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HBsAg subtypes among HBsAg carriers in Okinawa, Japan. Evidence of an important relationship in seroconversion from HBeAg to anti-HBe
Journal of Infection (1994) 28, I4I-I5O
HBsAg subtypes a m o n g HBsAg carriers in Okinawa, Japan. Evidence o f an important relationship in seroconversion f r o m HBeAg to anti-HBe Akinori Noguchi, Jun Hayashi, Koya Nakashima, Miki Hirata, Hideyuki Ikematsu and Seizaburo Kashiwagi Department of General Medicine, Kyushu University Hospital, Japan Accepted for publication 2I October I993
Summary In Okinawa, Japan i26i hepatitis B surface antigen (HBsAg) carriers were investigated for clinical differences among HBsAg subtypes. Among the 854 for whom they could be determined, subtype adw was found in 6o4 (70"7 %), adr in 232 (27"2 %) and others in I8 (2.I%). The overall prevalence of hepatitis B e antigen (HBeAg) was significantly lower in subtype adw (I I'9 %) than in adr (I7"7 %) (P < o.oi). The mean age of HBeAg-positive carriers was significantly lower in adw (20"4 years) than in adr (26"9 years) (P < 0"05). Seroconversion from HBeAg to antibody to HBeAg (antiHBe) occurred in 43 carriers. The seroconversion rate was higher in adw (43.I %) than in adr (29"3 %) and the seroconversion age was younger in adw (22" 7 years) than in adr (27' 9 years). These results suggest that carriers with subtype adw tend to seroconvert from HBeAg to anti-HBe at a higher rate and a younger age than do those with adr. HBsAg subtypes may be closely associated with the HBeAg/anti-HBe status.
Introduction Hepatitis B surface antigen (HBsAg) has a common determinant called 'a '1 and two additional pairs of mutually exclusive antigenic determinants named d / y and w / r , 2' 3 resulting in the following four major subtypes: adw, ayw, adr and ayr. Geographical distribution of HBsAg subtypes and their relationship with epidemiological factors have attracted a great deal of attention. 4-s In the Japanese population, it has been found that there are two main subtypes, adr and adw. 7 We identified HBsAg subtypes to determine the route of transmission.8-1° HBsAg subtypes have recently become more easily and accurately identified since an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against HBsAg subtypes has become available. 11 Shiina et al. 12'13 reported that HBsAg subtypes may be linked to the development of chronic liver disease, although it has been established that they correlate more directly with epidemiological factors than with disease. Since I968, we have investigated hepatitis B virus (HBV) infection in Okinawa, Japan. Infants and adults are afflicted and we found that HBV was endemic in this area. 14-16 In the present study, I26I HBsAg carriers were investigated with regard to HBsAg subtypes, hepatitis B e antigen (HBeAg), Address correspondence to: Jun Hayashi, Department of General Medicine, Kyushu University Hospital, Higashi-Ku, Fukuoka 812, Japan. oI63-4453/94/o2o14I + IO $08.00/0
© I994 The British Society for the Study of Infection
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A. NOGUCHI
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antibody to HBeAg (anti-HBe) status, liver function test ( L F T ) , age and sex in order to determine if there are clinical differences between HBsAg subtypes. Materials and methods Study population Inhabitants of the Yaeyama District of Okinawa, Japan were surveyed. Okinawa is in the subtropical zone about iooo k m south of the main islands of Japan. T h e Yaeyama District is in the south-western part of Okinawa near Taiwan (Figure I). All subjects studied were of the Japanese race and were born and raised in this area. T h e r e is some genetic variation between the Okinawans and the Japanese in the main islands due to differences in historical demographics. We surveyed over 9 0 % of the estimated population of Hateruma, Iriomote, Kohama, Taketomi, and Kuroshima Islands and over 60% of the people on Ishigaki Island, for H B V markers. A total of i 8 8 2 i inhabitants were tested for HBsAg in I98o. We found IZ6I HBsAg carriers (males 748, females 5 I3, age 1-87 years), and each was followed for one year or longer between I98O and I99O. T h e y were tested for H B V markers and L F T s were performed at least twice, at an interval of 3 m o n t h s or longer. Serum samples were stored at - 2 o °C until tested. Laboratory methods HBsAg was measured by reversed passive haemagglutination ( R P H A ; Revesecell, Meguroken, Osaka, Japan) and H B e A g / a n t i - H B e by radioimmunoassay (RIA; HBe RIA, Abbott Laboratories, I L , U.S.A.). HBsAg subtypes were determined by E L I S A , by means of monoclonal antibodies (HBsAg Subtype EIA, Institute of I m m u n o l o g y , Tokyo, Japan). 