THE JOURNAL OF INFECTIOUS DISEASES. VOL. 136, NO. I • JULY 1977
© 1977 by the University of Chicago. All rights reserved.
Serodiagnosis of Hepatitis B Virus Infection by Antibody to Core Antigen From the Departments of Virus Diseases and Preventive Medicine, Walter Reed Army Institute of Research, Washington, D.C.
Gilbert R. Irwin, Richard G. Allen, Herbert G. Segal, Alfred M. Allen,* J. Robert Putnak, Hubert G. Cannon, and Franklin H. Top, Jr.
port initial data on the detection of anti-H'B, in a military population in which hepatitis B was endemic. A radioimmunoassay (RIA) inhibition test for anti-H'B, was used; this test increased the efficiency of the serodiagnosis of clinical and subclinical HBV infections.
Sensitive and specific tests for presence of hepatitis B surface antigen (HB s Ag) and antibody to HB s Ag (anti-HB s) have significantly improved our understanding of the basic epidemiology of hepatitis B virus (HBV) infection. Recently, an additional marker of HBV infection, antibody to the core antigen (anti-Hb.), has been described [1, 2]. This antigen-antibody system appears to be of great potential in the further elucidation of HBV infection [3, 4]. Since anti-Hls, is produced early in the course of viral infection, as opposed to anti-Hls., which is detected at various times after infection, it provides evidence for HBV infection in the absence of HB s Ag and anti-Hb., Because of scarce quantities of hepatitis B core antigen (HB c Ag), the serologic analysis of epidemiologically important populations for anti-Hb, has been limited. In this paper we re-
Materials and Methods
Sera for this study were obtained from military personnel at Fort Hood, Texas. Four different subpopulations were studied. The first subpopulation consisted of soldiers hospitalized for treatment of hepatitis during 1973 and 1974 from whom samples of blood were collected at admission to obtain acute-phase sera. Samples of blood were also collected during the first week of jaundice. Previous surveys had demonstrated that this sustained outbreak of hepatitis was due to HBV [5]. Patients who lacked HB s Ag in acute-phase sera were epidemiologically associated with HB s Ag-positive cases. Eightyfive patients whose blood was found to be negative for HBr; Ag by RIA were tested for anti-Hb, as described below. In addition, 25 of 60 patients with HB s Ag in acute-phase sera were selected for anti-Hb, serology. The second subpopulation included patients with acute hepatitis during 1973 from whom acute- and convalescent-phase sera (obtained two to 33 months after infection) were available. All
Received for publication August 26, 1976, and in revised form December 23,1976. In conducting the research described in this report, the investigators adhered to the "Guide for Laboratory Animal Facilities and Care," as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. Please address requests for reprints to Dr. Gilbert R. Irwin, Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012. • Present address: Letterman Army Institute of Research, Presidio, San Francisco, California 94129.
31
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
In a military population antibody to hepatitis B core antigen (anti-H'Bj) was found in 39% of acute hepatitis cases negative for hepatitis B surface antigen (RB s Ag) and in 96% of RB s Ag-positive cases. Persistence of antibody to RB s Ag (anti-RB s ) in convalescent-phase sera was significantly greater (P < 0.001) in individuals with acute RB s Ag-positive hepatitis B than in patients with clinical RB s Ag-negative hepatitis B. The prevalence of anti-Hb; in the absence of RB s Ag, anti-Hb., and clinical disease was 3.2% in this military population. In longitudinal studies of hepatitis B infection, the presence of anti-Hb; preceded anti-H'B, and improved the ability to determine the onset of subclinical infection. Anti-Hb, is a useful serologic marker for the study of the epidemiology of hepatitis B and improves the efficiency of detection of hepatitis B virus infection.
