNA was detected in 7 subjects. In this group amplified HBV-DNA was found in 6 out of 24 subjects (25%) without detectable anti-HBs antibodies, and in only 1 (6%) out of 18 subjects positive for anti-HBs antibodies. HBV-DNA was detected in 4 (44%) and 18 (58%) of the patients with cirrhosis or PHCC respectively.

5. D I S C U S S I O N In Senegal, an area highly endemic for HBV, a high rate of detection of H B V g e n o m e was observed in HBsAg negative patients suffering from liver cirrhosis or primary liver cancer. This study confirmed the hypothesis of Paterlini et al. [13] that there is an etiologic link between H B V and PHC in HBsAg-negative patients. Moreover H B V was detected in the HBsAg-negative individuals from the general population, in accordance with previous results from Chemin et al. [12]., This indicates a persistence of the virus or a persistence of H B V - D N A in these subjects. However, one of these subjects will be considered to have been cured of H B V infection on the basis of the presence of anti-HBs antibodies. Therefore in HBsAg-negative subjects, a decline in the viral expression which is related to the age of the patient during the sequence leading from chronic

carrier state to the development of PHCC, may account for HBsAg serologic negativity and HBV-DNA detection. This is in accordance with the fact that PHCC has been observed to develop in subjects who were anti-HBs positive [1,17].

ACKNOWLEDGEMENTS We thank V. Thiers and C. Brechot for providing the sequence of primers and for their help with PCR technology.

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