Detection of Hepatitis B Surface Antigen in Patients with Acute Viral Hepatitis* Noreen I. Lehmann'i and I. D. Gust:
From the Fairfield Hospital for Communicable Diseases, Victoria
Summary: Detection of hepatitis B surface antigen in patients w i t h acute viral hepatitis. N I . Lehrnann and I . D Gust, Aust N Z J M e d . 1976. 6. pp. 7-9.
The relative value of solid phase radioimmunoassay and counter immunoelectrophoresis for the detection of hepatitis B surface antigen in the serum, was studied in 1401 patients with acute viral hepatitis. When specimens were obtained early in the illness, counter immunoelectrophoresis was found to be a reliable test for the diagnosis of hepatitis B, detecting 95.6% of patients found to be antigen positive by radioimm un oassay . The additional sensitivity of ra d i o i m m u no assay was of particular value for detecting antigen later in the illness and in patients with post-transfusion hepatitis in which the titre was frequently below the level detectable by counter immunoelectrophoresis. Radioimmunoassay was also of value in testing sera which had been stored for many years, detecting up to 3 . 3 times as many patients with hepatitis B as did counter immunoelectrophoresis.
Radioimmunoassay (RIA) is the most sensitive method currently available for detecting hepatitis B surface antigen (HB,Ag).' Whilst many studies have denionstrated the value of RIA for screening blood donors', ', less attention had been paid to its use in clinical laboratories. This paper describes the results obtained by radioimmunoassay and counter immunoelectrophoresis (CIEP) on sera collected from 1401 patients with acute viral hepatitis. _ _ _ ~ 'This project was assisted by a grant and Medical Research Council. +Virologist :Medical Virologist.
~
~-~
~
from t h e National Health
Correspondence D r . I . D. G u s t , Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield. Victoria 3078 Accepted for publication 22 October, 1975
Materials and Methods
Serum specimenb- A L Fairfield Hospital. after the c o n pletion o f routinc tests, all sera from patients with liver discasc are catalogued and irored at 20 C. Several groups of sera collected from patients with acutc viral hepatiLi, wcre selected fur the current study. -
Drtcc'riorr of Hepciritis H Surfirce Anligen Counter immunoelcctrophore~ib and solid phase radiuimmunoassay IAusria ' 2 s I ~ were performed as prebiously described.', The specificity of positive results obtained by KIA was confirmed by neutralization \kith specific antisera. Lirer Function Testc The lollowing biochemical tests of livcr function were performed, serum bilirubin and serum aapartate (AST). A result was considered abnormal if the serum bilirubin was greater than 17 mM(1 and/or the AST greater than 40 U. In patients with hepatitis. the duration of illness wab measurcd from the first objcetive symptom. namely the onset of dark urine. Results
Examination of Stored Sera The ability of CIEP and RIA to detect HB,Ag was initially compared on two batches of sera which had been stored frozen for up to 20 years. The results are shown in Table 1. Group (i) comprised 469 sera from 21 I patients admitted between November 1954 and November 1955. Hepatitis B surface antigen was detected by both techniques in a single serum from each of three patients, and in 13 sera from an additional seven patients by RIA alone. Group (ii) comprised 411 sera from 357 patients admitted between January 1963 and December 1966. Hepatitis B surface antigen was detected in 19 sera from 18 patients by both techniques and in 12 sera from an _ _ additional 11 patients by RIA alone. In these studies RIA was considerably more sensitive than CIEP, detecting HB,Ag in 3 . 3 times as many patients in group (1) and I ' 6 times as many in group (ii). Accordingly, doubts were raised whether the CIEP test used routinely at this hospital for the diagnosis
8
LLHMASN
~
-~~________~