32
RIA inhibition test for anti-Hb : The chimpanzee liver was used as a source of antigen for a RIA inhibition test for anti-Hb.; A 20% liver suspension was prepared in hypotonic (0.01 M) phosphate-buffered saline (PBS). After homogenization, the liver suspension was clarified by centrifugation, and the supernatant was used as a source of HB c Ag. Anti-Hb, IgG derived from an asymptomatic carrier of HB s Ag without anti-H'B, activity was labeled by the method of Purcell et al. [6]. The following reagents were added, in order, to a test tube: 20 ,ul of 0.2 M phosphate buffer, pH 7.4; 200 ,uCi of high-specific activity 125 1 (in 5,u1); 10,ug of the protein to be labeled (in 1-10 ,ul); 15 ,ul of a solution of chloramine T (3.5 ,ug/ ,ul); 20 ,ul of a solution of sodium metabisulfite (4.8 ,ug/ ,ul); and 20 ,ul of a solution of sucrose (22.5%) and potassium iodine (2 tug] ml), After the addition of chloramine T, the reaction was allowed to proceed for 15 sec before being terminated by the addition of sodium metabisulfite. The mixture was applied to the top of a column (0.9 X 15 em) packed with Sephadex G-200 and was equilibrated with PBS, pH 7.4, with 0.1% sodium azide. The protein was eluted with the same buffer. Fractions containing the first peak of radioactivity were pooled and diluted with an equal volume of I % bovine serum albumin. This stock mixture was stored at 4 C and diluted 1:2 with 1% bovine serum albumin just before use. The micro-solid-phase RIA inhibition test was a modification of the method of Purcell et al. [7]. The wells of polyvinyl microtiter plates were coated for 4 hr at 37 C with diluted (1:100) anti-H'B, (derived from the same human source as mentioned above) in PBS. After being washed with saline, the sensitized wells received 200 ,ul of 1% bovine serum albumin in saline and were incubated overnight at 4 C. The wells were then washed with PBS and inoculated with 50 ,ul of HB c Ag, and the plates were incubated at 4 C for 24-48 hr. After another washing in PBS, 50 ,ul of each patient's serum was added to duplicate wells. The plates were incubated at 4 C for 24 hr and washed with PBS, and 50 ,ul of radiolabeled anti-Hb, globulin was added to each well; the plates were then incubated at 37 C for 4-6 hr or at 4 C overnight.
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
samples were tested for HB s Ag, anti-Hfl., and anti-HBcThe third subpopulation consisted of 2,333 soldiers who were newly assigned to Fort Hood between February and April 1974, and from whom blood samples were obtained every four months for one year. The total number of soldiers available for collection of blood samples at four, eight, and 12 months was 1,915, 1,210, and 900, respectively. This population was tested for HB s Ag and anti-Hll., If any specimen was found to be positive for anti-Hls., all available sera for that subject were tested for anti-Hb., In addition, from this group 215 soldiers who were negative for HB s Ag and anti-H'B, were randomly selected for anti-H'B, testing. The fourth subpopulation consisted of military units in which the prevalence of HB s Ag and anti-H'B, was determined in February 1973. Included were units both with and without a hospitalized index case of hepatitis B. In this study, sera from 377 members of units with an index case and sera from 520 members of control units were tested for anti-Hh., Production of HB c Ag. Two male chimpanzees, weighing 22.7 and 25 kg, were isolated, and a course of immunosuppression with cyclophosphamide at a dosage of 15 mg/kg was begun. This drug was administered two to three times a week throughout the course of the experiment. Twenty-six days after initiation of treatment with cyclophosphamide, both of the animals received I ml of a I: 10 dilution of infectious human plasma containing HB s Ag. (The samples of plasma were provided by Dr. Jay Hoofnagle of the Bureau of Biologics, Food and Drug Administration [FDA], Bethesda, NId.) On day 32 after inoculation with HBV, HB s Ag was detected by RIA in both chimpanzees. Liver biopsies were performed on each animal twice a week during the period of antigenemia. The presence of HB c Ag in the liver tissue was determined by CF with use of human and chimpanzee anti-HB c . When it was apparent that the titers of HB c Ag in homogenates of serial liver biopsy specimens were not increasing, the animals were sacrificed, and the livers were removed and frozen at -70 C. The titers of HB c Ag in 20% liver homogenates from the two animals were 1:8 and I: 16, respectively.
Irwin et al,
Diagnosis of Hepatitis B with Anti-HB c
Results
An indication of the sensitivity of the RIA inhibition test for anti-Hb, as compared with a CF test (Bureau of Biologics, FDA) is found in table 1. The RIA inhibition test was more sensitive as evidenced by the positive results with RIA inhibi tion when the CF test was negative and by the higher titers with RIA inhibition when the CF test was positive. With use of RIA inhibition, anti-H'B, was found in 34 (39%) of 85 sera from patients with acute HB s Ag-negative hepatitis and in 24 (96c;Jo) of 25 samples of HB s Ag-positive acute-phase sera. Since it has been shown previously in this military population that 50% of the cases of acute hepatitis B were HB s Ag-positive (G. R. Irwin, unpublished observations), the detection of anti-Hls, in 39% of the acute cases lacking HB s Ag improved the efficiency of serodiagnosis of the total number of acute hepatitis B cases by --20%. Persistence of anti-Hls, after acute icteric hepatitis B is demonstrated in table 2. Most individuals (21 of 22) with acute HB s Ag-positive hepatitis had anti-H'B, initially, and 20 of these patients had anti-H'B, in convalescent-phase sera obtained three to 33 months after the onset of
Table 1. Comparison of C F and radioimmunoassay (RIA) inhibition tests for detection of antibody to hepatitis B core antigen (anti-HB c)' Titer of anti-HB c Sample no.
