Clinica Chimica Acta 430 (2014) 171–172
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Resolving a case of concurrent hepatitis B virus surface antigen (HBsAg) and surface antibody (HBsAb) Thomas Kampfrath a,⁎, Saeed A. Jortani b, Stanley S. Levinson b,c a b c
Department of Pathology and Laboratory Medicine, Santa Clara Valley Medical Center, San Jose, CA, United States Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY, United States Laboratory Service, Department of Veteran Affairs Medical Center, Robley Rex VA Medical Center, Louisville, KY, United States
a r t i c l e
i n f o
Article history: Received 2 November 2013 Received in revised form 10 January 2014 Accepted 10 January 2014 Available online 4 February 2014 Keywords: Hepatitis HBsAg HBsAb Infection
a b s t r a c t Background: In most cases, patients appear to recover from acute hepatitis B virus (HBV) infection and do not exhibit the surface antigen (HBsAg). Chronic carriers are positive for HBsAg but HBsAb is usually not present. After acute infection only HBsAb remains. The presence of both HBsAg and HBsAb is unusual. Methods: We report on a patient whose results were analytically and clinically discrepant — positive for HBsAg and HBsAb on one testing platform but only HBsAg on another platform. Results: Reasons for this result: 1) Interference from endogenous antibodies; 2) HBsAg is from one strain and HBsAb is from another: and 3) The presence of HBsAb and HBsAg. Growing evidence indicates that both may be present in many patients. Low HBsAb may be neutralized and not recognized by the solid-phase HBsAg in the assay. Likewise, low HBsAg may be neutralized by HBsAb. It remains unclear whether HBsAg is always cleared after acute infection. Conclusions: Testing indicated that both HBsAb and HBsAb were present. The data shows that different testing platforms may produce different results depending on the kinetics, the exposure of the capture HBsAg and the extent of endogenous HBAg/HBsAb. We demonstrate a simple way to rule out test interference to presumptively identify true HBsAb. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Case
Hepatitis B virus (HBV) is a hepadnavirus that contains a core or nucleocapsid, an enclosing DNA and an envelope or hepatitis B surface antigen (HBsAg), encoded by the S gene [1]. There are four major serotypes, adr, adw, ayr, and ayw, all of which contain the “a” determinant that is common to HBsAg. Prince and colleagues described the HBsAg confirmation by neutralization, laid down the foundations for understanding interference with two-site immunometric assays by heterophile antibodies [2]. A key to these discoveries was the biology of the virus that is unique since it is the only animal virus that produces its coat in great excess. Here, we report on a patient that tested positive for HBsAg on two different platforms and positive for HBsAb on one. In investigating these peculiarities, we present evidence that the positive HBsAb was not due to interference such as heterophile antibodies but true HBsAb. We also demonstrate a relatively simple way to help resolve this issue. The data also shows why different platforms might give rise to discrepant results. Moreover, this case brings attention to the growing pathophysiological view that apparent acute HBV infection may have long lasting occult effects.