17 We dispensed 5o #1 each of the serum samples and control sera to wells which were coated with monoclonal antibody against the c o m m o n determinant ' a ' of HBsAg and the microplate was incubated at 37 °C for 3 h. We then dispensed 50 #1 labelled monoclonal antibody against determinants ' d ' , ' y', ' w ' , o r ' r' to corresponding wells and the microplate was incubated at 37 °C for z h. T h e reaction was terminated by adding 50 #1 of 4 N H2SO4, and the intensity of developed colour was determined by the absorption at 492 nm. Absorption was measured within io m i n of stopping the colouring reaction. HBsAg subtypes could not be determined in 4o7 HBsAg carriers because of their low HBsAg titres (under 28 R P H A ) and they were excluded from analysis. T h e r e remained 854 HBsAg carriers (males 522, females 332, age 2-88 years) whose subtypes were available for analysis. T h e period of the observation was 6-6 ___2. 7 years. Conventional L F T s were performed by use of multiple autoanalyser. T h e presence of liver dysfunction was defined as an elevated serum alanine aminotransferase (above 40 IU/1). Statistical analysis Chi-square, Mantel-Haenszel and Student's t-tests were used to determine statistical significance. Results T h e distribution of HBsAg subtypes among carriers in the islands of Yaeyama District of Okinawa, Japan, in w h o m subtypes could be determined were adw
HBsAg subtypes and HBeAg/anti-HBe status Table I
I43
Geographical distribution of HBsAg subtypes among HBsAg carriers in the Yaeyama District of Okinawa, Japan, I98O HBsAg subtype
Island Ishigaki Iriomote Kohama Kuroshima Taketomi Hateruma Total
Number of carriers 728 74 24 5 5 18 854
Number adw (%)
Number adr (%)
523 (71.8) i92 (26-4) 44 (59'5) 28 (37"8) 14 (58'3) 9 (37"5) 5 (Ioo) o 4 (80) I (20) 14 (77"8) 2 (II.i) 604 (7o'7) 232 (27"2)
Number Number Number ayw ayr adyr (%) (%) (%) I (0"I) o o o o o I (o.I)
I (O'I)
I (i.4) o o o o 2 (0.2)
3 (0'4) I (I'4) o o o o 4 (o'5)
Number adwr (%) 8 (I'I) o I (4"2) o o 2 (ii.i) II (1"3)
HBsAg, hepatitis B surface antigen. Table II
Age-specific distribution of HBsAg subtypes among HBsAg carriers in the Yaeyama district of Okinawa, Japan, I 9 8 0 HBsAg subtype
Age (years)
Number of carriers
Number adw (%)
Number adr (%)
Number ayw (%)
Number ayr (%)
Number adyr (%)
Number adwr (%)
o-9 IO--I9 2o-29 30-39 4o-49 5° Total
33 ii2 14o 268 lO8 193 854
26 (78"8) 81 (72'3) 99 (7o'7) I9O (70'9) 70 (64'8) 138 (71"5) 604 (7o'7)
6 (I8-2) 3o (26.8) 37 (26"4) 72 (26'9) 36 (33'3) 51 (26'4) 232 (27'2)
o o I (o'7) o o o I (o'I)
o 1 (0'9) o o o I (0"5) 2 (0'2)
o o e (1-4) 1 (0'4) o I (0'5) 4 (o'5)
I (3"0) o
I (0'7) 5 (1'9) 2 (I'9) 2 (I'O) IX (I'3)
HBsAg, hepatitis B surface antigen. 604 (70"7 % ) , a d r 232 (27.2 % ) , a y w I ( o ' 1 % ) , a y r 2 (0"2 ~/o), a d y r 4 (0"5 % ) a n d a d w r 11 (I" 3 ~/o). T h e p r e v a l e n c e o f s u b t y p e a d w was significantly h i g h e r t h a n t h a t o f t h e o t h e r s ( P < o'ooz). T h e p r e v a l e n c e o f s u b t y p e a d w was h i g h e s t o n t h e islands, a l t h o u g h o n I r i o m o t e a n d K o h a m a its rate was less t h a n 6o % a n d t h a t o f a d r was m o r e t h a n 35 % ( T a b l e I). T h e overall rate o f s u b t y p e a d w was m o r e t h a n 7 ° % a n d a d r less t h a n 30 % in each age g r o u p , e x c e p t for the 40--49 y e a r g r o u p . T h e c o m p o u n d s u b t y p e s ( a d y r a n d a d w r ) i n c r e a s e d w i t h a d v a n c i n g age ( T a b l e II). I n m a l e s , s u b t y p e a d w was f o u n d in 366 ( 7 o ' 1 % ) , a d r in 148 (28"4 % ) , a y r in I (0"2 ~/o) a n d a d w r in 7 (1"3 % ) a n d in females, a d w was f o u n d in 238 ( 7 I ' 7 % ) , a d r in 84 (25"3 % ) , a y w in z (o'3 % ) , ayr in (o'3 % ) , a d y r in 4 (1'2 % ) a n d a d w r in 4 (1"2 % ) . N o significant d i f f e r e n c e s b y sex or age w e r e o b s e r v e d . O f t h e 854 H B s A g carriers, t h e p r e v a l e n c e o f H B e A g was 115 ( 1 3 5 % ) a n d t h a t o f a n t i - H B e was 714 (83"6 % ) ; t h e r e m a i n i n g 25 (2"9 % ) w e r e n e g a t i v e for