101 102 103 104 105 106 107 108 109
RIA inhibition
CF
1:64
1:8
1:512 1:2,048
1:32
1:4,096
1:1,024
1:2,048
110
111 112 113 114 115 116 117 118 119 120
1:2,048
1:4,096 1:1,024 1:256 1:32 1:4,096 1:4,096
1:10,384 1:16 1: 16
1:8
NOTE. Minus signs indicate negative results.
illness. Of 19 cases of acute hepatitis Blacking HB s Ag, seven (37%) had anti-H'B, initially. Four of these seven individuals had anti-H'B, persistent in their convalescent-phase sera. Of the remaining 12 individuals with neither HB s Ag nor anti-Hfs, present during acute illness, three seroconverted to anti-Hb, by three, four, and 15 months after illness, respectively. Seroconversion to anti-Hb.-positive without antiHB e was noted in two other patients. Positive and negative groups were comparable in the mean time of follow-up and in the range of time until convalescent-phase sera were obtained. The efficiency of anti-Hls, in defining the onset of subclinical hepatitis B is described in table 3. The data are derived from 2,333 soldiers followed for one year. Only individuals with follow-up sera available are included in the table. Of the soldiers tested, 48 developed anti-Hls, by four, eight, or 12 months after their arrival at Fort Hood. Twenty-two of these 48 soldiers had anti-HB e, and 18 of the 22 anti-HBe-positive soldiers had anti-H'B, present on arrival at Fort Hood. Thirty-eight soldiers had anti-Hls, upon
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
After the plates were given a final washing in PBS, the wells were cut with scissors and transferred to y-counting tubes; radioactive counts were measured with a Nuclear Chicago (Searle; Chicago, Ill.) y-counter for I min. Each plate contained four wells of anti-Hb.vnegative serum as a control for plate variation. The iodinated preparation of anti-H'B, did not react with normal human or chimpanzee liver by direct RIA when these antigens were substituted for HB c Ag. Serum that contained only anti-Hls, and gave high counts of radioactivity by the AusAb test (Abbott Laboratories, North Chicago, 111.) did not inhibit the reaction. Marked reduction in radioactivity with unlabeled anti-H'B, but not with normal serum was indicative of the specificity of the reaction. A sample was considered to have anti-Hb, if a ~ 50% «3 SD below the negative control mean) reduction in cpm of labeled anti-H'B, occurred. Negative and positive controls were tested on each plate. All sera were tested in duplicate.
33
34
Irwin et al.
Table 2. Persistence of antibody to hepatitis B core antigen [anti-Hb-] and seroconversion to antibody to hepatitis B surface antigen [anti-Hll.] in acute hepatitis B surface antigen (HB s Ag)-positive and -negative hepatitis. No. with Convalescent-phase sera anti-HB c Months in acuteuntil phase No. with No. with anti-HB c anti-HBs sera collection *
Group (no. of patients)
21
20
n
7
12t
8.5 (3-33)
6
9.4 (2-25)
*Data are given as medians (ranges). one patient who was anti-Hllg-positive and anti-Hlls-negative. :l:Four of these patients are included in the column for acute-phase sera.
ms, Ag persisted in
entering Fort Hood and remained pOSItIVe for anti-Hls, over the follow-up period: of these soldiers, 35 (92%) had anti-Hb, on arrival, and all of them remained anti-Hlk.-positive throughout the year. Among 37 individuals who lost antiHB s after being initially positive, anti-H'B, was present in only 17. The fourth group hsted in table 3 consists of 215 randomly selected soldiers who were negative for anti-Hb, and HB s Ag and were examined for anti-Hfs.; The only bias in the selection process was the necessity for a follow-up specimen at eight and/or 12 months. Of these 215 individuals, 22 had anti-Hb.: 15 of these 22 developed anti-Hb, while at Fon Hood, Table 3. Detection of antibody to hepatitis B core antigen (anti-HBd in soldiers with subclinical hepatitis B followed for one year. Anti-HBc-positive
Group (no. of subjects) *
No. detected while in No. detected endemic initially area
Ahti-HBs-negative to -positive (48) Anti-Hllg-positive for one year (38) Anti-HBs-positive to -negative (37) Never positive for anti-HB s or HB s Ag (215) *HB s Ag to HB s Ag.
