Interpretation of hepatitis B virus serology results by medical scientists

Interpretation of hepatitis B virus serology results by medical scientists S C Thompson 1, C G Ryan 2, R J Warren 3 1Epidemiology Unit, Macfarlane Burnet Centre for Medical Research, Fairfield VIC 3078; 2Microbiology Serology Department, Dorevitch Pathology, Camberwell VIC 3124; 3Microbiology Department, Victorian Infectious Diseases Reference Laboratory, Fairfield Hospital, Fairfield VIC 3078, Australia

Summary Medical laboratory scientists who had agreed to participate in an evaluation which required them to measure the level of anti-HBs in a panel of sera, were given a brief clinical scenario for seven of the results and asked to comment on the person's immune status. The interpretations of immune status were assessed against the matters considered by the authors to reflect the current state of knowledge about immunity to hepatitis B and recommendations from authorities. There were a number of common but not universal misconceptions in the interpretation of results and current guidelines, particularly regarding appropriate use of hepatitis B immune globulin (HBIG), equating the absence of anti-HBs in a previous seroconverter with no immunity or the need to institute a new course of vaccination, and inappropriate recommendations for further testing or retesting. Given that the cost of serology for anti-HBs now exceeds the cost of three doses of vaccine, more effective and rational testing of immune status to hepatitis B is required. Key words: Hepatitis B, serology, interpretation, immune status, anti-HBs Serodiagn. Immunother. Infect. Disease 1995, Vol. 7, 64-69, June

Introduction Less than a decade ago, a safe and effective vaccine against hepatitis B virus (HBV) became available. Initially the vaccine was derived from the plasma of chronic H B V carriers, was in limited supply and was very expensive, so its use was reserved for individuals who belonged to identified high-risk groups. The development of vaccines prepared by recombinant technology enabled the vaccine to be produced in far greater quantities and this was associated with a marked drop in the cost of a vaccine course. This has meant, first, that the cost of serological testing (e.g. for antibodies to the surface antigen of the hepatitis B virus (anti-HBs)) significantly exceeds the unit cost for a dose of the vaccine. Currently in

Received: 1 August 1994 Reviewed: 11 September 1994 Accepted: 4 November 1994 Correspondence and reprint

requests to: SC Thompson, Epidemiology Unit, Macfarlane Burnet Centre for Medical Research, PO Box 254, Fairfield VIC 3078, Australia 0888-0786/95/$09.50© 1995 Elsevier Science B.V. All rights reserved

Australia, the cost of testing for anti-HBs approximates the vaccine cost for a standard three-dose vaccination course against HBV. Second, the indications for vaccination have broadened. Universal childhood vaccination has already been introduced into a number of countries and the World Health Organisation has recommended universal H B V vaccination be implemented in all countries by 1997. The highly infectious nature of H B V has been recognized by the increasing provision of H B V vaccination in an occupational health and safety setting to personnel with a limited likelihood of blood exposure. Medicolegal and economic considerations influence policy in these circumstances, with the obligation on employers in Australia to take reasonable precautions to protect their employees and to compensate workers who develop illness caused through their work. Despite increasing use of the vaccine, some fundamental queries remain unanswered about its cost-effectiveness. Important among these are the need for serological testing to determine initial seroconversion, the adequacy of protection when antibody levels have fallen below a 'protective' level, the need for booster