A. NOGUCHI ET AL.
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I47
both. HBeAg and anti-HBe were not simultaneously present in any of the samples tested. T h e prevalence of HBeAg was z4"9 % in males and IZ'1% in females. T h e r e was no significant difference between the sexes. Age and HBsAg subtype distribution of HBeAg among H B V carriers is shown in Table III. T h e prevalence of HBeAg decreased with advancing age. Its prevalence was I7"7 % in 232 carriers with subtype adr, a value significantly higher than that in the 604 carriers with subtype adw (I I'9 %, P < o'o5). T h e prevalence of HBeAg in carriers with subtype adr was clearly higher than in those with subtype adw who were more than IO years old. In the 4o-49 year age group, the prevalence of HBeAg was significantly higher in carriers with subtype adr (z6"7 %) than in those with subtype adw (o %) (P < o.oI). Moreover, the mean age of HBeAg-positive carriers with subtype adw was 20"4+I5"2, an age significantly younger than those with subtype adr (26"9__+z5"8, P < 0"05). Of the I 15 HBsAg carriers with HBeAg, 43 (37"4 %) seroconverted to antiHBe during the observation period. Of 43 carriers, 23 seroconverted to antiHBe within 4 years of starting the investigation, z 7 within 5-9 years and three within zo-I I years. T h e rate of seroconversion was higher in females (44"2 %) than in males (33"3 %) and the age of seroconversion was younger in females (22" 4 -]- I3"8 years) than in males (25"z _ 18"5 years). These differences however were not statistically significant. T h e rate of seroconversion was higher and occurred at a younger age in carriers with subtype adw (43"I %, 22"7__+i6.6 years) than in those with subtype adr (29"3 %, 27"9__+I5"8 years). However, neither difference was statistically significant (Table IV). T h e prevalence of liver dysfunction in 854 HBsAg carriers was significantly higher in HBeAg-positive carriers (33"0 %) than in HBeAg-negative carriers (9"z %): a d w + 3 I ' 9 % , a d w - 9 " 6 % ; a d r + 3 4 " 1 % , a d r - 8 " 4 % . T h e r e was no statistically significant difference in prevalence of liver dysfunction between carriers with subtypes adr and adw (Table V). Discussion
HBsAg subtypes show distinct geographical distribution. 4-8 T h e p r e d o m i n a n t HBsAg subtype is adr on the Asian continent, while adw is p r e d o m i n a n t in the Philippines, Indonesia and Taiwan. Japan is apparently at a geographical boundary of adr and adw, forming a south to north gradient in which adr is more frequent in the south and adw is more frequent in the north. 7 In Kyushu, the most southern of the four main islands, adr is present in 82.o % of the HBsAg carriers. 8 We previously reported the prevalence of subtype adw to be between 73"8 % and 76"7 % in Okinawa in a small scale survey)' 10 T h e present large scale study confirms that adw is the p r e d o m i n a n t subtype in Okinawa, with an overall prevalence of 70"7 % and p r e d o m i n a n t on all islands and in all age groups. We previously reported that the prevalence of HBeAg was significantly higher in K y u s h u (36"4 %) than in Okinawa (2o-o %), that HBeAg-positive carriers seroconverted to anti-HBe at a younger age in Okinawa than in K y u s h u 17 and that subtype adr was p r e d o m i n a n t in K y u s h u as was adw in Okinawa. 8 T h e s e results suggested that differences in the rate and age of seroconversion between K y u s h u and Okinawa reflect differences between
I48
A. NOGUCHI
ET AL.
N
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Kyushu
o
Kuroshima Hateruma
F i g . I.
subtypes adr and adw. However, there is a great deal of difference between Kyushu and Okinawa in terms of history, race and environment. T h e present study revealed that the prevalence of HBeAg was higher in carriers with adr than in those with adw, and that HBeAg seroconverted to anti-HBe at a younger age in carriers with adr than in those with adw, even though all were of the same race and were born and raised in the same area. We also reported previously that the prevalence of liver dysfunction was significantly higher in HBeAg-positive carriers than in anti-HBe-positive carriers. 18 However, the present study revealed no difference in the prevalence of liver dysfunction between the two subtypes. Liver dysfunction directly reflects the positivity of HBeAg, but not the identity of the subtype. It has been reported that adr is found more frequently than adw in chronic liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma). 12 Okinawa has the highest prevalence of HBsAg in Japan 8 yet the mortality rates for liver cirrhosis and hepatocellular carcinoma are the lowest, 19 while in contrast, in Kyushu they are the highest. As HBsAg carriers with subtype adr have HBeAg for a longer duration than do those with subtype adw, the former are prone to develop chronic liver diseases. This is one reason why there were few patients in this area with severe chronic HBV related liver disease, despite the high prevalence of HBsAg. In this area there were 18 HBsAg carriers with subtypes other than adw and adr. Although subtype ay is rare in Japan, v' 8 we found seven carriers with it.
H B s A g subtypes a n d H B e A g / a n t i - H B e
status
149
B e c a u s e t h e y h a d n e v e r left J a p a n a n d h a d n e v e r r e c e i v e d b l o o d t r a n s f u s i o n , t h e o r i g i n o f t h e i r s u b t y p e is u n c l e a r . M o r e o v e r , I5 c a r r i e r s w e r e c o m p o u n d s u b t y p e s a d y r a n d a d w r . T h e s e p r o b a b l y r e p r e s e n t m i x e d infections. O n t h e o t h e r h a n d , O k a m o t o et al. 2° f o u n d t h a t t h e d / y o r w / r s u b t y p e c h a n g e was d u e to a p o i n t m u t a t i o n in t h e S g e n e a n d c o - i n f e c t i o n o f h e p a t o c y t e s w i t h an H B V g e n o m e a n d its m u t a n t . W e i n v e s t i g a t e d c h r o n i c h e p a t i t i s B p a t i e n t s w h o r e c e i v e d i n t e r f e r o n t h e r a p y 21 a n d in w h o m t h e H B s A g s u b t y p e c h a n g e d f r o m a d r to a d w r . 11 T h e H B s A g c o m p o u n d s u b t y p e s w e r e d e t e r m i n e d a m o n g p a t i e n t s w i t h c h r o n i c liver disease. ~2 H o w e v e r , t h e r e l a t i o n s h i p o f c o m p o u n d s u b t y p e s a n d liver d y s f u n c t i o n was u n c e r t a i n , b e c a u s e t h e r e w e r e f e w c a r r i e r s w i t h this s u b t y p e in t h e p r e s e n t s t u d y . I n c o n c l u s i o n , H B s A g s u b t y p e a d w is p r e d o m i n a n t in O k i n a w a a n d c a r r i e r s o f it s e r o c o n v e r t f r o m H B e A g to a n t i - H B e at a h i g h e r rate a n d at a y o u n g e r age t h a n t h o s e w i t h s u b t y p e adr.
