THE JOURNAL OF INFECTIOUS DISEASES VOL. 134, NO.3 SEPTEMBER 1976 1976 by the University f Chicag. All rights reserved. Radiimmunassay fr Antibdy t Hepatitis B Surface Antigen Shws Transmissin f Hepatitis B Virus amng Husehld Cntacts c. J. Peters,* R. H. PureeD, J. J. Lander, t and K. M. Jhnsn Frm the Middle America Research Unit, Balba Heights, Canal Zne; and the Labratry f Infectius Diseases, Natinal Institute f Allergy and Infectius Diseases, Natinal Institutes f Health, Bethesda, Maryland The epidemilgy f infectins with hepatitis B virus was studied by measurement f serum antibdies t hepatitis B surface antigen ( Ag) with the radiimmunprecipitatin test. Prspective studies f husehld cntacts f Ag-psitive patients with acute hepatitis demnstrated that fur f 41 children and ne f 60 adults experienced anicteric sercnversins. Tw f the 60 adults als had mild icteric hepatitis and became chrnically antigenemic. Neither Ag nr antibdy t Ag was detected in fllw-up bld samples frm 8 sernegative cntacts f Ag-negative patients with hepatitis, althugh seven icteric secndary cases were identified. Husehld cntacts f Ag carriers frequently had serlgic evidence f expsure t hepatitis B virus (44% f adults), but the remainder had neither detectable antigen nr antibdy. Hepatitis B surface antigen ( Ag) has prvided a useful marker f infectin with ne strain f human hepatitis virus, even thugh the exact relatin between viremia and antigenemia is still unclear. The presence f Ag in acute-phase sera frm ne-quarter f the adults hspitalized with spradic hepatitis in Panama City, Panama [1] demnstrated active transmissin f hepatitis B virus (HBV), but in many cases it was nt pssible t implicate husehld carriers [1], cntact with anther patient with hepatitis, r recgnized parenteral expsure as a surce f infectin. 1 Received fr publicatin September, 197, and in revised frm January 19, 1976. This paper is publicatin n. 1036 frm the Department f Immunpathlgy, Scripps Clinic and Research Fundatin, La Jlla, Califrnia. Please address requests fr reprints t Dr. R. H. Purcell, Labratry f Infectius Diseases, Natinal Institute f Allergy and Infectius Diseases, Natinal Institutes f Health, Bethesda, Maryland 0014. * Present address: Scripps Clinic and Research Fundatin, La Jlla, Califrnia. t Present address: Washingtn University Medical Schl, Department f Medicine, Barnes Hspital Plaza, St. Luis, Missuri. 1 Fifty-eight cnsecutive Ag-psitive patients [1] were queried cncerning expsure t jaundiced persns r injectins in the previus six mnths, and 16 (8%) denied either (C. J. Peters, unpublished bservatins). In an attempt t understand the mechanisms f HBV transmissin in the cmmunity, we studied husehld cntacts f patients with hepatitis and f carriers f Ag with use f the sensitive radiimmunprecipitatin test fr detectin f antibdy t Ag (anti-hb,'). Materials and Methds Technical prcedures. Sera were separated frm bld clts within 48 hr f cllectin and were stred in cded vials at - 0 C. All sera were tested fr Ag by cunterelectrphresis [] and by CF [3] at the Middle America Research Unit. Radiimmunprecipitatin tests fr anti were perfrmed in the Labratry f Infectius Diseases, Natinal Institute f Allergy and Infectius Diseases [4]. Sera were rutinely tested at a 1: 4 dilutin with gat antiserum t human y-glbulin as a precipitating reagent. Sera with binding f ~ 1% were cnsidered psitive if results were cnfirmed in repeat tests. Separate aliquts frm all apparent sercnversins were retested and were accepted as valid if the first sample had less than 10% binding and the secnd had ~1%. Drs. Harvey Alter and Paul Hlland (Bld Bank, Clinical Center, Natinal Institutes f Health, Bethesda, Md.) perfrmed the radiim- 18
