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TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1995) 89, 330

Significance of anti-hepatitis B surface antigen as the only serological marker of hepatitis B in non-vaccinated subjects in Gabon

Ousmane Traorel, Alain Goudeau*, Philippe Roin- geard*, Maryvonne Kombilas, Fr&d&ic Dubois* and Dominique Richard-Leaoblel ~Dkpartenumt de Parasi- tologie et Mt+decine Tropicale, Fact& de MLdecitw de Tours, Francei *Unit& de Virologie, DQartement de Micro- biologie Mddtcale et Molkulaire, URA CNRS 1334, CHU Bretonneau, 37044 Tours, France; 3Dt?partement de Parasitologie, Fact& de Mbdectne de Libreville, Gabon

Keywords: hepatitis B, anti-HBsAg, Gabon

Seroepidemiological studies in Africa have shown that between 70% and 90% of the adult population have sero- logical evidence of past hepatitis B infection (BAFUN et al., 1982). Antibodies to hepatitis B surface antigen (anti- HBs) as the sole marker of mfection are considered to be unusual. However, anti-HBs seroconversion without antibodies to heDatitis B core antieen (anti-HBc) has been observed 6 studies in both ‘Africa and Europe (BOWRY, 1983; DRISS et al., 1991). It is not yet known whether-this is the result of immunization with he atitis B surface an&en (HBsAd from heDatitis B virus ( R BV) or an HBV m&ant (FOR&N et al.; 1994), or whether it is the result of exposure to either some other infection or naturally occurring antigens which cross react with a subdeterminant of HBsAg.

The present study determined the prevalence of HBsAg, anti-HBs and anti-HBc in healthy rural subjects in Gabon. Anti-HBs as the only HBV marker was fre- quently observed. In an attempt to clarify the signific- ance of this, we tested these sera for antibodies to preS2 epitopes (anti-preS2). Although anti-preS2 activity is transient (BUDKOWSKA et al., 1986), a positive response would suggest that anti-HBs without anti-HBc indicated true seroconversion following exposure to HBsAg.

Serum samples were collected between December 1991 and June 1992 from 303 healthy subjects (158 males, 145 females) living in 3 rural villages in Gabon which are involved in an onchocerciasis control project. These 3 areas were selected because their populations are stable and sedentary. After individual informed consent was obtained, blood from the entire population (aged 2- 80 years, mean 29f20 years) was collected by venepunc- ture and sera were stored at -20°C. No donor had a his- tory of anti-hepatitis B immunization. All sera were tested using enzyme immunoassay (EIA) test kits for HBsAg (Hepanostike@, Organon), anti-HBs (Ausab@, Abbott) and anti-HBc (Eti AB Corek@, Sorin). For the detection of anti-preS2 antibodies, we used a specific anti-nreS2 EIA based on a 55 amino acid svnthetic nen- tide kindly provided by F. Aubrit (Diagno&s Transfu- sion, Marnes la Coquette, France). This peptide, cover- ing the entire preS2 sequence of the HBsAg (subtype aw), was used as coating material. Microtitre plates (kunc) were coated with 160 uL of a solution containing 10 ua/mL of ores2 neotide diluted in nhosnhate-buf-

I” I s 1~- 1 .

Address for correspondence: Professeur D. Richard-Lenoble, Parasitologie et Medecine Tropicale, 2 bis Boulevard Tonne& BP. 3223,37032 Tours cedex, France.

fered saline (PBS, pH 7.4), incubated overnight at +4”C and subsequently washed 3 times with PBS-Tween 2Od (0.5%). A post-coating step was performed by incubation of the plates in 3OOu.L of PBS with 2% foetal calf serum. Briefly, the preS2 EIA procedure was as follows. Incuba- tion of diluted samples or controls (l/100 with PBS Tween-2@) for 1 h at room temperature, 5 washes with PBS Tween-20@, incubation with anti-human immuno- ,globulin G-peroxidase conjugate for 1 h at 37”C, 5 washes, and development for 30 min at room tempera- ture in the dark; the reaction was stopped with 1~ sul- phuric acid and optical densities were determined at 490 nm. Normal human sera were used as ne ative controls and vaccinated patients’ sera (GenHevac % @, Pasteur) as positive controls. Samples were tested in triplicate.

HBsAg was found m 59 subjects (19%), anti-HBc in 234 (78%), and anti-HBs in 218 (72%); 279 (92%) pa- tients were seropositive for at least one HBV marker and 34 (11%) sera had anti-HBs as a sin le marker. Sera were considered to be positive for anti-H % s when the level was >lO mitt/ml and/or samples were repeatedly reactive. None of these subiects had been nreviouslv immunized with anti-HBV vaccine. Sera from 7 patienis were again collected one year later; 6 showed the same serological pattern (anti-HBs alone) and one had anti-HBs and de- tectable anti-HBc. Anti-preS2 was searched for by EIA in 25 of the 34 patients who had anti-HBs alone; 4 were positive, 3 being subjects with high anti-HBs levels (>50 miu/mL) and one havine a low level of anti-HBs (18 miuhLj.

The percentage of individuals positive for anti-HBs alone in this highly HBV infected population was elev- ated. When a check one vear later was Dossible. these patterns had remained stable. A study hong French blood donors reported that the prevalence of anti-HBs alone, measured by the same EIA kits we used, was very low and none of these subjects had anti-preS2 (DIUSS et al., 1991). Moreover, the isolated anti-HBs antibodies were labile, since 4 months later only one subject still had detectable anti-HBs. The sianificance of this unusual profile may be different in Euro-pe and in Africa. In some of these African sera, the presence of anti-HBs as the only serological marker undoubtedly indicated previous infection by HBV or an HBV mutant and did not result from a cross reaction with another microorganism.

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Received 21 November 1994; revised 2 Februay 1995; accepted fm publication 3 Februa y 1995