of hepatitis B since October 1970, may have failed to detect a considerable numbcr of patients with this disease. To evaluate this, a batch of sera from patients with acute viral hepatitis, which had previously been tested by CIEP. was re-tested by RIA. Group (iii) comprised 641 patients admitted to hospital between 1971-73. The serum sample obtained from each patient on admission, was re-examined by both techniques ; antigen was detected by CIEP in 217 patients (33 89,) and in all these plus an additional 12 (35.7",) by RIA (see Table 1). Prospectice S t u d y of Patierits Mith Hepatitis Between November 1973 and June 1974, a further 192 patients with acute viral hepatitis were admitted (Group iv) and 986 sera from them were available for testing. In 73 patients (38 7;)the first serum specimen obtained after admission to hospital was positive by CIEP ; RIA detected all these patients and one more (38.5%) (see Table 1). When sera which had been collected later in the illness were examined, the difference in sensitivity of the two tcchniques became more apparent. Overall, HB,Ag was detected by,both methods in 170 (17.2%) of the 986 sera and in a further 117 (total 29.1 %) sera by RIA alone. Examination of sera collected weekly from 60 of the 74 patients with hepatitis B, showed that RIA usually detected antigen in the blood for two weeks longer than CIEP. The mean duration of antigenaemia (as measured from the onset of dark urine) being 43 and 29 days respectively. The mean duration of abnormal liver function tests in these patients was 36 days, thus almost all were antigen-positive and thus still infectious, after biochemical recovery was complete and they had been discharged from hospital. Discussion
As the majority of tests for HB,Ag are performed by blood transfusion services, most studies on the relative sensitivity of difl'erent detection methods have been carried out in blood donors. Based on serial dilutions, the commercially-available radioimmunoassay is about 75-350 times as sensitive as CIEP'. 6 , ' '3
TmLt 1 Detection of HB,A@ in patients with acute \ i r a l hcp;ititi\ HB,.A@ dctection
Group
ClEP
Year ~~~~
(11
199-5
(iii
1963 6
(iii! iiv)
i971-3 1973 4
RIA
~
Patients Sera Pariznn Sera Patients Patients Sera
21 1 469 357 411
641 192
986
3 ( I 3 '>,,I 3 (0.6u,,~ 18 ( S . O ~ , , I 1') (3.6",,I 21: (33.8",,1 73 138 0 " " l 170 ( 1 7 . 2 ""I
10(4-7",,) I h 1?.3",,1 19 i x - I '?,'I 7 1 17 i " , l 229 (35.7",,1 ?4 (38. 5 ",,I 2x7 (29 I" J
and regularly identifies up to 50:); more blood donors as chronic carriers of hepatitis B than CIEP. When sera from patients with acute viral hepatitis have been examined for HB,Ag by CIEP and RIA: RIA has detected more positives. Hacker er described 18 patients with sporadic acute viral hepatitis. Hepatitis B surface antigen was detected by CIEP and RIA in five patients and by RIA alone in another five ; the remaining eight patients were antigennegative by both techniques. Two studies examined groups of patients who have been regarded as having hepatitis A on the basis of negative ClEP tests for HB,Ag. When sera from these patients were re-examined by RIA, Ling and Overby' found 41 out of 245 (17%) antigen positive. However, as no neutralization tests were performed to check the specificity of these findings, the real incidence was probably lower. Hansson and Johnsson' examined a group of 138 CIEP-negative patients and found 23 (17%) had antigen detectable by RIA. These results were confirmed by neutralization with specific antiserum. The present findings with sera which had been collected recently, are in conflict with those described above. Radioimmunoassay olTered little advantage over CIEP in the diagnosis of patients with acute hepatitis. In 833 patients admitted between 1971 and 1974 (Groups (iii) and (iv)), 290 were diagnosed as hepatitis B by CIEP. Radioimmunoassay detected antigen in each of these patients and in a further 13, a gain of only 4 . 4 There are several possible explanations for these apparently contradictory results. It is possible that the CIEP test used in the laboratory
z.