References I. Levene C, Blumberg BS. Additional specificities of Australia antigen and the possible identification of hepatitis carriers. Nature 1969; 22I: I95-I96. 2. Bancroft WH, Mundon FK, Russel PK. Detection of additional antigenic determinants of hepatitis B antigen. J Immunol 1972; IO9: 842-848. 3. Le Bouvier GL. The heterogeneity of Australia antigen. ,7 Infect Dis I971 ; I23 : 671-675. 4. Mazzur S, Burgert S, Blumberg BS. Geographical distribution of Australia antigen determinants d, y and w. Nature I974; 247: 38-40. 5. Feinman SV, Berris B, Sinclair JC, Wrobel DM, Alter HJ, Holland PV. Relation of hepatitis-B-antigen subtypes in symptom-free carriers to geographical origin and liver abnormalities. Lancet I973; 2:867-869. 6. Dodd RY, Holland PV, Ni LY, Smith HM, Greenwalt TJ. Hepatitis B antigen: regional variation in incidence and subtype ratio in the American Red Cross donor population. Am J Epidemiol 1973; 97: 111-115. 7- Yamashita Y, Kurashima S, Miyakawa Y, Mayumi M. South-to-north gradient in distribution of the r determinant of hepatitis B surface antigen in Japan. J Infect Dis I975; I31 : 567-569. 8. Kashiwagi S, Hayashi J, Nomura H, et al. Subtypes of hepatitis B surface antigen in familial clusters of hepatitis B virus carriers. Am J Epidemiol 1983; x 18:795-798. 9. Kashiwagi S, HayashiJ, IkematsuH, etal. An outbreak ofhepatitis B ifl members of a high school sumo wrestling club. J A M A 1982; 248: 213-214. io. Hayashi J, Noguchi A, Nakashima K, Morofuji M, Kashiwagi S. Frequency of concurrence of hepatitis B surface antigen and antibody in a large number of carriers in Okinawa, Japan. Gastroenterol Jpn 199o; 25 : 593-597I I. Usuda S, Tsuda F, Gotanda T, et al. A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies. J Immunol Methods 1986; 2o3-21o. 12. Shiina S, Fujimoto H, Uta Y, et al. Relationship of HBsAg subtypes with HBeAg/antiHBe status and chronic liver disease. Part I: Analysis of 1744 HBsAg carriers. Am J Gastroenterol 1991 ; 86 : 866-871. 13. Shiina S, Fujimoto H, Kawabe T, et al. Relationship ofHBsAg subtypes with HBeAg/antiHBe status and chronic liver disease. Part 2: Evaluation of epidemiological factors and suspected risk factors of liver dysfunction. Am J Gastroenterol 1991; 86: 872-875. 14. Kashiwagi S, Hayashi J, Ikematsu H~ et al. An epidemiologic study of hepatitis B virus in Okinawa and Kyushu, Japan. Am J Epidemiol 1983 ; 118: 787-794. 15. Hayashi J, Kashiwagi S, Nomura H, et al. Hepatitis B virus transmission in nursery schools. Am J Epidemiol 1987; 125: 492-498.
~5o
A. N O G U C H I E T A L .
I6. Noguchi A, Hayashi J, Nakashima K, Ikematsu H, Hirata M, Kashiwagi S. Decrease of hepatitis A and B virus infections in the population of Okinawa, Japan. ff Infect I99I ; 23: 255- 262. X7. Kashiwagi S, Hayashi J, Nomura H, et al. Age-specific prevalence of hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) among asymptornatic hepatitis B surface antigen (HBsAg) carriers in Okinawa and Kyushu, Japan. Microbiol Immunol 1986; 3o: 675-682. 18. Hayashi J, Kashiwagi S, Ikematsu H, et al. Sex- and age-specific prevalences of HBeAg and anti-HBe among HBsAg carriers with or without liver function abnormalities in Okinawa, Japan. Microbiol Imrnunol 1986; 32: 843-85o. 19. Sakugawa H, Ohwan T, Yamashiro A, et al. Natural seroconversion from hepatitis Be antigen to antibody among hepatitis B virus carriers in Okinawa islands. ] Med Virol I99I ; 34: I22-I26. 20. Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol I988; 69: 2575--2583 • 2I. Hayashi J, Kajiyama W, Noguchi A, et al. Glycyrrhizin withdrawal followed by human lymphoblastoid interferon in the treatment of chronic hepatitis B. Gastroenterol ]pn I99I; 26: 742-746. 22. Kobayashi M, Kumada H, Ikeda K, et al. Influence of subtype on the prognosis of chronic type B hepatitis. (Japanese) Acta Hepatol ]pn I988; 29: 468-475-
HBsAg subtypes a m o n g HBsAg carriers in Okinawa, Japan. Evidence o f an important relationship in seroconversion f r o m HBeAg to anti-HBe Akinori Noguchi, Jun Hayashi, Koya Nakashima, Miki Hirata, Hideyuki Ikematsu and Seizaburo Kashiwagi Department of General Medicine, Kyushu University Hospital, Japan Accepted for publication 2I October I993
Summary In Okinawa, Japan i26i hepatitis B surface antigen (HBsAg) carriers were investigated for clinical differences among HBsAg subtypes. Among the 854 for whom they could be determined, subtype adw was found in 6o4 (70"7 %), adr in 232 (27"2 %) and others in I8 (2.I%). The overall prevalence of hepatitis B e antigen (HBeAg) was significantly lower in subtype adw (I I'9 %) than in adr (I7"7 %) (P < o.oi). The mean age of HBeAg-positive carriers was significantly lower in adw (20"4 years) than in adr (26"9 years) (P < 0"05). Seroconversion from HBeAg to antibody to HBeAg (antiHBe) occurred in 43 carriers. The seroconversion rate was higher in adw (43.I %) than in adr (29"3 %) and the seroconversion age was younger in adw (22" 7 years) than in adr (27' 9 years). These results suggest that carriers with subtype adw tend to seroconvert from HBeAg to anti-HBe at a higher rate and a younger age than do those with adr. HBsAg subtypes may be closely associated with the HBeAg/anti-HBe status.