= hepatitis
Total no.
18
4
22
35
o
35
17
o
17
7
15
22
B surface antigen; anti-Hlt, "" antibody
Discussion
The data from this survey confirm the usefulness of the detection of anti-H'B, in the serologic diagnosis of HBV infection, particularly in individuals who are negative for the classical surface antigen and antibody markers. The data from this study are derived from several serologic surveys of soldiers in an area endemic for hepatitis B. Anti-H'B, was present in 39% of single specimens from patients with acute icteric hepatitis B without HB s Ag. In our experience with military populations in which --50% of acute icteric HBV infections are HE s Ag-positive,
Table 4. Prevalence of hepatitis B surface antigen (HB s Ag), antibody to HB s Ag (anti-HB s), and antibody to hepatitis B core antigen (anti-Hh., Ag) in military units with a current hospitalized index case of acute hepatitis B and in control units. Platoon group (rio. of subjects)
No. of subjects with HBs Ag
Anti-HB s
With index case (377) Control (520)
5 5
53
46 (13)*
70
57 (7)
*Numbers in. parentheses anti-Hb, or HB s Ag.
indicate
individuals without
Anti-HB c
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
HBs Agpositive (22) HB s Agnegative (19)
and the remalmng seven soldiers (3.2%> represent the prevalence of anti-HB, without HB s Ag or anti-Hfs, among individuals entering the endemic area. Additional evidence for HBV infection beyond that which can be obtained from HB s Ag or antiHB s was noted in the cross-sectional survey of military units either with or without a hospitalized index case of hepatitis B (index and control units; 897 soldiers) (table 4). In this study 103 (11.5
© 1977 by the University of Chicago. All rights reserved.
Serodiagnosis of Hepatitis B Virus Infection by Antibody to Core Antigen From the Departments of Virus Diseases and Preventive Medicine, Walter Reed Army Institute of Research, Washington, D.C.
Gilbert R. Irwin, Richard G. Allen, Herbert G. Segal, Alfred M. Allen,* J. Robert Putnak, Hubert G. Cannon, and Franklin H. Top, Jr.
port initial data on the detection of anti-H'B, in a military population in which hepatitis B was endemic. A radioimmunoassay (RIA) inhibition test for anti-H'B, was used; this test increased the efficiency of the serodiagnosis of clinical and subclinical HBV infections.
Sensitive and specific tests for presence of hepatitis B surface antigen (HB s Ag) and antibody to HB s Ag (anti-HB s) have significantly improved our understanding of the basic epidemiology of hepatitis B virus (HBV) infection. Recently, an additional marker of HBV infection, antibody to the core antigen (anti-Hb.), has been described [1, 2]. This antigen-antibody system appears to be of great potential in the further elucidation of HBV infection [3, 4]. Since anti-Hls, is produced early in the course of viral infection, as opposed to anti-Hls., which is detected at various times after infection, it provides evidence for HBV infection in the absence of HB s Ag and anti-Hb., Because of scarce quantities of hepatitis B core antigen (HB c Ag), the serologic analysis of epidemiologically important populations for anti-Hb, has been limited. In this paper we re-
Materials and Methods
Sera for this study were obtained from military personnel at Fort Hood, Texas. Four different subpopulations were studied. The first subpopulation consisted of soldiers hospitalized for treatment of hepatitis during 1973 and 1974 from whom samples of blood were collected at admission to obtain acute-phase sera. Samples of blood were also collected during the first week of jaundice. Previous surveys had demonstrated that this sustained outbreak of hepatitis was due to HBV [5]. Patients who lacked HB s Ag in acute-phase sera were epidemiologically associated with HB s Ag-positive cases. Eightyfive patients whose blood was found to be negative for HBr; Ag by RIA were tested for anti-Hb, as described below. In addition, 25 of 60 patients with HB s Ag in acute-phase sera were selected for anti-Hb, serology. The second subpopulation included patients with acute hepatitis during 1973 from whom acute- and convalescent-phase sera (obtained two to 33 months after infection) were available. All
Received for publication August 26, 1976, and in revised form December 23,1976. In conducting the research described in this report, the investigators adhered to the "Guide for Laboratory Animal Facilities and Care," as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council. Please address requests for reprints to Dr. Gilbert R. Irwin, Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, D.C. 20012. • Present address: Letterman Army Institute of Research, Presidio, San Francisco, California 94129.