Thompson et al.: Interpretation of anti-HBs testing doses of vaccine and whether this should be based upon determination of antibody status or upon mass standardized estimates of the likely duration of protection in those with an initially protective level of antibody 1-7. The need for initial seroconversion to an anti-HBs level of 10 m l U ml -I in order to ensure immunity to H B V has been clearly demonstrated 8, and increasingly public health bodies have been recommending that post-vaccination serological testing be performed 7,9-1°. In conjunction with the documented p o o r e r seroconversion rate in older adults H, testing for anti-HBs has become a significant add-on cost to adult H B V immunization programmes. In addition to more confidently knowing the immune status of the individual, the performance of laboratory tests quantitating antiHBs seems central to increasing our knowledge about post-vaccination immune status. Studies conducted in research settings will eventually enable these questions to be definitively answered with greater confidence. Meanwhile, many doctors and o t h e r s c o n c e r n e d with occupational health, must make decisions about serological testing and vaccination based upon their clinical experience with the vaccine and interpretation of the tests available. In these circumstances, laboratories are often called upon to provide interpretation of the antibody levels which they have measured. Not uncommonly, the accompanying clinical information is sketchy. Whilst the information provided on a pathology report form is signed out by an accredited pathologist or senior scientist, informal queries over the telephone are regularly addressed by the laboratory staff who perform the test. This group may not have received updates on the interpretation of hepatitis serology in association with clinical information, or be acquainted with recent literature regarding duration of immunity following H B V vaccination. Since there are no simple tests which measure cellular immunity, measurement of antibody levels has been used as the measure of 'protection'. Given the importance of anti-HBs as a measure of immunity, a panel of sera was prepared and sent to laboratories which had expressed a willingness to participate in a quality assurance exercise. This paper examines the interpretation of anti-HBs provided for seven results where an accompanying brief clinical scenario was supplied. It must however be recognized that many of the responding scientists would in normal practice refer enquiries, particularly those regarding vaccine and immunoglobul.in administration, to a clinician and in fact it is probable that in formulating the replies given, the scientists would have collaborated with clinicians. Methods

Included within a larger panel for anti-HBs testing were seven serum specimens with accompanying case scenarios. The scientist was invited to test the specimens and make comments about the person's H B V immune status based on the test results. Participants

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were not invited to make any comments about management with respect to other bloodborne viruses. Case scenarios Case 1. A laboratory worker who completed a three dose im course of recombinant H B V vaccine 6 weeks previously. Case 2. A doctor who had been vaccinated against H B V as a medical student 3 yr previously and had just sustained a needlestick injury. No serology had been performed post-vaccination. Case 3. A doctor vaccinated against H B V as a medical student 3 yr ago who had just sustained a needlestick injury. Serological testing 4 weeks after the third vaccination had shown that she had seroconverted. Case 4. An ethnic health worker with no clinical history of past infection who was being tested as part of prevaccination screening. Case 5. A healthcare worker involved in a needlestick accident who had not completed the full vaccination course at the time of the injury. Case 6. An ambulance officer who finished a three dose im course of H B V vaccine 4 weeks previously. Case 7. An 18 yr-old injecting drug user (IDU) with a history of possible jaundice. Results

A total of 19 laboratories participated in the study; not all laboratories provided answers for each scenario. Case 1. The mean anti-HBsAg of the different tests was 24mlUm1-1 (so =7.3; range 18-45). Seventeen laboratories made comments, and all except one (94%) noted that the person had seroconverted. The one exception commented that the person required a booster, without commenting on the level of immunity. A total of 10 (59%) of the respondents noted that the antibody level was low and eight respondents (47%) advised a booster. One of those respondents who noted the low level of immunity commented that the response could just be slow, and advised retesting in 3 months followed by a booster if the level remained low. A n o t h e r response also suggested the need to retest in 3 months to see if levels had declined, stating that the need for this depended upon the age of the worker. Case 2. The mean anti-HBs value was 0 m l U ml-1; the only laboratories with values not equal to zero reported results of 0.14 and 1.0 mIU ml-L Nineteen laboratories made comments related to this case study,

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16 (84%) noting that there was no evidence of immunity. Seven respondents noted the need for HBIG. Twelve respondents suggested further vaccination, two of these suggesting a new course. One respondent commented only to "consider revaccination". Seven laboratories noted the need to check the person's serology following further vaccination. Two laboratories m a d e comments suggesting that serum from the doctor be retained or further serological testing be performed. T w o laboratories suggested performing testing for antibody to hepatitis core antigen (anti-HBc), with the result influencing whether a booster was given in one instance or whether further testing for H B s A g was undertaken. Case 3. All laboratories reported the result as negative with the exception of one kit which gave a value of 8 m l U m1-1. O f 17 respondents, 16 c o m m e n t e d that there was no protective level of antibody and 11 that the doctor should receive further vaccination, with two responding "consider revaccination". Five respondents suggested that H B I G be given. Five responses suggested that the absence of antibody in this situation equated with no immunity, two even suggesting that a whole new course of vaccine be given. T w o respondents suggested further serological testing for H B s A g and anti-HBc. Case 4. The m e a n a n t i - H B s A g of the different tests was 103mlUm1-1 (SD = 21; range 80-139). O f 18 respondents, 14 c o m m e n t e d that the result was consistent with past exposure, and nine of these suggested confirming this with anti-HBc. Thirteen respondents noted that the person was i m m u n e so that there was no further need for vaccination, and two suggested the need to recheck antibody levels. Case 5. The a n t i - H B s A g m e a n of the tests was 75 m l U m1-1 (so = 27; range 41-123). All (100%) of the 15 respondents noted that there was evidence of a protective level of immunity, and 12 of these noted that it was at a low level and/or suggested finishing the p r i m a r y course. One respondent, where the antibody level had b e e n quantitated at 92 m l U m1-1, c o m m e n t e d that the worker was " p r o b a b l y i m m u n e " and suggested further testing after the third dose of vaccine. Case 6. The anti-HBs value on testing was 0 m l U m1-1, a value obtained by all participating laboratories. O f 17 respondents to this case scenario, 15 noted that there was no seroconversion or evidence of immunity, and 12 that there was a need for additional doses of vaccine. O f those advising further vaccine, only seven c o m m e n t e d on the need for testing to confirm seroconversion following the extra dose(s). T h e r e were two respondents who suggested waiting a longer period and rechecking the antibody levels, and one respondent suggested to k e e p on vaccinating until the ambulance driver did seroconvert. Explicit advice