Transmissin f Hepatitis B Virus 19 munassay fr antigen using the Ausria (Abbtt Labratries, Nrth Chicag, Ill. ) system and determined the subtype f the antigens by cunterelectrphresis using absrbed human r guinea pig reagents []. We measured serum aspartate amintransferase by a clrimetric methd (Sigma Chemical C., St. Luis, M.) using a cmmercial lyphilized standard (Enzatrl, Dade Labratries, Miami, Fla.). Case selectin. Between September 1969 and September 1971, cases f acute hepatitis were sught by regular visits t the majr hspitals in Panama [1]. During the early mnths f the study, we attempted t demnstrate transmissin f HBV by sampling asymptmatic cntacts f patients and searching fr Ag by CF testing r fr liver damage by determining levels f amintransferase in serum. When these methds failed, we began t examine fllw-up sera frm cntacts fr antibdy cnversin. Husehlds were chsen fr study n the basis f ease f access, willingness t cperate, and availability f the investigatr's time. A "cntact" was a persn wh slept r ate in the same huse as the index patient n the majrity f days; 91 % f the cntacts were permanent residents f the same huse as an index patient. The study included 1 index subjects; amng them were patients with acute Ag-psitive and Ag-negative hepatitis as well as carriers f Ag. Thirty-six subjects were males; grups represented were Negr (14 subjects), Mestiz (36), and Caucasian (ne). Subjects resided in Panama City (44) r elsewhere in Panama (five), and tw were frm the Canal Zne. Thirty families had purified water and mdern tilet facilities in their hmes. The characteristics f the tw grups f patients with hepatitis and the carriers f Ag were similar (table 1). RB s Ag-psitive patients and their cntacts. Ag was detectable in acute sera frm all 0 index patients by CF tests and cuntere1ectrphresis. The index patients had been sick fr five t 37 days befre the initial sample was taken (median, 14 days). Fur patients had Ag f subtype ay, and 16 had antigen f subtype ad. Tw cases f hepatitis with antigen f subtype ad and tw with subtype ay fllwed bld transfusin, but the remainder f the cases were spradic. All index patients were adults. Eight f Table 1. Characteristics f the index persns wh were included in the study. Characteristic Age <1 years ~1 years Sex Male Female Ethnic grup Negr Mestiz Caucasian Residence Panama City 19 Interir f Panama 1 Canal Zne 0 Tilet Mdern, individual 13 Mdern, shared 4 Latrine 3 Water supply City water, individual 14 City water, shared with ther families 4 Well water N. f subjects with indicated characteristic Ag-psitive hepatitis 0 1 7 13 Ag carrier 11 Ag-negative hepatitis 8 10 the 1 men were heads f husehlds, and all five wmen were married t a head f husehld. The remaining seven men were sns r nephews f a head f husehld. We tested paired sera frm 107 cntacts f these 0 index patients. The first sample frm the cntacts was btained within tw mnths f the nset f disease in the index patient, with the exceptin f seven patients wh were sampled within three mnths and wh were all initially negative fr antibdy. The secnd sample was btained three t five mnths after the nset f the disease in the index patient in 34 subjects (median, fur mnths) and six mnths r lnger after nset in 73. The interval between samples was three t five mnths in 8 persns (median, fur mnths) and six mnths r lnger in 49. RB s Ag carriers and their cntacts. Agpsitive subjects were lcated by tests f sera frm asymptmatic persns, principally bld 11 4 9 10 1 7 4 7 6 10 8 3 14 1 1 1 8 9 4