I EBRL AKY. __-~ ~
1976
DETECTION OF H 3 4 g lh AC'UTE VIRAL HEPATITIS
9
~-
where the present work was carried out is more sensitive than that used by other workers or that the RIA tcst is less sensitive. Recent collaborative studies with other centres suggest that this is not SO.^ The second explanation relates to the time in the patient's illness in which specimens were obtained. Most patients with acute viral hepatitis are referred to Fairfield Hospital within 1-2 weeks of thc onset of dark urine. At this time HB,Ag is at peak levels in the blood and thus readily detected by even relatively insensitive techniques. Previous studies" have shown that the antigen titre then falls gradually and that the subsequent detection rate and hence the duration of antigenaemia, depends on the sensitivity of the detection system used. In the present study. the average duration of antigenaemia was four weeks when measured by CIEP and six weeks by RIA. Neither Ling and Overby8 or Hansson and Johnsson7 describe at what stage of the illness the sera they tested were obtained and it is possible that a proportion were collected at a stage when the antigen level had fallen below that detectable by CIEP. When the patients in Groups (iii) and (iv) were classified according to the probable mode of infection it was found that the most marked difference between RIA and CIEP occurred in post-transfusion hepatitis. Of 2 I patients with this disease, HB,Ag was detected by both techniques in 11 and in a further seven by RIA alone. It seems therefore, that the majority of post-transfusion hepatitis in Melbourne is hepatitis Type B and that a considerable proportion is associated with low levels of antigen. The results of comparative testing of stored sera are of interest, and in marked contrast
to the results obtained with fresh sera. It appears that the longer sera are stored, the greater the value of RIA in detecting antigen. It seems that the antigen gradually declines in titre and will often fail to be detected, with other than the most sensitive techniques. Whilst the CIEP tcst has been shown to be quite reliable in the serological diagnosis of hepatitis B when specimens are obtained early, RIA has a marked advantage later in the illness. When serial sera collected from 74 patients with hepatitis B were tested, RIA detected 69 7; more positives and extended the period of antigenaemia from four to six weeks. The RIA test is also of particular value in examining sera in which the antigen titre may be low, for example, sera which have been stored for many years. RIA detected 3 . 3 times more patients with hepatitis B than CIEP in sera which had been stored for 19-20 years and 1 . 6 times as many in sera stored for 8- 11 years. References I M I I i ru. 1 . P Third generdtion tests for the detection ol'hepatitir
2
3 4. 5 b
B antirrn (HB,Ag] Hcpatms B. Logan. ti F b I O R G I N I . GINAI... HOI I l \ L l R. F B.. B L ~ ( ~ ~A, ~ \ , .rH,,YER. w n ( of hepatitis B antigen: A prviprctivu s t u d y in hluod d u n o r < a n d r r c ~ p ~ c n t ~ . J Am?, rnrd. A i c 222, 1514. H A ~ U I I E, . I. and A A CH. R. D. (19??). Delcction 01 hepdlitir dsaociated anrigen and a n l t - H A 4 J A m o mcd A u 223. 414 N A V A \M I , ~ i iCi ,~PmIvLLt, R C and G i r l . I D (1972). The detection of Australia antigsn and antibody by cruss-owr Iinmunoclc'tiupliore,l\. A u ~ rJ M d Trchncil 3, I I 1 ti~sr.I D and LEHMAHN. N I (1975) J h e d e t e c t i n i i o i I ~ ~ p d t iHi iw~i f a c e antigen by rddwimmunoauay. Pniho1oi.i. 1, -. ALTtR. H J . HOLLAND.P V , PURCELL.K H dlld G F K I N1. L (1973) The Auaria Test: Critical eialuation of sensitivity and ?pccificity. Blood 42, 947
IIANISClh. 8. G . and J l l l i h h ~ l ~ hT. . (1974) E\.aluar,on of a solid phdTe radioimmunoassay (Aosria 125) fur rhc dctcction 01 hcpat!t,a B d n t l g t n (.4uslralia antigen) in twu clinical malerials, J . cl!rr. Pnrh 27. 15 8. LINC C. M and Ovtaim, L. R. (1972): Prevalance of hepatitis B virus antigen as ievealed by diiect :adio-inimune as%) with ' ' ~ lantrhody. J hnmuno! 109, 831 9 TAYLOR. PATKICIAE.: W.H.O. collaborarrw evaluarioii of !elei.mce hepatitis B antigen and annbody rcagenti, subrypc D. Bul!. W'ld Nlrh Orp. ( i n press) I0 "IUST.1 D . WHITE.J and KALDOK, J (19711: Duration of antigenaemta in Au-poaitive wrdl hepatitis. Mud J . A u i r 2, I01 F
7.