31
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
In a military population antibody to hepatitis B core antigen (anti-H'Bj) was found in 39% of acute hepatitis cases negative for hepatitis B surface antigen (RB s Ag) and in 96% of RB s Ag-positive cases. Persistence of antibody to RB s Ag (anti-RB s ) in convalescent-phase sera was significantly greater (P < 0.001) in individuals with acute RB s Ag-positive hepatitis B than in patients with clinical RB s Ag-negative hepatitis B. The prevalence of anti-Hb; in the absence of RB s Ag, anti-Hb., and clinical disease was 3.2% in this military population. In longitudinal studies of hepatitis B infection, the presence of anti-Hb; preceded anti-H'B, and improved the ability to determine the onset of subclinical infection. Anti-Hb, is a useful serologic marker for the study of the epidemiology of hepatitis B and improves the efficiency of detection of hepatitis B virus infection.
32
RIA inhibition test for anti-Hb : The chimpanzee liver was used as a source of antigen for a RIA inhibition test for anti-Hb.; A 20% liver suspension was prepared in hypotonic (0.01 M) phosphate-buffered saline (PBS). After homogenization, the liver suspension was clarified by centrifugation, and the supernatant was used as a source of HB c Ag. Anti-Hb, IgG derived from an asymptomatic carrier of HB s Ag without anti-H'B, activity was labeled by the method of Purcell et al. [6]. The following reagents were added, in order, to a test tube: 20 ,ul of 0.2 M phosphate buffer, pH 7.4; 200 ,uCi of high-specific activity 125 1 (in 5,u1); 10,ug of the protein to be labeled (in 1-10 ,ul); 15 ,ul of a solution of chloramine T (3.5 ,ug/ ,ul); 20 ,ul of a solution of sodium metabisulfite (4.8 ,ug/ ,ul); and 20 ,ul of a solution of sucrose (22.5%) and potassium iodine (2 tug] ml), After the addition of chloramine T, the reaction was allowed to proceed for 15 sec before being terminated by the addition of sodium metabisulfite. The mixture was applied to the top of a column (0.9 X 15 em) packed with Sephadex G-200 and was equilibrated with PBS, pH 7.4, with 0.1% sodium azide. The protein was eluted with the same buffer. Fractions containing the first peak of radioactivity were pooled and diluted with an equal volume of I % bovine serum albumin. This stock mixture was stored at 4 C and diluted 1:2 with 1% bovine serum albumin just before use. The micro-solid-phase RIA inhibition test was a modification of the method of Purcell et al. [7]. The wells of polyvinyl microtiter plates were coated for 4 hr at 37 C with diluted (1:100) anti-H'B, (derived from the same human source as mentioned above) in PBS. After being washed with saline, the sensitized wells received 200 ,ul of 1% bovine serum albumin in saline and were incubated overnight at 4 C. The wells were then washed with PBS and inoculated with 50 ,ul of HB c Ag, and the plates were incubated at 4 C for 24-48 hr. After another washing in PBS, 50 ,ul of each patient's serum was added to duplicate wells. The plates were incubated at 4 C for 24 hr and washed with PBS, and 50 ,ul of radiolabeled anti-Hb, globulin was added to each well; the plates were then incubated at 37 C for 4-6 hr or at 4 C overnight.