A 40-year-old female with Central African origin tested positive for hepatitis B surface antigen (HBsAg) with a signal intensity of 7440 (reference interval (RI) N 5.00 is considered positive). The patient was also positive for antibody to hepatitis surface antigen (HBsAb) with a titer of 26.7 mIU/ml (RI ≥ 12.0 mIU/ml is considered positive) when assayed by the Vitros ECi (Ortho Clinical Diagnostics). This instrument uses a 2-site immunoassay with purified human source HBsAg to bind HBsAb. Examination of the patient record showed that hepatitis B virus DNA (HBV DNA) measured by RT-PCR (Quest Diagnostics) resulted in 821 copies/ml (RI N 116 copies/ml). This test was performed to assess the certainty of infection. The hepatitis B e antigen (HBeAg) and hepatitis B e antibody (HBeAb) were both nonreactive. Also, IgM antibody against hepatitis B core antigen (HBcAb) was negative. A follow-up evaluation about 1.5 year after the first visit revealed that the aspartate and alanine aminotransferases were normal at 27 U/l (RI = 10–40 U/l) and 29 U/l (RI = 10–50 U/l), respectively. The result of the hepatitis B total core antibody test was reactive.7 Because of the unusual result that the patient was positive for both HBsAg and HBsAb, we retested HBsAg and HBsAb from the same sample on a similar platform (Vitros 3600, Ortho) and on a different platform in duplicate (Advia Centaur XP, Siemens Diagnostics). The Siemens platform
⁎ Corresponding author. E-mail address: [email protected] (T. Kampfrath). 0009-8981/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2014.01.016
172
T. Kampfrath et al. / Clinica Chimica Acta 430 (2014) 171–172
Table 1 Experiment results showing absorption of HBsAb by pre-incubation in HBsAg coated wells. Sample
Test
Result (quantitative)
Result (qualitative)
Patient prior to incubations HBsAb Patient (incubated for 45′ at 37 °C) HBsAb
20.1 mIU/ml 13 mIU/ml
Patient (incubated overnight at 4 °C) HBsAb HBsAg Control (incubation in HCV wells) HBsAb
4.3 ± 0.3 mIU/ml 7985 ± 65 19.5 ± 0.7 mIU/ml
Positive (Meaningfully less positive) Negative Positive Positive
also uses a two-site immunoassay for HBsAb with HBsAg. Both the Vitros ECi and the Siemens Advia Centaur use the same HBsAg subtypes Ad and Ay. The result for HBsAg was positive on both platforms while the result for the HBsAb test from the Vitros 3600 was positive but that from the Siemens was negative.
3. Discussion
3.2. Occult infection as an explanation for concomitant HBsAg and HBsAb In most cases, patients appear to recover from acute HBV infection and do not exhibit HBsAg but do exhibit HBsAb. Nevertheless, about 5% of infected persons develop chronic hepatitis and are at increased risk for cirrhosis and HCC. Of these, there are asymptomatic HBsAg carriers who are positive for HBsAg for more than 6 months but who exhibit no clinical evidence of disease and normal aminotransferases [4]. Some of these chronic carriers become HBsAg negative over a period of time called delayed clearance. Although the presence of HBsAg and HBsAb in the same sample is unusual there is growing evidence for “occult” infection in persons with acute hepatitis and apparent recovery [5]. Occult HBV infection is associated with very low levels of HBV DNA [6,7]. In this case, very low level 101–103 copies of HBV viral DNA are found in the liver or in blood mononuclear cells [7,8] and even in serum [7]. Although still controversial, it appears that in some people recovery from HBV may not be complete and that the immune system keeps the virus well controlled. Still, there is no evidence that persons with apparently normal recovery from acute HBV infection but who harbor very low levels of viral DNA are at increased risk for cirrhosis or hepatocellular carcinoma (HCC) [7]. But active infection may reoccur in cases of immunosuppression.
3.1. Testing for true positive HBsAb 3.3. Resolution of this case The HBsAb result was the same (positive) on both Ortho platforms but was negative on the Siemens even though it uses the same HBsAg subtypes for binding. Because of this discrepancy, we chose a simple work-up that causes little additional laboratory expense for presumptive identification of true HBsAb. We pre-incubated the patient's serum (100 μl per well) in the wells of the Vitros that are coated with HBsAg, either at 37 °C for 45 min, mimicking the assay procedure or overnight at 4 °C (Table 1). Afterwards the sample supernatant was transferred and reassayed on the Vitros. Although HBsAb titer in the patient's serum incubated at 37 °C for 45 min decreased, it was still considered positive. Due to antigen–antibody reaction kinetics, the ideal protocol to work up this case is to pre-incubate the patient's serum overnight at 4 °C [3]. This successfully absorbed out the HBsAb in the sample. The follow-up measurement for HBsAb appeared negative. As a control, the serum was also pre-incubated in wells coated with hepatitis C virus (HCV) recombinant antigens. In this case, the removal of HBsAb by pre-incubation indicates that it was true HBsAb so this patient's serum contained both antibody and antigen. Since there may be varying amounts of HBsAg and HBsAb in a sample, depending on the amount and accessibility of the capture antigen/antibody, different tests could provide different results, depending on exposure to different ratios of endogenous antigen/antibody. Thus, with one assay, if a HBsAg–HBsAb complex is exposed to a higher concentration or for a longer exposure to bound HBsAb, more antibodies may dissociate from a complex. This is very different from hepatitis C virus and human immunodeficiency virus infection where the antibody is in great excess and the presence of active infection virus can be best determined by molecular genetic techniques.