about the need to counsel the worker about his continued susceptibility was not given by any laboratory and nor was there any suggestion that this was anything other than a vaccine failure, neglecting the possibility that this person could be a H B V carrier. Case 7. This specimen also had an anti-HBs of 0 m l U ml -a, a value obtained by all laboratories with the exception of R I A - A U S A B 2 which obtained a value of 1 mlUm1-1. O f 18 respondents, 11 c o m m e n t e d that there was no evidence of immunity in this I D U , 15 suggested that the person should be checked for H B s A g and one suggested testing for anti-HBc. Only three respondents noted that if the person was not a carrier, H B V vaccination should be performed.

Discussion A u t h o r s ' interpretation The responses which the authors considered important in advising a course, based upon the serological results, are outlined below and are based upon r e c o m m e n d a tions of public health authorities 1z,13. Case 1. Since antibody levels decline, this person would likely have undetectable or a 'non-protective' level of antibody within a short period of time, so m a n y experts would r e c o m m e n d that the person receive another dose of vaccine now to boost antibody levels further. As the person has seroconverted, one could argue that there is no need to recheck the antibody level after the extra booster. The time interval between the final dose of the vaccine course and testing was adequate. Case 2. Since there was no knowledge of whether this doctor had ever seroconverted and therefore whether immunological m e m o r y had been induced, immunity against hepatitis B m a y not exist. Ideally, the source of the needlestick injury should be tested to ascertain H B V infectivity. In the absence of this information maximal protection would be achieved by the doctor receiving H B I G as well as a booster dose of vaccine. However, given the risk of acquiring H B V from an unknown source in a country with a low level of chronic H B V carriage, availability and cost constraints m e a n that this extra prophylaxis is not generally administered. If the source of blood contaminating the needle/syringe was not HBsAg+ve, then a H B V booster would suffice. It would also be prudent to retain serum from the doctor to check for H B s A g if necessary. Serum should be taken in 4 weeks time to check for seroconversion following the booster. Case 3. In such an instance of past seroconversion but no current protective level of anti-HBsAg, it was considered that the current advice was that a booster dose of vaccine should be given immediately, but that there was no need to give H B I G .