Introduction Hepatitis B surface antigen (HBsAg) has a common determinant called 'a '1 and two additional pairs of mutually exclusive antigenic determinants named d / y and w / r , 2' 3 resulting in the following four major subtypes: adw, ayw, adr and ayr. Geographical distribution of HBsAg subtypes and their relationship with epidemiological factors have attracted a great deal of attention. 4-s In the Japanese population, it has been found that there are two main subtypes, adr and adw. 7 We identified HBsAg subtypes to determine the route of transmission.8-1° HBsAg subtypes have recently become more easily and accurately identified since an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies against HBsAg subtypes has become available. 11 Shiina et al. 12'13 reported that HBsAg subtypes may be linked to the development of chronic liver disease, although it has been established that they correlate more directly with epidemiological factors than with disease. Since I968, we have investigated hepatitis B virus (HBV) infection in Okinawa, Japan. Infants and adults are afflicted and we found that HBV was endemic in this area. 14-16 In the present study, I26I HBsAg carriers were investigated with regard to HBsAg subtypes, hepatitis B e antigen (HBeAg), Address correspondence to: Jun Hayashi, Department of General Medicine, Kyushu University Hospital, Higashi-Ku, Fukuoka 812, Japan. oI63-4453/94/o2o14I + IO $08.00/0
© I994 The British Society for the Study of Infection
I42
A. NOGUCHI
ET AL.
antibody to HBeAg (anti-HBe) status, liver function test ( L F T ) , age and sex in order to determine if there are clinical differences between HBsAg subtypes. Materials and methods Study population Inhabitants of the Yaeyama District of Okinawa, Japan were surveyed. Okinawa is in the subtropical zone about iooo k m south of the main islands of Japan. T h e Yaeyama District is in the south-western part of Okinawa near Taiwan (Figure I). All subjects studied were of the Japanese race and were born and raised in this area. T h e r e is some genetic variation between the Okinawans and the Japanese in the main islands due to differences in historical demographics. We surveyed over 9 0 % of the estimated population of Hateruma, Iriomote, Kohama, Taketomi, and Kuroshima Islands and over 60% of the people on Ishigaki Island, for H B V markers. A total of i 8 8 2 i inhabitants were tested for HBsAg in I98o. We found IZ6I HBsAg carriers (males 748, females 5 I3, age 1-87 years), and each was followed for one year or longer between I98O and I99O. T h e y were tested for H B V markers and L F T s were performed at least twice, at an interval of 3 m o n t h s or longer. Serum samples were stored at - 2 o °C until tested. Laboratory methods HBsAg was measured by reversed passive haemagglutination ( R P H A ; Revesecell, Meguroken, Osaka, Japan) and H B e A g / a n t i - H B e by radioimmunoassay (RIA; HBe RIA, Abbott Laboratories, I L , U.S.A.). HBsAg subtypes were determined by E L I S A , by means of monoclonal antibodies (HBsAg Subtype EIA, Institute of I m m u n o l o g y , Tokyo, Japan). 17 We dispensed 5o #1 each of the serum samples and control sera to wells which were coated with monoclonal antibody against the c o m m o n determinant ' a ' of HBsAg and the microplate was incubated at 37 °C for 3 h. We then dispensed 50 #1 labelled monoclonal antibody against determinants ' d ' , ' y', ' w ' , o r ' r' to corresponding wells and the microplate was incubated at 37 °C for z h. T h e reaction was terminated by adding 50 #1 of 4 N H2SO4, and the intensity of developed colour was determined by the absorption at 492 nm. Absorption was measured within io m i n of stopping the colouring reaction. HBsAg subtypes could not be determined in 4o7 HBsAg carriers because of their low HBsAg titres (under 28 R P H A ) and they were excluded from analysis. T h e r e remained 854 HBsAg carriers (males 522, females 332, age 2-88 years) whose subtypes were available for analysis. T h e period of the observation was 6-6 ___2. 