A Population-based Seroprevalence Study of Hepatitis A Virus Using Oral Fluid in England and Wales

M. C. Morris-Cunnington1,2 , W. J. Edmunds1, E. Miller1 and D. W. G. Brown3

1 Communicable Disease Surveillance Centre, Health Protection Agency, London, United Kingdom.
2 Infectious Disease Epidemiology Unit, London School of Hygiene and Tropical Medicine, London, United Kingdom.
3 Enteric, Respiratory, and Neurological Virus Laboratory, Central Public Health Laboratory, Health Protection Agency, London, United Kingdom.

Received for publication July 8, 2003; accepted for publication November 18, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Population-based seroprevalence studies provide important data on susceptible groups and the potential for future outbreaks. However, the invasive nature of serum collection has limited studies. This paper describes the first postal population-based survey using noninvasive oral fluid technology to collect antibody prevalence data in conjunction with extensive risk factor data to assess the distribution of immunity to common viral infections in England and Wales. These results pertain to hepatitis A virus (HAV). Approximately 5,500 oral fluid samples were collected between August 2001 and May 2002, as well as individual risk factor data through a questionnaire, from persons aged less than 45 years randomly sampled from general practices countrywide. Samples were tested for immunoglobulin G-specific antibody marking a past infection or immunity to HAV using an antibody-capture enzyme-linked immunosorbent assay. The age-specific HAV seroprevalences indicated a low incidence of infection (overall seroprevalence of 18.9% (95% confidence interval: 17.0, 20.9) and of 9.2% (95% confidence interval: 7.1, 11.3) after the exclusion of vaccinees). Vaccination proved the most important determinant of seropositivity. Ethnic minority groups were underrepresented, and adjustment increased the overall prevalence to 20.1% and to 12.1% in unvaccinated individuals. The availability of comprehensive risk factor data allowed the description of two risk profiles related to natural infection and vaccination.

hepatitis A virus; immunity; saliva; seroepidemiologic studies

Abbreviations: Abbreviations: HAV, hepatitis A virus; GPRF, General Practice Research Framework.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Data on the proportion of the population that is immune or has been infected with a specific virus have many important epidemiologic applications. These include the identification of susceptible groups in the population, the evaluation of health programs (e.g., vaccine uptake), and the use of these data in mathematical modeling to predict outbreaks (1, 2).

The most accurate way to monitor population immunity is through serologic surveillance. Sera can be collected from a randomly selected population-based sample, or residual serum banks can be accessed. Unfortunately, both methods have serious drawbacks. The former technique is expensive requiring trained individuals to visit participants to collect a serum sample, and low response rates are common in Europe given the invasive nature of serum collection (3, 4). The use of residual sera (e.g., from routine health-care testing) overcomes many of these problems, but samples are usually anonymous, meaning very few background data on the patient are available (usually only age and sex), and may not be representative of the general population (5).

The development of laboratory tests able to detect virus-specific antibody markers in oral fluid offers a solution to many of these problems. The minimally invasive nature of oral fluid sample collection makes the self-taking of samples possible, reducing costs and potentially increasing response rates. This paper reports the first population-based postal survey in England and Wales (and to our knowledge only the second such study worldwide) and the first study anywhere to collect oral fluid samples in conjunction with detailed demographic and social data through a questionnaire. The results presented here pertain to immunity to hepatitis A virus (HAV).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The study aimed to select a representative sample of individuals aged 0–44 years in England and Wales (after this age there are relatively few new HAV infections (6)). This was achieved through the Medical Research Council General Practice Research Framework (GPRF), which allowed access to individual children and adults through the registers of practices that had agreed to participate in research studies (7). The Medical Research Council GPRF consists of over 1,000 practices covering about 10 percent of the population in the United Kingdom. Although practices in the GPRF are not representative of general practices (tending to be larger than the average), their patients are broadly typical of United Kingdom norms (7). Further, there are sufficient practices across different geographic and socioeconomic locations to allow practices of all types to be represented in a recruited sample, giving access to a representative sample of the general population (3, 7).

A stratified and clustered study design was used, sampling the general practices as the primary/cluster unit. During recruitment, the practices were stratified first by location (north, mid, south, or London according to the Royal College of General Practitioners’ sentinel surveillance boundaries (8)) and then by the Carstairs’ deprivation score (deprivation tertiles from 1991 electoral ward census data) (9, 10). Between two and four practices were recruited within each of the 12 strata. Lower deprivation tertiles were oversampled, as such areas are often less well represented in surveys (11, 12). A total of 40 general practices were sampled nationally.