Thompson et al.: Interpretation of anti-HBs testing Case 4. This person had evidence of past exposure to H B V , indicating past infection or vaccination. Demonstration of anti-HBc would confirm previous infection with HBV. Approximately two-thirds of those who contract H B V do not have classical symptoms of H B V infection and for m a n y of those who are infected, intensive questioning will fail to define any risk factor or reveal the source of their infection. Since in this case there is evidence of immunity to H B V , vaccination is not required. H o w e v e r given the relative costs of serological testing and vaccination, very few groups in Australia justify prevaccination screening. Case 5. Although this person had not completed the course of H B V vaccine, there is evidence of immunity to H B V , and further t r e a t m e n t or vaccination to protect against the immediate risk of H B V infection is not required. However, because of the relatively low levels of anti-HBs, it would be reasonable to give a further dose of vaccine immediately and the course should be completed. Case 6. This person had failed to seroconvert after the standard immunization protocol. Approximately onethird of such cases would seroconvert after additional dose(s) of vaccine, although there is no consensus as to how this should be achieved, i.e. with one or two standard doses of vaccine at least 1 m o n t h apart or by giving a double dose of antigen. Since an ambulance officer is at high risk of exposure to H B V , further vaccination should be undertaken in an attempt to induce immunity and the officer must be counselled regarding susceptibility to infection with H B V and should receive H B I G in the event of known potential exposure. T h e r e is a possibility that people who fail to seroconvert may be carriers so if no anti-HBs is detected following appropriate additional vaccine doses the vaccine recipient should be checked for HBsAg. Case 7. This person showed no evidence of immunity to hepatitis and although the rationale for performing the test was not clear, belongs to an identified high risk group for H B V infection. The history of possible jaundice is not helpful one way or the other, although people who develop clinical disease in association with acute H B V infection are less likely to become p e r m a n e n t carriers. The serum could be checked for evidence of hepatitis A or C infection; jaundice may also be a result of noninfective causes of liver pathology. The fact that this person showed no evidence of immunity to H B V m a y rule out previous exposure, but the possibility of the carrier state should be ruled out by determining H B s A g status. Nonexposed, noninfected I D U who share needles should be vaccinated against HBV. Discussion o f responses T h e r e a p p e a r e d to be some misconceptions over the appropriate use of H B I G . The need to administer H B I G was stated by seven laboratories in Case 2,

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where the scenario described a needlestick injury in a healthcare worker who had never been tested to demonstrate production of protective antibody. The need for H B I G in such circumstances depends upon the source of the blood. It might be argued that where this information is unknown maximal protection occurs if H B I G is given, since no evidence of protection against H B V has been demonstrated. H o w e v e r the risk of acquiring H B V from a source of unknown status in a person who had completed a course of H B V vaccination is very low 14. In fact, in the UK, even in an unvaccinated healthcare worker, an accelerated course of vaccination is the r e c o m m e n d e d procedure 15.16. In the third case, five laboratories suggested H B I G be given and in a person with previously d o c u m e n t e d adequate protection following vaccination, this is unnecessary and wasteful of a blood product which is expensive and in limited supply. Case 3 also d e m o n s t r a t e d that some respondents equated the absence of antibody with no immunity despite the history of past seroconversion. Although antibody levels wane, there is minimal evidence that a seroconverter is at risk of the serious sequelae of H B V even after antibody levels are undetectable or have fallen below 10 m l U ml-L Although antibody levels have been used as the surrogate measure for a person's i m m u n e status, active immunization stimulates both humoral and cellular immunity and the anamnestic response can be rapid even in the absence of persisting antibody 17. A single booster is required and serological follow-up is not necessary. T h e r e was a tendency (Cases 1 and 5) to suggest that a low antibody level was a result of an inadequate time interval before serological testing. In the scenarios given, this was not a reasonable explanation for a low antibody level nor was there any reason on finding a low antibody level (say < 1 0 0 m l U m l -~) to suggest rechecking anti-HBs levels in a few months, since almost certainly the levels will be lower. The relative cost of providing vaccination c o m p a r e d to antibody testing should be taken into account in the advice given and currently it is cheaper to give a booster vaccination rather than to k e e p re-assaying antibody levels until a person needs vaccine because of inadequate immunity. Similarly, in a person who has (presumably) recovered from a natural infection, there is no point in continuing to recheck their anti-HBs levels since ongoing vaccination is not necessary. With a n o n r e s p o n d e r after a primary course (such as Case 6), a n u m b e r of the serologists c o m m e n t e d that the vaccinee required a whole new course. Although there is uncertainty as to how nonresponders should be treated, reasonable advice would be to administer additional doses and then retest for seroconversion 2. (The need for follow-up serology should be stressed.) Others have advocated giving a double dose of vaccine, a protocol favoured for renal dialysis patients ~8-2°. The degree of risk of H B V to which an individual is exposed will influence how persistent one is in trying to achieve seroconversion and it appears that some