7 years. Conventional L F T s were performed by use of multiple autoanalyser. T h e presence of liver dysfunction was defined as an elevated serum alanine aminotransferase (above 40 IU/1). Statistical analysis Chi-square, Mantel-Haenszel and Student's t-tests were used to determine statistical significance. Results T h e distribution of HBsAg subtypes among carriers in the islands of Yaeyama District of Okinawa, Japan, in w h o m subtypes could be determined were adw
HBsAg subtypes and HBeAg/anti-HBe status Table I
I43
Geographical distribution of HBsAg subtypes among HBsAg carriers in the Yaeyama District of Okinawa, Japan, I98O HBsAg subtype
Island Ishigaki Iriomote Kohama Kuroshima Taketomi Hateruma Total
Number of carriers 728 74 24 5 5 18 854
Number adw (%)
Number adr (%)
523 (71.8) i92 (26-4) 44 (59'5) 28 (37"8) 14 (58'3) 9 (37"5) 5 (Ioo) o 4 (80) I (20) 14 (77"8) 2 (II.i) 604 (7o'7) 232 (27"2)
Number Number Number ayw ayr adyr (%) (%) (%) I (0"I) o o o o o I (o.I)
I (O'I)
I (i.4) o o o o 2 (0.2)
3 (0'4) I (I'4) o o o o 4 (o'5)
Number adwr (%) 8 (I'I) o I (4"2) o o 2 (ii.i) II (1"3)
HBsAg, hepatitis B surface antigen. Table II
Age-specific distribution of HBsAg subtypes among HBsAg carriers in the Yaeyama district of Okinawa, Japan, I 9 8 0 HBsAg subtype
Age (years)
Number of carriers
Number adw (%)
Number adr (%)
Number ayw (%)
Number ayr (%)
Number adyr (%)
Number adwr (%)
o-9 IO--I9 2o-29 30-39 4o-49 5° Total
33 ii2 14o 268 lO8 193 854
26 (78"8) 81 (72'3) 99 (7o'7) I9O (70'9) 70 (64'8) 138 (71"5) 604 (7o'7)
6 (I8-2) 3o (26.8) 37 (26"4) 72 (26'9) 36 (33'3) 51 (26'4) 232 (27'2)
o o I (o'7) o o o I (o'I)
o 1 (0'9) o o o I (0"5) 2 (0'2)
o o e (1-4) 1 (0'4) o I (0'5) 4 (o'5)
I (3"0) o
I (0'7) 5 (1'9) 2 (I'9) 2 (I'O) IX (I'3)
HBsAg, hepatitis B surface antigen. 604 (70"7 % ) , a d r 232 (27.2 % ) , a y w I ( o ' 1 % ) , a y r 2 (0"2 ~/o), a d y r 4 (0"5 % ) a n d a d w r 11 (I" 3 ~/o). T h e p r e v a l e n c e o f s u b t y p e a d w was significantly h i g h e r t h a n t h a t o f t h e o t h e r s ( P < o'ooz). T h e p r e v a l e n c e o f s u b t y p e a d w was h i g h e s t o n t h e islands, a l t h o u g h o n I r i o m o t e a n d K o h a m a its rate was less t h a n 6o % a n d t h a t o f a d r was m o r e t h a n 35 % ( T a b l e I). T h e overall rate o f s u b t y p e a d w was m o r e t h a n 7 ° % a n d a d r less t h a n 30 % in each age g r o u p , e x c e p t for the 40--49 y e a r g r o u p . T h e c o m p o u n d s u b t y p e s ( a d y r a n d a d w r ) i n c r e a s e d w i t h a d v a n c i n g age ( T a b l e II). I n m a l e s , s u b t y p e a d w was f o u n d in 366 ( 7 o ' 1 % ) , a d r in 148 (28"4 % ) , a y r in I (0"2 ~/o) a n d a d w r in 7 (1"3 % ) a n d in females, a d w was f o u n d in 238 ( 7 I ' 7 % ) , a d r in 84 (25"3 % ) , a y w in z (o'3 % ) , ayr in (o'3 % ) , a d y r in 4 (1'2 % ) a n d a d w r in 4 (1"2 % ) . N o significant d i f f e r e n c e s b y sex or age w e r e o b s e r v e d . O f t h e 854 H B s A g carriers, t h e p r e v a l e n c e o f H B e A g was 115 ( 1 3 5 % ) a n d t h a t o f a n t i - H B e was 714 (83"6 % ) ; t h e r e m a i n i n g 25 (2"9 % ) w e r e n e g a t i v e for