From the Fairfield Hospital for Communicable Diseases, Victoria
Summary: Detection of hepatitis B surface antigen in patients w i t h acute viral hepatitis. N I . Lehrnann and I . D Gust, Aust N Z J M e d . 1976. 6. pp. 7-9.
The relative value of solid phase radioimmunoassay and counter immunoelectrophoresis for the detection of hepatitis B surface antigen in the serum, was studied in 1401 patients with acute viral hepatitis. When specimens were obtained early in the illness, counter immunoelectrophoresis was found to be a reliable test for the diagnosis of hepatitis B, detecting 95.6% of patients found to be antigen positive by radioimm un oassay . The additional sensitivity of ra d i o i m m u no assay was of particular value for detecting antigen later in the illness and in patients with post-transfusion hepatitis in which the titre was frequently below the level detectable by counter immunoelectrophoresis. Radioimmunoassay was also of value in testing sera which had been stored for many years, detecting up to 3 . 3 times as many patients with hepatitis B as did counter immunoelectrophoresis.
Radioimmunoassay (RIA) is the most sensitive method currently available for detecting hepatitis B surface antigen (HB,Ag).' Whilst many studies have denionstrated the value of RIA for screening blood donors', ', less attention had been paid to its use in clinical laboratories. This paper describes the results obtained by radioimmunoassay and counter immunoelectrophoresis (CIEP) on sera collected from 1401 patients with acute viral hepatitis. _ _ _ ~ 'This project was assisted by a grant and Medical Research Council. +Virologist :Medical Virologist.
~
~-~
~
from t h e National Health
Correspondence D r . I . D. G u s t , Fairfield Hospital for Communicable Diseases, Yarra Bend Road, Fairfield. Victoria 3078 Accepted for publication 22 October, 1975
Materials and Methods
Serum specimenb- A L Fairfield Hospital. after the c o n pletion o f routinc tests, all sera from patients with liver discasc are catalogued and irored at 20 C. Several groups of sera collected from patients with acutc viral hepatiLi, wcre selected fur the current study. -
Drtcc'riorr of Hepciritis H Surfirce Anligen Counter immunoelcctrophore~ib and solid phase radiuimmunoassay IAusria ' 2 s I ~ were performed as prebiously described.', The specificity of positive results obtained by KIA was confirmed by neutralization \kith specific antisera. Lirer Function Testc The lollowing biochemical tests of livcr function were performed, serum bilirubin and serum aapartate (AST). A result was considered abnormal if the serum bilirubin was greater than 17 mM(1 and/or the AST greater than 40 U. In patients with hepatitis. the duration of illness wab measurcd from the first objcetive symptom. namely the onset of dark urine. Results
Examination of Stored Sera The ability of CIEP and RIA to detect HB,Ag was initially compared on two batches of sera which had been stored frozen for up to 20 years. The results are shown in Table 1. Group (i) comprised 469 sera from 21 I patients admitted between November 1954 and November 1955. Hepatitis B surface antigen was detected by both techniques in a single serum from each of three patients, and in 13 sera from an additional seven patients by RIA alone. Group (ii) comprised 411 sera from 357 patients admitted between January 1963 and December 1966. Hepatitis B surface antigen was detected in 19 sera from 18 patients by both techniques and in 12 sera from an _ _ additional 11 patients by RIA alone. In these studies RIA was considerably more sensitive than CIEP, detecting HB,Ag in 3 . 3 times as many patients in group (1) and I ' 6 times as many in group (ii). Accordingly, doubts were raised whether the CIEP test used routinely at this hospital for the diagnosis
8
LLHMASN
~
-~~________~