Clinically, there are 2 concerns with chronically infected patients. Might the patient develop cirrhosis? If so, should treatment with antiviral therapy be initiated? Second, patients who are chronically infected are at a higher risk of developing HCC and should be monitored for early identification of this condition by ultrasound and alphafetoprotein elevations. In the patient described here, the normal aminotransferases and low viral DNA levels suggest that treatment is not necessary. The positive HBsAb did not appear to be due to an interference. It is possible that the patient will eventually appear to clear the HBsAg. Until then, periodic screening for HCC is advised [5,9]. References [1] Lee W. Hepatitis B, virus infection. N Engl J Med 1997;337:1733–45. [2] Prince AMD, Brotman JB, Ikram H. Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis B antigen. Lancet 1973:1346–50. [3] Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960;39:1157–9. [4] Liaw YF, Sheen IS, Chen TJ, Chu CM, Pao CC. Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study. Hepatology 1991;13:627–31. [5] Lok AS, McMahon BJ. Chronic hepatitis B: AASLD practice guidelines. Hepatology 2007;45:507–39. [6] Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely “occult”. Hepatology 2001;34:194–203. [7] Conjeevaram HS, Lok AS. Occult hepatitis B virus infection: a hidden menace. Hepatology 2001;34:204–6. [8] Michalak TI, Pasquinelli C, Guilhot S, Chisari FV. Hepatitis B virus persistence after recovery from acute viral hepatitis. J Clin Invest 1994;93:230–9. [9] Nam SW, Jung JJ, Bae SH, et al. Clinical outcomes of delayed clearance of serum HBsAg in patients with chronic HBV infection. Korean J Intern Med 2007;22:73–6.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Resolving a case of concurrent hepatitis B virus surface antigen (HBsAg) and surface antibody (HBsAb) Thomas Kampfrath a,⁎, Saeed A. Jortani b, Stanley S. Levinson b,c a b c
Department of Pathology and Laboratory Medicine, Santa Clara Valley Medical Center, San Jose, CA, United States Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY, United States Laboratory Service, Department of Veteran Affairs Medical Center, Robley Rex VA Medical Center, Louisville, KY, United States
a r t i c l e
i n f o
Article history: Received 2 November 2013 Received in revised form 10 January 2014 Accepted 10 January 2014 Available online 4 February 2014 Keywords: Hepatitis HBsAg HBsAb Infection
a b s t r a c t Background: In most cases, patients appear to recover from acute hepatitis B virus (HBV) infection and do not exhibit the surface antigen (HBsAg). Chronic carriers are positive for HBsAg but HBsAb is usually not present. After acute infection only HBsAb remains. The presence of both HBsAg and HBsAb is unusual. Methods: We report on a patient whose results were analytically and clinically discrepant — positive for HBsAg and HBsAb on one testing platform but only HBsAg on another platform. Results: Reasons for this result: 1) Interference from endogenous antibodies; 2) HBsAg is from one strain and HBsAb is from another: and 3) The presence of HBsAb and HBsAg. Growing evidence indicates that both may be present in many patients. Low HBsAb may be neutralized and not recognized by the solid-phase HBsAg in the assay. Likewise, low HBsAg may be neutralized by HBsAb. It remains unclear whether HBsAg is always cleared after acute infection. Conclusions: Testing indicated that both HBsAb and HBsAb were present. The data shows that different testing platforms may produce different results depending on the kinetics, the exposure of the capture HBsAg and the extent of endogenous HBAg/HBsAb. We demonstrate a simple way to rule out test interference to presumptively identify true HBsAb. © 2014 Elsevier B.V. All rights reserved.