A. NOGUCHI ET AL.
144
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HBsAg subtypes and HBeAg/anti-HBe status
I47
both. HBeAg and anti-HBe were not simultaneously present in any of the samples tested. T h e prevalence of HBeAg was z4"9 % in males and IZ'1% in females. T h e r e was no significant difference between the sexes. Age and HBsAg subtype distribution of HBeAg among H B V carriers is shown in Table III. T h e prevalence of HBeAg decreased with advancing age. Its prevalence was I7"7 % in 232 carriers with subtype adr, a value significantly higher than that in the 604 carriers with subtype adw (I I'9 %, P < o'o5). T h e prevalence of HBeAg in carriers with subtype adr was clearly higher than in those with subtype adw who were more than IO years old. In the 4o-49 year age group, the prevalence of HBeAg was significantly higher in carriers with subtype adr (z6"7 %) than in those with subtype adw (o %) (P < o.oI). Moreover, the mean age of HBeAg-positive carriers with subtype adw was 20"4+I5"2, an age significantly younger than those with subtype adr (26"9__+z5"8, P < 0"05). Of the I 15 HBsAg carriers with HBeAg, 43 (37"4 %) seroconverted to antiHBe during the observation period. Of 43 carriers, 23 seroconverted to antiHBe within 4 years of starting the investigation, z 7 within 5-9 years and three within zo-I I years. T h e rate of seroconversion was higher in females (44"2 %) than in males (33"3 %) and the age of seroconversion was younger in females (22" 4 -]- I3"8 years) than in males (25"z _ 18"5 years). These differences however were not statistically significant. T h e rate of seroconversion was higher and occurred at a younger age in carriers with subtype adw (43"I %, 22"7__+i6.6 years) than in those with subtype adr (29"3 %, 27"9__+I5"8 years). However, neither difference was statistically significant (Table IV). T h e prevalence of liver dysfunction in 854 HBsAg carriers was significantly higher in HBeAg-positive carriers (33"0 %) than in HBeAg-negative carriers (9"z %): a d w + 3 I ' 9 % , a d w - 9 " 6 % ; a d r + 3 4 " 1 % , a d r - 8 " 4 % . T h e r e was no statistically significant difference in prevalence of liver dysfunction between carriers with subtypes adr and adw (Table V). Discussion
HBsAg subtypes show distinct geographical distribution. 4-8 T h e p r e d o m i n a n t HBsAg subtype is adr on the Asian continent, while adw is p r e d o m i n a n t in the Philippines, Indonesia and Taiwan. Japan is apparently at a geographical boundary of adr and adw, forming a south to north gradient in which adr is more frequent in the south and adw is more frequent in the north. 7 In Kyushu, the most southern of the four main islands, adr is present in 82.o % of the HBsAg carriers. 8 We previously reported the prevalence of subtype adw to be between 73"8 % and 76"7 % in Okinawa in a small scale survey)' 10 T h e present large scale study confirms that adw is the p r e d o m i n a n t subtype in Okinawa, with an overall prevalence of 70"7 % and p r e d o m i n a n t on all islands and in all age groups. We previously reported that the prevalence of HBeAg was significantly higher in K y u s h u (36"4 %) than in Okinawa (2o-o %), that HBeAg-positive carriers seroconverted to anti-HBe at a younger age in Okinawa than in K y u s h u 17 and that subtype adr was p r e d o m i n a n t in K y u s h u as was adw in Okinawa. 8 T h e s e results suggested that differences in the rate and age of seroconversion between K y u s h u and Okinawa reflect differences between
I48
A. NOGUCHI
ET AL.
N
• oo
Kyushu
o
Kuroshima Hateruma
F i g . I.
subtypes adr and adw. However, there is a great deal of difference between Kyushu and Okinawa in terms of history, race and environment. T h e present study revealed that the prevalence of HBeAg was higher in carriers with adr than in those with adw, and that HBeAg seroconverted to anti-HBe at a younger age in carriers with adr than in those with adw, even though all were of the same race and were born and raised in the same area. We also reported previously that the prevalence of liver dysfunction was significantly higher in HBeAg-positive carriers than in anti-HBe-positive carriers. 18 However, the present study revealed no difference in the prevalence of liver dysfunction between the two subtypes. Liver dysfunction directly reflects the positivity of HBeAg, but not the identity of the subtype. It has been reported that adr is found more frequently than adw in chronic liver disease (chronic hepatitis, liver cirrhosis and hepatocellular carcinoma). 12 Okinawa has the highest prevalence of HBsAg in Japan 8 yet the mortality rates for liver cirrhosis and hepatocellular carcinoma are the lowest, 19 while in contrast, in Kyushu they are the highest. As HBsAg carriers with subtype adr have HBeAg for a longer duration than do those with subtype adw, the former are prone to develop chronic liver diseases. This is one reason why there were few patients in this area with severe chronic HBV related liver disease, despite the high prevalence of HBsAg. In this area there were 18 HBsAg carriers with subtypes other than adw and adr. Although subtype ay is rare in Japan, v' 8 we found seven carriers with it.
H B s A g subtypes a n d H B e A g / a n t i - H B e
status
149
B e c a u s e t h e y h a d n e v e r left J a p a n a n d h a d n e v e r r e c e i v e d b l o o d t r a n s f u s i o n , t h e o r i g i n o f t h e i r s u b t y p e is u n c l e a r . M o r e o v e r , I5 c a r r i e r s w e r e c o m p o u n d s u b t y p e s a d y r a n d a d w r . T h e s e p r o b a b l y r e p r e s e n t m i x e d infections. O n t h e o t h e r h a n d , O k a m o t o et al. 2° f o u n d t h a t t h e d / y o r w / r s u b t y p e c h a n g e was d u e to a p o i n t m u t a t i o n in t h e S g e n e a n d c o - i n f e c t i o n o f h e p a t o c y t e s w i t h an H B V g e n o m e a n d its m u t a n t . W e i n v e s t i g a t e d c h r o n i c h e p a t i t i s B p a t i e n t s w h o r e c e i v e d i n t e r f e r o n t h e r a p y 21 a n d in w h o m t h e H B s A g s u b t y p e c h a n g e d f r o m a d r to a d w r . 11 T h e H B s A g c o m p o u n d s u b t y p e s w e r e d e t e r m i n e d a m o n g p a t i e n t s w i t h c h r o n i c liver disease. ~2 H o w e v e r , t h e r e l a t i o n s h i p o f c o m p o u n d s u b t y p e s a n d liver d y s f u n c t i o n was u n c e r t a i n , b e c a u s e t h e r e w e r e f e w c a r r i e r s w i t h this s u b t y p e in t h e p r e s e n t s t u d y . I n c o n c l u s i o n , H B s A g s u b t y p e a d w is p r e d o m i n a n t in O k i n a w a a n d c a r r i e r s o f it s e r o c o n v e r t f r o m H B e A g to a n t i - H B e at a h i g h e r rate a n d at a y o u n g e r age t h a n t h o s e w i t h s u b t y p e adr.