of hepatitis B since October 1970, may have failed to detect a considerable numbcr of patients with this disease. To evaluate this, a batch of sera from patients with acute viral hepatitis, which had previously been tested by CIEP. was re-tested by RIA. Group (iii) comprised 641 patients admitted to hospital between 1971-73. The serum sample obtained from each patient on admission, was re-examined by both techniques ; antigen was detected by CIEP in 217 patients (33 89,) and in all these plus an additional 12 (35.7",) by RIA (see Table 1). Prospectice S t u d y of Patierits Mith Hepatitis Between November 1973 and June 1974, a further 192 patients with acute viral hepatitis were admitted (Group iv) and 986 sera from them were available for testing. In 73 patients (38 7;)the first serum specimen obtained after admission to hospital was positive by CIEP ; RIA detected all these patients and one more (38.5%) (see Table 1). When sera which had been collected later in the illness were examined, the difference in sensitivity of the two tcchniques became more apparent. Overall, HB,Ag was detected by,both methods in 170 (17.2%) of the 986 sera and in a further 117 (total 29.1 %) sera by RIA alone. Examination of sera collected weekly from 60 of the 74 patients with hepatitis B, showed that RIA usually detected antigen in the blood for two weeks longer than CIEP. The mean duration of antigenaemia (as measured from the onset of dark urine) being 43 and 29 days respectively. The mean duration of abnormal liver function tests in these patients was 36 days, thus almost all were antigen-positive and thus still infectious, after biochemical recovery was complete and they had been discharged from hospital. Discussion
As the majority of tests for HB,Ag are performed by blood transfusion services, most studies on the relative sensitivity of difl'erent detection methods have been carried out in blood donors. Based on serial dilutions, the commercially-available radioimmunoassay is about 75-350 times as sensitive as CIEP'. 6 , ' '3
TmLt 1 Detection of HB,A@ in patients with acute \ i r a l hcp;ititi\ HB,.A@ dctection
Group
ClEP
Year ~~~~
(11
199-5
(iii
1963 6
(iii! iiv)
i971-3 1973 4
RIA
~
Patients Sera Pariznn Sera Patients Patients Sera
21 1 469 357 411
641 192
986
3 ( I 3 '>,,I 3 (0.6u,,~ 18 ( S . O ~ , , I 1') (3.6",,I 21: (33.8",,1 73 138 0 " " l 170 ( 1 7 . 2 ""I
10(4-7",,) I h 1?.3",,1 19 i x - I '?,'I 7 1 17 i " , l 229 (35.7",,1 ?4 (38. 5 ",,I 2x7 (29 I" J
and regularly identifies up to 50:); more blood donors as chronic carriers of hepatitis B than CIEP. When sera from patients with acute viral hepatitis have been examined for HB,Ag by CIEP and RIA: RIA has detected more positives. Hacker er described 18 patients with sporadic acute viral hepatitis. Hepatitis B surface antigen was detected by CIEP and RIA in five patients and by RIA alone in another five ; the remaining eight patients were antigennegative by both techniques. Two studies examined groups of patients who have been regarded as having hepatitis A on the basis of negative ClEP tests for HB,Ag. When sera from these patients were re-examined by RIA, Ling and Overby' found 41 out of 245 (17%) antigen positive. However, as no neutralization tests were performed to check the specificity of these findings, the real incidence was probably lower. Hansson and Johnsson' examined a group of 138 CIEP-negative patients and found 23 (17%) had antigen detectable by RIA. These results were confirmed by neutralization with specific antiserum. The present findings with sera which had been collected recently, are in conflict with those described above. Radioimmunoassay olTered little advantage over CIEP in the diagnosis of patients with acute hepatitis. In 833 patients admitted between 1971 and 1974 (Groups (iii) and (iv)), 290 were diagnosed as hepatitis B by CIEP. Radioimmunoassay detected antigen in each of these patients and in a further 13, a gain of only 4 . 4 There are several possible explanations for these apparently contradictory results. It is possible that the CIEP test used in the laboratory
z.