1. Introduction
2. Case
Hepatitis B virus (HBV) is a hepadnavirus that contains a core or nucleocapsid, an enclosing DNA and an envelope or hepatitis B surface antigen (HBsAg), encoded by the S gene [1]. There are four major serotypes, adr, adw, ayr, and ayw, all of which contain the “a” determinant that is common to HBsAg. Prince and colleagues described the HBsAg confirmation by neutralization, laid down the foundations for understanding interference with two-site immunometric assays by heterophile antibodies [2]. A key to these discoveries was the biology of the virus that is unique since it is the only animal virus that produces its coat in great excess. Here, we report on a patient that tested positive for HBsAg on two different platforms and positive for HBsAb on one. In investigating these peculiarities, we present evidence that the positive HBsAb was not due to interference such as heterophile antibodies but true HBsAb. We also demonstrate a relatively simple way to help resolve this issue. The data also shows why different platforms might give rise to discrepant results. Moreover, this case brings attention to the growing pathophysiological view that apparent acute HBV infection may have long lasting occult effects.
A 40-year-old female with Central African origin tested positive for hepatitis B surface antigen (HBsAg) with a signal intensity of 7440 (reference interval (RI) N 5.00 is considered positive). The patient was also positive for antibody to hepatitis surface antigen (HBsAb) with a titer of 26.7 mIU/ml (RI ≥ 12.0 mIU/ml is considered positive) when assayed by the Vitros ECi (Ortho Clinical Diagnostics). This instrument uses a 2-site immunoassay with purified human source HBsAg to bind HBsAb. Examination of the patient record showed that hepatitis B virus DNA (HBV DNA) measured by RT-PCR (Quest Diagnostics) resulted in 821 copies/ml (RI N 116 copies/ml). This test was performed to assess the certainty of infection. The hepatitis B e antigen (HBeAg) and hepatitis B e antibody (HBeAb) were both nonreactive. Also, IgM antibody against hepatitis B core antigen (HBcAb) was negative. A follow-up evaluation about 1.5 year after the first visit revealed that the aspartate and alanine aminotransferases were normal at 27 U/l (RI = 10–40 U/l) and 29 U/l (RI = 10–50 U/l), respectively. The result of the hepatitis B total core antibody test was reactive.7 Because of the unusual result that the patient was positive for both HBsAg and HBsAb, we retested HBsAg and HBsAb from the same sample on a similar platform (Vitros 3600, Ortho) and on a different platform in duplicate (Advia Centaur XP, Siemens Diagnostics). The Siemens platform
⁎ Corresponding author. E-mail address: [email protected] (T. Kampfrath). 0009-8981/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2014.01.016
172
T. Kampfrath et al. / Clinica Chimica Acta 430 (2014) 171–172
Table 1 Experiment results showing absorption of HBsAb by pre-incubation in HBsAg coated wells. Sample
Test
Result (quantitative)
Result (qualitative)
Patient prior to incubations HBsAb Patient (incubated for 45′ at 37 °C) HBsAb
20.1 mIU/ml 13 mIU/ml
Patient (incubated overnight at 4 °C) HBsAb HBsAg Control (incubation in HCV wells) HBsAb
4.3 ± 0.3 mIU/ml 7985 ± 65 19.5 ± 0.7 mIU/ml
Positive (Meaningfully less positive) Negative Positive Positive
also uses a two-site immunoassay for HBsAb with HBsAg. Both the Vitros ECi and the Siemens Advia Centaur use the same HBsAg subtypes Ad and Ay. The result for HBsAg was positive on both platforms while the result for the HBsAb test from the Vitros 3600 was positive but that from the Siemens was negative.
3. Discussion
3.2. Occult infection as an explanation for concomitant HBsAg and HBsAb In most cases, patients appear to recover from acute HBV infection and do not exhibit HBsAg but do exhibit HBsAb. Nevertheless, about 5% of infected persons develop chronic hepatitis and are at increased risk for cirrhosis and HCC. Of these, there are asymptomatic HBsAg carriers who are positive for HBsAg for more than 6 months but who exhibit no clinical evidence of disease and normal aminotransferases [4]. Some of these chronic carriers become HBsAg negative over a period of time called delayed clearance. Although the presence of HBsAg and HBsAb in the same sample is unusual there is growing evidence for “occult” infection in persons with acute hepatitis and apparent recovery [5]. Occult HBV infection is associated with very low levels of HBV DNA [6,7]. In this case, very low level 101–103 copies of HBV viral DNA are found in the liver or in blood mononuclear cells [7,8] and even in serum [7]. Although still controversial, it appears that in some people recovery from HBV may not be complete and that the immune system keeps the virus well controlled. Still, there is no evidence that persons with apparently normal recovery from acute HBV infection but who harbor very low levels of viral DNA are at increased risk for cirrhosis or hepatocellular carcinoma (HCC) [7]. But active infection may reoccur in cases of immunosuppression.