The sample size calculations, which incorporated a design effect, have been described elsewhere (13). These aimed to estimate the age-specific seroprevalences of HAV (based on a 1996 serosurvey (14)) with adequate absolute precision (±1.5–3.0 percent). A higher precision was required for the children 0–4 years of age who may represent a peak in transmission (1, 14). After accounting for the expected nonresponse (50 percent) (4) and practice register inaccuracies (10 percent) (3), we found that the calculations gave a sample size of 372 randomly selected individuals per practice, giving 14,800 individuals overall.

The sample collection has been described in detail elsewhere (13). Briefly, the practice nurses generated a random patient sample by computer and scanned the relevant patient notes to ensure eligibility (no terminal or psychiatric illness or recent bereavement). Selected individuals were sent a letter of invitation and an information leaflet explaining the study, as well as an oral fluid sample kit and a questionnaire. The targeted individuals were asked to self-take an oral fluid sample using an Oracol swab (Malvern Medical, Worcestershire, United Kingdom), a sponge swab which is rubbed along the teeth and gums for approximately 1 minute, and to complete a questionnaire collecting demographic and social information (age, sex, ethnicity, household size, occupation, time spent living abroad as a child, and HAV vaccination status). Samples and questionnaires were returned in the packaging provided to a free postal address at the Central Public Health Laboratory (now Health Protection Agency) in London, United Kingdom. Nonrespondents received up to two postal reminders. Note that participants were not given any financial inducement to take part nor were they offered the results of their tests.

The contents of the Oracol swab were recovered using a method previously described (15). Two different anti-HAV immunoglobulin G assays (an in-house enzyme-linked immunosorbent assay and an in-house radioimmunoassay) were used to ascertain antibody serostatus, both based on an antibody-capture technique. Given the complexities of the radioimmunoassay (16), the availability of a large number of oral fluid samples was used to develop a more time-efficient and practical antibody-capture enzyme-linked immunosorbent assay (16). Both assays were validated (or revalidated) using 120 paired serum-oral fluid samples with the oral fluid results compared with those from a "gold standard" serum competitive radioimmunoassay (17, 18). The enzyme-linked immunosorbent assay had a sensitivity that was slightly superior to that of the oral fluid radioimmunoassay (92.5 percent vs. 87 percent, respectively; both had 100 percent specificity), but the difference proved nonsignificant at the 5 percent statistical level ({chi}2 = 1.28, p = 0.26) and all data were analyzed together. As vaccinated individuals may have lower anti-HAV immunoglobulin G concentrations, the assay cutoff value was reviewed within vaccinated individuals using mixture modeling (19). The distribution of anti-HAV immunoglobulin G reactivities (adjusted absorbance values) within vaccinated and nonvaccinated individuals was very similar, indicating that the same cutoff was appropriate for both groups (data available upon request).

All statistical analyses were performed in STATA 6.0 (Stata Corporation, College Station, Texas) software. Seroprevalence estimates and risk factor analyses were adjusted for the stratified and clustered survey design (20). Population weightings were allocated to each individual to correct estimates for differential response and seroprevalence by age, geographic location, and social deprivation. Final seroprevalence estimates were also adjusted for the test sensitivity and specificity. Risk factor analyses were completed using a logistic regression model with HAV seropositivity as the dependent variable. All risk factors that proved significant at the 10 percent statistical level in single-variable models were entered into a multivariable analysis where the 5 percent level was taken as the significance cutoff. All possible two-way interactions were investigated.

Ethical approval for the study was granted by the North Thames Multi-Regional Ethics Committee and by the ethics committees of the Public Health Laboratory Service and the London School of Hygiene and Tropical Medicine.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The population-based sample
A total of 14,398 individuals from 40 general practices were invited to participate. Of those 5,457 returned samples, there were 43 without a questionnaire, 10 with a blank questionnaire, and 13 from individuals over 44 years of age. This left 5,391 individuals with complete data for analysis and an overall survey design adjusted response of 40 percent. Response rates were lowest in adult males and in urban areas (13).

The main characteristics of the sample are described in table 1. Comparisons with the general population indicated a generally good agreement. However, there was an underrepresentation of ethnic minority groups (4.4 percent non-White vs. 8.7 percent in the 2001 census in England and Wales), as well as an overrepresentation of the higher social classes (40 percent with managerial occupation vs. 27 percent in the 2001 census). Very few individuals had lived abroad in childhood (5 percent), and knowledge of HAV vaccination status was relatively poor with over 30 percent of respondents unsure of their status.