I EBRL AKY. __-~ ~
1976
DETECTION OF H 3 4 g lh AC'UTE VIRAL HEPATITIS
9
~-
where the present work was carried out is more sensitive than that used by other workers or that the RIA tcst is less sensitive. Recent collaborative studies with other centres suggest that this is not SO.^ The second explanation relates to the time in the patient's illness in which specimens were obtained. Most patients with acute viral hepatitis are referred to Fairfield Hospital within 1-2 weeks of thc onset of dark urine. At this time HB,Ag is at peak levels in the blood and thus readily detected by even relatively insensitive techniques. Previous studies" have shown that the antigen titre then falls gradually and that the subsequent detection rate and hence the duration of antigenaemia, depends on the sensitivity of the detection system used. In the present study. the average duration of antigenaemia was four weeks when measured by CIEP and six weeks by RIA. Neither Ling and Overby8 or Hansson and Johnsson7 describe at what stage of the illness the sera they tested were obtained and it is possible that a proportion were collected at a stage when the antigen level had fallen below that detectable by CIEP. When the patients in Groups (iii) and (iv) were classified according to the probable mode of infection it was found that the most marked difference between RIA and CIEP occurred in post-transfusion hepatitis. Of 2 I patients with this disease, HB,Ag was detected by both techniques in 11 and in a further seven by RIA alone. It seems therefore, that the majority of post-transfusion hepatitis in Melbourne is hepatitis Type B and that a considerable proportion is associated with low levels of antigen. The results of comparative testing of stored sera are of interest, and in marked contrast
to the results obtained with fresh sera. It appears that the longer sera are stored, the greater the value of RIA in detecting antigen. It seems that the antigen gradually declines in titre and will often fail to be detected, with other than the most sensitive techniques. Whilst the CIEP tcst has been shown to be quite reliable in the serological diagnosis of hepatitis B when specimens are obtained early, RIA has a marked advantage later in the illness. When serial sera collected from 74 patients with hepatitis B were tested, RIA detected 69 7; more positives and extended the period of antigenaemia from four to six weeks. The RIA test is also of particular value in examining sera in which the antigen titre may be low, for example, sera which have been stored for many years. RIA detected 3 . 3 times more patients with hepatitis B than CIEP in sera which had been stored for 19-20 years and 1 . 6 times as many in sera stored for 8- 11 years. References I M I I i ru. 1 . P Third generdtion tests for the detection ol'hepatitir
2
3 4. 5 b
B antirrn (HB,Ag] Hcpatms B. Logan. ti F b I O R G I N I . GINAI... HOI I l \ L l R. F B.. B L ~ ( ~ ~A, ~ \ , .rH,,YER. w n ( of hepatitis B antigen: A prviprctivu s t u d y in hluod d u n o r < a n d r r c ~ p ~ c n t ~ . J Am?, rnrd. A i c 222, 1514. H A ~ U I I E, . I. and A A CH. R. D. (19??). Delcction 01 hepdlitir dsaociated anrigen and a n l t - H A 4 J A m o mcd A u 223. 414 N A V A \M I , ~ i iCi ,~PmIvLLt, R C and G i r l . I D (1972). The detection of Australia antigsn and antibody by cruss-owr Iinmunoclc'tiupliore,l\. A u ~ rJ M d Trchncil 3, I I 1 ti~sr.I D and LEHMAHN. N I (1975) J h e d e t e c t i n i i o i I ~ ~ p d t iHi iw~i f a c e antigen by rddwimmunoauay. Pniho1oi.i. 1, -. ALTtR. H J . HOLLAND.P V , PURCELL.K H dlld G F K I N1. L (1973) The Auaria Test: Critical eialuation of sensitivity and ?pccificity. Blood 42, 947
IIANISClh. 8. G . and J l l l i h h ~ l ~ hT. . (1974) E\.aluar,on of a solid phdTe radioimmunoassay (Aosria 125) fur rhc dctcction 01 hcpat!t,a B d n t l g t n (.4uslralia antigen) in twu clinical malerials, J . cl!rr. Pnrh 27. 15 8. LINC C. M and Ovtaim, L. R. (1972): Prevalance of hepatitis B virus antigen as ievealed by diiect :adio-inimune as%) with ' ' ~ lantrhody. J hnmuno! 109, 831 9 TAYLOR. PATKICIAE.: W.H.O. collaborarrw evaluarioii of !elei.mce hepatitis B antigen and annbody rcagenti, subrypc D. Bul!. W'ld Nlrh Orp. ( i n press) I0 "IUST.1 D . WHITE.J and KALDOK, J (19711: Duration of antigenaemta in Au-poaitive wrdl hepatitis. Mud J . A u i r 2, I01 F
7.