3.1. Testing for true positive HBsAb 3.3. Resolution of this case The HBsAb result was the same (positive) on both Ortho platforms but was negative on the Siemens even though it uses the same HBsAg subtypes for binding. Because of this discrepancy, we chose a simple work-up that causes little additional laboratory expense for presumptive identification of true HBsAb. We pre-incubated the patient's serum (100 μl per well) in the wells of the Vitros that are coated with HBsAg, either at 37 °C for 45 min, mimicking the assay procedure or overnight at 4 °C (Table 1). Afterwards the sample supernatant was transferred and reassayed on the Vitros. Although HBsAb titer in the patient's serum incubated at 37 °C for 45 min decreased, it was still considered positive. Due to antigen–antibody reaction kinetics, the ideal protocol to work up this case is to pre-incubate the patient's serum overnight at 4 °C [3]. This successfully absorbed out the HBsAb in the sample. The follow-up measurement for HBsAb appeared negative. As a control, the serum was also pre-incubated in wells coated with hepatitis C virus (HCV) recombinant antigens. In this case, the removal of HBsAb by pre-incubation indicates that it was true HBsAb so this patient's serum contained both antibody and antigen. Since there may be varying amounts of HBsAg and HBsAb in a sample, depending on the amount and accessibility of the capture antigen/antibody, different tests could provide different results, depending on exposure to different ratios of endogenous antigen/antibody. Thus, with one assay, if a HBsAg–HBsAb complex is exposed to a higher concentration or for a longer exposure to bound HBsAb, more antibodies may dissociate from a complex. This is very different from hepatitis C virus and human immunodeficiency virus infection where the antibody is in great excess and the presence of active infection virus can be best determined by molecular genetic techniques.
Clinically, there are 2 concerns with chronically infected patients. Might the patient develop cirrhosis? If so, should treatment with antiviral therapy be initiated? Second, patients who are chronically infected are at a higher risk of developing HCC and should be monitored for early identification of this condition by ultrasound and alphafetoprotein elevations. In the patient described here, the normal aminotransferases and low viral DNA levels suggest that treatment is not necessary. The positive HBsAb did not appear to be due to an interference. It is possible that the patient will eventually appear to clear the HBsAg. Until then, periodic screening for HCC is advised [5,9]. References [1] Lee W. Hepatitis B, virus infection. N Engl J Med 1997;337:1733–45. [2] Prince AMD, Brotman JB, Ikram H. Specificity of the direct solid-phase radioimmunoassay for detection of hepatitis B antigen. Lancet 1973:1346–50. [3] Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960;39:1157–9. [4] Liaw YF, Sheen IS, Chen TJ, Chu CM, Pao CC. Incidence, determinants and significance of delayed clearance of serum HBsAg in chronic hepatitis B virus infection: a prospective study. Hepatology 1991;13:627–31. [5] Lok AS, McMahon BJ. Chronic hepatitis B: AASLD practice guidelines. Hepatology 2007;45:507–39. [6] Brechot C, Thiers V, Kremsdorf D, Nalpas B, Pol S, Paterlini-Brechot P. Persistent hepatitis B virus infection in subjects without hepatitis B surface antigen: clinically significant or purely “occult”. Hepatology 2001;34:194–203. [7] Conjeevaram HS, Lok AS. Occult hepatitis B virus infection: a hidden menace. Hepatology 2001;34:204–6. [8] Michalak TI, Pasquinelli C, Guilhot S, Chisari FV. Hepatitis B virus persistence after recovery from acute viral hepatitis. J Clin Invest 1994;93:230–9. [9] Nam SW, Jung JJ, Bae SH, et al. Clinical outcomes of delayed clearance of serum HBsAg in patients with chronic HBV infection. Korean J Intern Med 2007;22:73–6.