0 Peters et al. dnrs. Twelve subjects with Ag f subtype ad and ne with subtype ay were tested again tw t 1 mnths later and were still antigenemic, with n change in titer and n symptms f acute hepatitis. In n case was there any indicatin f when the persn had first becme antigenemic. Bld was btained frm cntacts within six weeks f identificatin f the index persn. RB s Ag-negative patients with hepatitis and their cntacts. A patient with hepatitis was cnsidered "negative" if acute serum was nnreactive in cunterelectrphresis, CF, and radiimmunassay tests fr Ag and if a cnvalescent sample btained at least tw mnths later did nt have significant Ag binding in the radiimmunprecipitatin test. The 18 index patients had been ill tw t days (median, 10 days) befre they were first tested. Nne f the infectins fllwed bld transfusin. Ten patients were adults (~1 years f age), and eight were children. Paired sera were available frm 90 cntacts f the 18 patients. All cntacts were initially sampled within tw mnths f nset f illness in the index patient. Fllw-up sera were btained within three t five mnths f the first sample in 40 cntacts and after six mnths r mre in 0 cntacts. Results Cntacts f RR s Ag-psitive patients. Six persns had antibdy in their initial sample and in subsequent sera. Amng the 101 sernegative cntacts, five sercnversins and tw antigenemic infectins were detected in five families (tables and 3). Ag was nt detected by CF tests r radiimmunassay in any f the sera frm cntacts wh sercnverted. Levels f serum aspartate amintransferase were als nrmal. The actual percentage f radilabeled Ag precipitated varied between - 3 and +7 in initial specimens f serum (mean, 3%) and between 1 and 68 (mean, 46 %) in later samples (table 4). There was cnsiderable variatin in the duplicate determinatins, but the tests were perfrmed under cde n different days, ften with different lts f radiactive antigen. In n case was there disagreement in the classificatin f the serum as "psitive" r "negative." Fur sercnversins ccurred in yung bys; these subjects were all asymptmatic. Bth antigenemic infectins were in adults, were assciated with mild cases f hepatitis, and resulted in chrnic antigenemia. One f the symptmatic adults and the nly adult wh experienced subclinical sercnversin were spuses f index patients. Eight ther spuses were initially ser- Table. Evidence f infectin with hepatitis B virus in cntacts f patients with hepatitis and carriers f hepatitis B surface antigen ( Ag). Initial test Final test Ttal Ttal Index case categry, Anti- infected Anti- infected N. f new age f cntacts (n.) * HBsAgt (%) HBgAg (%) infectins Acute hepatitis, Ag-psitive Child* (43) 0 () 6 0 6(14) 4 Adult (64) 4 0 4(6) 7(11) 3 Acute hepatitis, HBg Ag-negative Child-(0) 1 0 1() 1 0 1() 0 Adult (40) 4 0 4(10) 4 0 4(10) 0 Carrier f Ag Child (43) 8 3 11(3) Adult (39) 11 6 17(44) * Antibdy t hepatitis B surface antigen (anti-hbg) detected by radiimmunassay. t Ag detected by CF test. *Less than 1 years f age.
Transmissin f Hepatitis B Virus 1 Table 3. Evidence f infectin with hepatitis B virus in sernegative cntacts f hepatitis B antigenpsitive patients with acute hepatitis. Age (years) 0-4 -9 10-14 1-19 0-9 30-39 40+ N. with evidence f infectin/ n. tested Males Females /7* 0/1 /6* 0/6 0/7 0/3 0/6 0/8 l/6t 0/1 0/8 1/7* l/6t 0/7 Ttal 6/46 1/ * Anicteric sercnversin. t Antigenemic hepatitis. negative and remained s n fllw-up study. In five cases nt included in this study, we tested a single sample btained mre than six mnths after the nset f acute hepatitis in the spuse; three samples had detectable antibdy. In this small study, we culd nt crrelate the frequency f secndary infectins with antigen subtype, rute f infectin, age, race, r sex f the index patient. Transmissin ccurred at apprximately the same rate regardless f size f family, sciecnmic class, water surce, methd f waste dispsal, r density f habitatin. Cntacts f Ag carriers. Bth antibdy t Ag and chrnic antigenemia ccurred with a relatively high prevalence amng cntacts f carriers; hwever, a substantial fractin had n evidence f infectin (table ). Only tw f the nine spuses tested had serum antibdy, and nne were antigenemic. Table 4. Percentage f radilabeled hepatitis B antigen precipitated by serum samples frm sercnverters. Time interval Initial sample * (weeks) Last sample* 0 (0, 1) t 41 68 (61, 7)t (0,3) 14 61 (47,7) (-3,6) 16 44 (34,) 7 (7,7) 14 34 (33,34) (0,) 14 1 (0,) * Average percentage binding when tested with radilabeled hepatitis B antigen. t Values frm tw different tests. Cntacts f Ag-negative patients with hepatitis. Anti-Hfl, was detected in initial sera frm five f 90 cntacts. There was n change in the antibdy status f the 90 persns, althugh seven f them develped secndary icteric hepatitis; all were negative fr Ag. Discussin The develpment f a radiimmunprecipitatin technique fr measurement f antibdy t Ag has permitted us t study patterns f HBV infectins in family cntacts f persns acutely r chrnically antigenemic withut reliance n the detectin f transient antigenemia by blind sampling r the develpment f clinical illness. The specificity f the radiimmunprecipitatin test in detectin f secndary infectins was supprted by the absence f sercnversins in cntacts f Ag-negative subjects, including seven persns with clinical and labratry evidence f hepatitis. Althugh the test is exceedingly sensitive in cmparisn with ther available methds, we knw relatively little abut ccurrence r persistence f antibdy after icteric r subclinical infectins. In ur experience with 37 antigenemic patients with icteric hepatitis frm Panama, nly 81% develped antibdy after the disappearance f Ag frm serum (C. J. Peters and R. H. Purcell, unpublished bservatins). Others have fund a similar rate f sercnversin amng institutinalized children (S. Krugman, persnal cmmunicatin) and adult vlunteers experimentally infected with HBV [6]. Fur f 41 children in cntact with patients with acute Ag-psitive hepatitis develped antibdy t Ag, but n hepatitis-like illness was recgnized by the families. The index patient was always a family member and resident f the husehld (parent, brther, r cusin). The frequency f infectins was smewhat higher in bys than in girls. Three f 60 adult cntacts had evidence f infectin. One f the three develped anti-hfl, withut clinical illness. Tw had mild cases f icteric hepatitis; Ag was detectable in sera f bth, and bth subsequently became chrnic carriers. The pattern f transmissin between adults was distinctive. Tw f the secndary infectins ccurred in spuses, a finding that cnfirmed
Peters et al. impressins based n bservatins f icteric cases [8]. Whether the key factr is sexual relatins r anther f the intimacies f cnjugal life is nt clear. Only tw f 10 prspectively studied sexual partners were actually infected. A grup f 34 prstitutes studied in Panama City had a smewhat higher frequency f antibdy (31 %) than an age-matched grup f nuns (17 % ), but the differences were nt s great as t suggest that sexual experience was the sle factr perative (C. J. Peters and R. H. Purcell, unpublished bservatins). Once the index patient was infected, transmissin seemed t ccur with similar frequency regardless f sciecnmic status r hygienic cnditins. This type f mdel fr intrafamilial spread f HBV is supprted by the clustering f subtypes amng immigrants [9, 10]. Hwever, studies f antibdy in several ppulatins have indicated that envirnmental influences are imprtant in mdifying the frequency f infectin [11, 1]. This apparent cntradictin may well be due t the small size f the study r t sme artifact f case selectin. In additin, there is n reasn t expect that a single mde f transmissin will predminate in all eclgical settings. We had n systematic survey f the prevalence f anti-hls, in different sciecnmic grups living within Panama City fr cmparisn. Hwever, fur (16%) f sera frm randmly chsen, adult male, vlunteer bld dnrs at a hspital serving all strata f sciety (Hspital Sant Tmas) were psitive. This ppulatin f dnrs has a prevalence f Ag f 1.3% [1]. This infectin rate was similar t that bserved amng adult cntacts f patients with Agpsitive hepatitis sn after the nset f disease and amng adult cntacts f Ag-negative patients (table ). Fr this reasn, and because the