References I. Levene C, Blumberg BS. Additional specificities of Australia antigen and the possible identification of hepatitis carriers. Nature 1969; 22I: I95-I96. 2. Bancroft WH, Mundon FK, Russel PK. Detection of additional antigenic determinants of hepatitis B antigen. J Immunol 1972; IO9: 842-848. 3. Le Bouvier GL. The heterogeneity of Australia antigen. ,7 Infect Dis I971 ; I23 : 671-675. 4. Mazzur S, Burgert S, Blumberg BS. Geographical distribution of Australia antigen determinants d, y and w. Nature I974; 247: 38-40. 5. Feinman SV, Berris B, Sinclair JC, Wrobel DM, Alter HJ, Holland PV. Relation of hepatitis-B-antigen subtypes in symptom-free carriers to geographical origin and liver abnormalities. Lancet I973; 2:867-869. 6. Dodd RY, Holland PV, Ni LY, Smith HM, Greenwalt TJ. Hepatitis B antigen: regional variation in incidence and subtype ratio in the American Red Cross donor population. Am J Epidemiol 1973; 97: 111-115. 7- Yamashita Y, Kurashima S, Miyakawa Y, Mayumi M. South-to-north gradient in distribution of the r determinant of hepatitis B surface antigen in Japan. J Infect Dis I975; I31 : 567-569. 8. Kashiwagi S, Hayashi J, Nomura H, et al. Subtypes of hepatitis B surface antigen in familial clusters of hepatitis B virus carriers. Am J Epidemiol 1983; x 18:795-798. 9. Kashiwagi S, HayashiJ, IkematsuH, etal. An outbreak ofhepatitis B ifl members of a high school sumo wrestling club. J A M A 1982; 248: 213-214. io. Hayashi J, Noguchi A, Nakashima K, Morofuji M, Kashiwagi S. Frequency of concurrence of hepatitis B surface antigen and antibody in a large number of carriers in Okinawa, Japan. Gastroenterol Jpn 199o; 25 : 593-597I I. Usuda S, Tsuda F, Gotanda T, et al. A solid-phase enzyme immunoassay for the common and subtypic determinants of hepatitis B surface antigen with monoclonal antibodies. J Immunol Methods 1986; 2o3-21o. 12. Shiina S, Fujimoto H, Uta Y, et al. Relationship of HBsAg subtypes with HBeAg/antiHBe status and chronic liver disease. Part I: Analysis of 1744 HBsAg carriers. Am J Gastroenterol 1991 ; 86 : 866-871. 13. Shiina S, Fujimoto H, Kawabe T, et al. Relationship ofHBsAg subtypes with HBeAg/antiHBe status and chronic liver disease. Part 2: Evaluation of epidemiological factors and suspected risk factors of liver dysfunction. Am J Gastroenterol 1991; 86: 872-875. 14. Kashiwagi S, Hayashi J, Ikematsu H~ et al. An epidemiologic study of hepatitis B virus in Okinawa and Kyushu, Japan. Am J Epidemiol 1983 ; 118: 787-794. 15. Hayashi J, Kashiwagi S, Nomura H, et al. Hepatitis B virus transmission in nursery schools. Am J Epidemiol 1987; 125: 492-498.
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I6. Noguchi A, Hayashi J, Nakashima K, Ikematsu H, Hirata M, Kashiwagi S. Decrease of hepatitis A and B virus infections in the population of Okinawa, Japan. ff Infect I99I ; 23: 255- 262. X7. Kashiwagi S, Hayashi J, Nomura H, et al. Age-specific prevalence of hepatitis B e antigen (HBeAg) and antibody to HBeAg (anti-HBe) among asymptornatic hepatitis B surface antigen (HBsAg) carriers in Okinawa and Kyushu, Japan. Microbiol Immunol 1986; 3o: 675-682. 18. Hayashi J, Kashiwagi S, Ikematsu H, et al. Sex- and age-specific prevalences of HBeAg and anti-HBe among HBsAg carriers with or without liver function abnormalities in Okinawa, Japan. Microbiol Imrnunol 1986; 32: 843-85o. 19. Sakugawa H, Ohwan T, Yamashiro A, et al. Natural seroconversion from hepatitis Be antigen to antibody among hepatitis B virus carriers in Okinawa islands. ] Med Virol I99I ; 34: I22-I26. 20. Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis B virus by homology in nucleotide sequence: comparison of surface antigen subtypes. J Gen Virol I988; 69: 2575--2583 • 2I. Hayashi J, Kajiyama W, Noguchi A, et al. Glycyrrhizin withdrawal followed by human lymphoblastoid interferon in the treatment of chronic hepatitis B. Gastroenterol ]pn I99I; 26: 742-746. 22. Kobayashi M, Kumada H, Ikeda K, et al. Influence of subtype on the prognosis of chronic type B hepatitis. (Japanese) Acta Hepatol ]pn I988; 29: 468-475-