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individuals, perhaps because of their genotype, will never seroconvert. One or two further boosters will result in seroconversion in - 3 0 - 5 0 % of those who failed to respond to the primary courseZl.2L Somewhat surprising was the advice given by a couple of respondents to measure hepatitis core antibody in situations where it was not appropriate. For example, although it might be reasonable to check whether Cases 2 or 7 were H B s A g carriers, there would seem to be little benefit in checking for core antibody as a prelude to looking for persistent HBsAg; this only results in extra serological testing before performing the test which will ascertain the person's immune status. Three other remarks are worthy of comment. First, people are not automatically (re)tested following a needlestick injury, as was stated by one respondent. A few respondents prefaced advice with "consider", but clearly consideration does not necessarily lead to action and where an intervention is indicated, this should be stated without such qualification. Finally, there is the use of terminology such as 'low level of immunity' and 'probable immunity'. This raises the question of what level of antibody indicates immunity, whether clinical details need to be taken into account when reporting results, or whether reporting reflects only the objective antibody level as determined by the assay of anti-HBs. Clearly, the clinical context must influence the action required in response to a particular test result and there would be benefit in ongoing training of laboratory scientists in the interpretation of tests in association with clinical case scenarios. This analysis of responses received from laboratory scientists who perform serological testing does not in any representative way address the extent of their knowledge about H B V or their competence in performing serological testing. Many laboratory staff do not give advice but refer queries to senior laboratory personnel or medical staff in either the microbiology d e p a r t m e n t or the staff clinic. However, in so far as the participants have responded to these questions, the analysis does highlight where there are certain misconceptions in interpretation, or where the advice which an enquiring doctor might receive by 'phone could fail to address some key factor which ensures a person's most appropriate m a n a g e m e n t with respect to adequate protection against HBV. T h e r e is no reason to think that many doctors who graduated before or subsequent to these tests becoming widely available, would be any better at interpreting the results of the tests and many would in fact rely upon the advice given by the laboratory. It should also be recognized that there is no unanimity of opinion as to what constitutes adequate immunity and when booster doses of vaccine are required. However, where advice from the laboratory differs from current advice recomm e n d e d by the expert committees who decide these public health policies based upon current knowledge, it may lead either to failure to ensure adequate protection of a person at risk of H B V , or unnecessary

testing, vaccination or other prophylaxis which increases expenditure in the health budget with no beneficial outcome.

Acknowledgements We would like to thank the members of the Victorian Branch of the Serology Special Interest Group of the Australian Society of Microbiology who participated in the study and the following pathology laboratories: Alfred Hospital, Amersham Pty Ltd, Ballaarat Base Hospital, Box Hill Hospital, Fairfield Hospital, Geelong Hospital, Gippsland Base Hospital, Gippsland Pathology, Goulburn Valley Base Hospital, LaTrobe Regional Hospital, Mercy Hospital, Microbiological Diagnostic Unit, Monash Medical Centre, Red Cross Blood Bank, St Vincent's Hospital, Warrnambool Base Hospital, Western Hospital. We also thank Fairfield Hospital and Dorevitch Pathology for material assistance and CSL Ltd. for distribution of serum panels.

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Thompson et al.: Interpretation of anti-HBs testing 15 PHLS Hepatitis Subcommittee. Exposure to hepatitis B virus: guidance on post-exposure prophylaxis. CDR Review 1992; 2:R97-R101 16 Morris D J, O'Neil EM, McCloy EC et al. Hepatitis B surface antibody responses following accelerated vaccination. J Hosp Infect 1993; 23:317-19 17 Thompson SC, Darlington R, Tallent D, Forsyth JRL. Booster doses of hepatitis B vaccine: response to low dose inoculations. Med J Aust 1993; 158:375--8 18 Bergami F, Zanetti AR, Ferroni P e t al. Immune response to hepatitis B vaccine in staff and patients in renal dialysis units. J Infect 1983; 7 (Suppl 1): 35-40

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19 Desmyler J, Coalaert EE, Reerink-Brongers et al. Efficacy of heat-inactivated hepatitis B vaccine in haemodialysis patients and staff. Lancet 1983; 2:1323-7 20 Stevens EE, Alter H J, Taylor PE, Zang EA, Harlet E J, Smuzness W. Hepatitis B vaccine in patients receiving hemodialysis. N Engl J Med 1984; 311:496-501 21 Wismans P, van Hattum J, Stelling T, Poel J, de Gast GC. Effect of supplementary vaccination in healthy non-responders to hepatitis B vaccination. Hepatogastroenterol 1988; 35:78-9 22 Craven DE, Awdeh ZL, Kunches LM et al. Non responsiveness to hepatitis B vaccine in health care workers. Ann Intern Med 1986; 105:356-60