different categries f index patients were similar with respect t imprtant epidemilgical variables, the data in table prvided an estimate f cmparative rates f infectin. The lw prevalence f anti-hls, in the initial sera frm cntacts f patients with hepatitis B suggested that transmissin was infrequent befre cntacts entered the study. Subsequent sera frm cntacts f patients psitive fr Ag had a higher frequency f antibdy, and cntacts f carriers had still mre evidence f infectin. Fewer than half f the cntacts f carriers had detectable Ag r anti-hfs., Similar results were reprted by Szmuness et al. [10], wh used passive hemagglutinatin t detect antibdy. We fund n evidence fr parenteral expsure, althugh subtle breaches f the skin barrier culd have ccurred. In this cntext, minute abrasins during sexual intercurse might be imprtant in explaining the spread between spuses. Msquites and ther hematphagus arthrpds are present in Panama City, but we think it is unlikely that hematphagus arthrpds were imprtant in the spread f HBV in these families. Neutralizing antibdies t several insect-brne viruses knwn t be present in Panama have a lw prevalence amng residents f Panama City. Furthermre, malaria r arbviral disease wuld be distinctly unusual in the specific lcatins f six f the seven secndary infectins. The pattern f secndary infectins is incmpatible with bilgical transmissin invlving an extrinsic incubatin perid, althugh presumably mechanical transmissin with interrupted feeding wuld be pssible. Thus, persn-t-persn spread f HBV infectin t cntacts f patients with acute hepatitis B is relatively cmmn. In this study the infectin had rarely been identified as "hepatitis," particularly in children. References 1. Peters, C. J., Reeves, W. C., Rivera, V., Jhnsn, K. M. Epidemilgy f hepatitis B antigen in Panama. Am. J. Epidemil, 98:301-310, 1973.. Alter, H. J., Hlland, P. V., Purcell, R. H. Cunterelectrphresis fr detectin f hepatitis-assciated antigen: methdlgy and cmparisn with gel diffusin and cmplement fixatin. J. Lab. CHn. Med. 77: 100D-1010, 1971. 3. Purcell, R. H., Hlland, P. V., Walsh, J. H., Wng, D. C., Mrrw, A. G., Chanck, R. M. A cmplement-fixatin test fr measuring Australia antigen and antibdy. J. Infect. Dis. 10:383-386, 1969. 4. Lander, J. J., Alter, H. J., Purcell, R. H. Frequency f antibdy t hepatitis-assciated antigen as measured by a new radiimmunassay technique. J. Immunl. 106: 1166-1171, 197.. Hlland, P. V., Purcell, R. H., Smith, H., Alter, H. J. Subtyping f hepatitis-assciated antigen (HB Ag) : simplified technique with cunterelectrphresis. J. Immunl, 109:40-4, 197.
Transmissin f Hepatitis B Virus 3 6. Barker, L. F., Petersn, M. R., Shulman, N. R., Murray, R. Antibdy respnses in viral hepatitis, type B. J.A.M.A. 3: 100-1008, 1973. 7. Cherubin, C. E., Purcell, R. H., Lander, J. J., McGinn, T. G., Cne, L. A. Acquisitin f antibdy t hepatitis B antigen in three sciecnmically different medical ppulatins. Lancet : 149-11, 197. 8. Szmuness, W., Much, M. I., Prince, A. M., Hfnagle, J. H., Cherubin, C. E., Harley, E. J., Blck, G. H. On the rle f sexual behavir in the spread f hepatitis B infectin. Ann. Intern. Med. 83: 489-49, 197. 9. Feinman, S. V., Berris, B., Sinclair, J. C., Wrbel, D. M., Alter, H. J., Hlland, P. V. Relatin f hepatitis-b-antigen subtypes in symptm-free carriers t gegraphical rigin and liver abnrmalities. Lancet :867-869, 1973. 10. Szmuness, W., Prince, A. M., Hirsch, R. L., Brtman, B. Familial clustering f hepatitis B infectin. N. Eng!. J. Med. 89: 116-1166, 1973. 11. Reeves, W. C., Peters, C. J., Lander, J. J., Purcell, R. H., Jhnsn, K. M. Differences in the prevalence f hepatitis B antigen and antibdy amng Panamanian Indian ppulatins. J. Infect. Dis. 18:6-70, 1973. 1. Bar-Shany, S., Edwards, V. M., Msley, J. W., Bancrft, W. H. Subtypes f hepatitis B antigen amng Israeli bld dnrs. Vx Sang. : 10 11, 1973.