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
samples were tested for HB s Ag, anti-Hfl., and anti-HBcThe third subpopulation consisted of 2,333 soldiers who were newly assigned to Fort Hood between February and April 1974, and from whom blood samples were obtained every four months for one year. The total number of soldiers available for collection of blood samples at four, eight, and 12 months was 1,915, 1,210, and 900, respectively. This population was tested for HB s Ag and anti-Hll., If any specimen was found to be positive for anti-Hls., all available sera for that subject were tested for anti-Hb., In addition, from this group 215 soldiers who were negative for HB s Ag and anti-H'B, were randomly selected for anti-H'B, testing. The fourth subpopulation consisted of military units in which the prevalence of HB s Ag and anti-H'B, was determined in February 1973. Included were units both with and without a hospitalized index case of hepatitis B. In this study, sera from 377 members of units with an index case and sera from 520 members of control units were tested for anti-Hh., Production of HB c Ag. Two male chimpanzees, weighing 22.7 and 25 kg, were isolated, and a course of immunosuppression with cyclophosphamide at a dosage of 15 mg/kg was begun. This drug was administered two to three times a week throughout the course of the experiment. Twenty-six days after initiation of treatment with cyclophosphamide, both of the animals received I ml of a I: 10 dilution of infectious human plasma containing HB s Ag. (The samples of plasma were provided by Dr. Jay Hoofnagle of the Bureau of Biologics, Food and Drug Administration [FDA], Bethesda, NId.) On day 32 after inoculation with HBV, HB s Ag was detected by RIA in both chimpanzees. Liver biopsies were performed on each animal twice a week during the period of antigenemia. The presence of HB c Ag in the liver tissue was determined by CF with use of human and chimpanzee anti-HB c . When it was apparent that the titers of HB c Ag in homogenates of serial liver biopsy specimens were not increasing, the animals were sacrificed, and the livers were removed and frozen at -70 C. The titers of HB c Ag in 20% liver homogenates from the two animals were 1:8 and I: 16, respectively.
Irwin et al,
Diagnosis of Hepatitis B with Anti-HB c
Results
An indication of the sensitivity of the RIA inhibition test for anti-Hb, as compared with a CF test (Bureau of Biologics, FDA) is found in table 1. The RIA inhibition test was more sensitive as evidenced by the positive results with RIA inhibi tion when the CF test was negative and by the higher titers with RIA inhibition when the CF test was positive. With use of RIA inhibition, anti-H'B, was found in 34 (39%) of 85 sera from patients with acute HB s Ag-negative hepatitis and in 24 (96c;Jo) of 25 samples of HB s Ag-positive acute-phase sera. Since it has been shown previously in this military population that 50% of the cases of acute hepatitis B were HB s Ag-positive (G. R. Irwin, unpublished observations), the detection of anti-Hls, in 39% of the acute cases lacking HB s Ag improved the efficiency of serodiagnosis of the total number of acute hepatitis B cases by --20%. Persistence of anti-Hls, after acute icteric hepatitis B is demonstrated in table 2. Most individuals (21 of 22) with acute HB s Ag-positive hepatitis had anti-H'B, initially, and 20 of these patients had anti-H'B, in convalescent-phase sera obtained three to 33 months after the onset of
Table 1. Comparison of C F and radioimmunoassay (RIA) inhibition tests for detection of antibody to hepatitis B core antigen (anti-HB c)' Titer of anti-HB c Sample no.
101 102 103 104 105 106 107 108 109
RIA inhibition
CF
1:64
1:8
1:512 1:2,048
1:32
1:4,096
1:1,024
1:2,048
110
111 112 113 114 115 116 117 118 119 120
1:2,048
1:4,096 1:1,024 1:256 1:32 1:4,096 1:4,096
1:10,384 1:16 1: 16
1:8
NOTE. Minus signs indicate negative results.
illness. Of 19 cases of acute hepatitis Blacking HB s Ag, seven (37%) had anti-H'B, initially. Four of these seven individuals had anti-H'B, persistent in their convalescent-phase sera. Of the remaining 12 individuals with neither HB s Ag nor anti-Hfs, present during acute illness, three seroconverted to anti-Hb, by three, four, and 15 months after illness, respectively. Seroconversion to anti-Hb.-positive without antiHB e was noted in two other patients. Positive and negative groups were comparable in the mean time of follow-up and in the range of time until convalescent-phase sera were obtained. The efficiency of anti-Hls, in defining the onset of subclinical hepatitis B is described in table 3. The data are derived from 2,333 soldiers followed for one year. Only individuals with follow-up sera available are included in the table. Of the soldiers tested, 48 developed anti-Hls, by four, eight, or 12 months after their arrival at Fort Hood. Twenty-two of these 48 soldiers had anti-HB e, and 18 of the 22 anti-HBe-positive soldiers had anti-H'B, present on arrival at Fort Hood. Thirty-eight soldiers had anti-Hls, upon
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
After the plates were given a final washing in PBS, the wells were cut with scissors and transferred to y-counting tubes; radioactive counts were measured with a Nuclear Chicago (Searle; Chicago, Ill.) y-counter for I min. Each plate contained four wells of anti-Hb.vnegative serum as a control for plate variation. The iodinated preparation of anti-H'B, did not react with normal human or chimpanzee liver by direct RIA when these antigens were substituted for HB c Ag. Serum that contained only anti-Hls, and gave high counts of radioactivity by the AusAb test (Abbott Laboratories, North Chicago, 111.) did not inhibit the reaction. Marked reduction in radioactivity with unlabeled anti-H'B, but not with normal serum was indicative of the specificity of the reaction. A sample was considered to have anti-Hb, if a ~ 50% «3 SD below the negative control mean) reduction in cpm of labeled anti-H'B, occurred. Negative and positive controls were tested on each plate. All sera were tested in duplicate.
33
34
Irwin et al.
Table 2. Persistence of antibody to hepatitis B core antigen [anti-Hb-] and seroconversion to antibody to hepatitis B surface antigen [anti-Hll.] in acute hepatitis B surface antigen (HB s Ag)-positive and -negative hepatitis. No. with Convalescent-phase sera anti-HB c Months in acuteuntil phase No. with No. with anti-HB c anti-HBs sera collection *
Group (no. of patients)
21
20
n
7
12t
8.5 (3-33)
6
9.4 (2-25)
*Data are given as medians (ranges). one patient who was anti-Hllg-positive and anti-Hlls-negative. :l:Four of these patients are included in the column for acute-phase sera.
ms, Ag persisted in
entering Fort Hood and remained pOSItIVe for anti-Hls, over the follow-up period: of these soldiers, 35 (92%) had anti-Hb, on arrival, and all of them remained anti-Hlk.-positive throughout the year. Among 37 individuals who lost antiHB s after being initially positive, anti-H'B, was present in only 17. The fourth group hsted in table 3 consists of 215 randomly selected soldiers who were negative for anti-Hb, and HB s Ag and were examined for anti-Hfs.; The only bias in the selection process was the necessity for a follow-up specimen at eight and/or 12 months. Of these 215 individuals, 22 had anti-Hb.: 15 of these 22 developed anti-Hb, while at Fon Hood, Table 3. Detection of antibody to hepatitis B core antigen (anti-HBd in soldiers with subclinical hepatitis B followed for one year. Anti-HBc-positive
Group (no. of subjects) *
No. detected while in No. detected endemic initially area
Ahti-HBs-negative to -positive (48) Anti-Hllg-positive for one year (38) Anti-HBs-positive to -negative (37) Never positive for anti-HB s or HB s Ag (215) *HB s Ag to HB s Ag.
= hepatitis
Total no.
18
4
22
35
o
35
17
o
17
7
15
22
B surface antigen; anti-Hlt, "" antibody
Discussion
The data from this survey confirm the usefulness of the detection of anti-H'B, in the serologic diagnosis of HBV infection, particularly in individuals who are negative for the classical surface antigen and antibody markers. The data from this study are derived from several serologic surveys of soldiers in an area endemic for hepatitis B. Anti-H'B, was present in 39% of single specimens from patients with acute icteric hepatitis B without HB s Ag. In our experience with military populations in which --50% of acute icteric HBV infections are HE s Ag-positive,
Table 4. Prevalence of hepatitis B surface antigen (HB s Ag), antibody to HB s Ag (anti-HB s), and antibody to hepatitis B core antigen (anti-Hh., Ag) in military units with a current hospitalized index case of acute hepatitis B and in control units. Platoon group (rio. of subjects)
No. of subjects with HBs Ag
Anti-HB s
With index case (377) Control (520)
5 5
53
46 (13)*
70
57 (7)
*Numbers in. parentheses anti-Hb, or HB s Ag.
indicate
individuals without
Anti-HB c
Downloaded from http://jid.oxfordjournals.org/ at University of Sussex on September 5, 2015
HBs Agpositive (22) HB s Agnegative (19)
and the remalmng seven soldiers (3.2%> represent the prevalence of anti-HB, without HB s Ag or anti-Hfs, among individuals entering the endemic area. Additional evidence for HBV infection beyond that which can be obtained from HB s Ag or antiHB s was noted in the cross-sectional survey of military units either with or without a hospitalized index case of hepatitis B (index and control units; 897 soldiers) (table 4). In this study 103 (11.5