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TABLE 1. Characteristics of the survey sample and factor-specific hepatitis A virus seroprevalence, England and Wales, 2001–2002
 
HAV antibody prevalence
The age-specific HAV antibody prevalence estimates are shown in figure 1. The overall prevalence in the present study rose steadily from 4.0 percent in children 1–4 years of age to 10.6 percent and 14.1 percent in persons aged 5–14 and 15–19 years, respectively. After a further increase to 23.8 percent in persons aged 20–24 years, the increase leveled off to a prevalence of 26.2 percent in the oldest age group (figure 1). The antibody prevalence in males and females (table 1) was very similar until age 20–24 years, when the female prevalence appeared higher than the male prevalence (28.0 percent in females vs. 17.6 percent in males).



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FIGURE 1. Comparison of hepatitis A virus seroprevalence estimates in England and Wales from the 2001–2002 oral fluid survey with a 1996 population-based seroprevalence survey by Morris et al. (14). Point seroprevalence estimates are shown with their 95% confidence intervals.

 
As ethnic minority groups were underrepresented in this survey and appeared to exhibit distinct HAV seroprevalence trends (tables 1 and 2), a sensitivity analysis was completed. When the overall HAV antibody prevalence was standardized to the ethnic distribution of the 2001 census, the estimate rose from 18.9 percent to 20.1 percent. Social deprivation was adjusted for in the analysis using weightings, but these were derived from practice, not individual, level information. However, when the overall antibody prevalence estimate was standardized to the occupational class (measure of socioeconomic class) distribution of the 2001 census, the estimate remained the same. The weighting adjustment therefore appeared adequate for social class.


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TABLE 2. Factors independently associated with hepatitis A virus seropositivity in a multivariable logistic regression, England and Wales, 2001–2002
 
Risk factors for immunity to HAV
The initial single-variable logistic regression models confirmed increasing antibody prevalence with age and in females. HAV antibody prevalence was also higher in London than in the north of England and Wales (27.8 percent vs. 16.9 percent), in all ethnic minority groups compared with Whites (41.1 percent in Blacks vs. 18.2 percent in Whites), in individuals who had lived abroad as children (36.3 percent "yes" vs. 17.6 percent "no"), and in those self-reporting vaccination (any type) compared with nonvaccinees (56.1 percent "yes" vs. 9.2 percent "no"). The prevalence was lower in individuals with manual occupations compared with managers (13.2 percent vs. 22.9 percent) and in those living in council rented property compared with homeowners (13.9 percent vs. 18.8 percent). All these trends proved significant at the 10 percent statistical level, and the factors were entered into a multivariable model (table 2).

There was a significant interaction between age and sex (p < 0.001), and the odds of seropositivity rose more steeply in females than in males, although the sex difference held only for adults (table 2). The occupational, ethnicity, and vaccination trends remained significant at the 5 percent level in the multivariable model. When the type of HAV vaccination was analyzed, seropositivity was greatest among recipients of the inactivated vaccine (56 percent). The high percentage who were seropositive among individuals reporting receipt of human normal immunoglobulin was surprising, given the short-term immunity conferred (4–6 months).

The antibody prevalence among unvaccinated individuals was 12.1 percent after adjustment for the underrepresentation of ethnic minority groups (compared with 20.1 percent in the total data set), indicating a low incidence of infection in the population and the increasing importance of vaccination in determining serostatus. Figure 2 compares the age-specific antibody prevalence of HAV with the age-specific self-reporting of vaccination receipt.



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FIGURE 2. Comparison of the percentage immune to hepatitis A virus (HAV) with the percentage self-reporting HAV vaccination in England and Wales in 2001–2002. HNIG, human normal immunoglobulin.

 
The percentage immune to HAV closely matched the percentage reporting receipt of the inactivated vaccine in most age groups. Relatively few individuals reported receipt of human normal immunoglobulin or did not know which vaccine they had received. The age group of those less than 1 year of age was the only group in which the percentage immune was higher than the percentage reporting any type of vaccination (~10 percent vs. 5 percent).

The risk of natural HAV infection (measured as seropositivity in nonvaccinees), determined through a further logistic regression analysis, increased with age and female gender. South Asian and mixed ethnic groups were also independently associated with seropositivity (table 3). Vaccination was independently associated with a wide range of variables in a multivariable logistic regression: the percentage vaccinated increased with age and was significantly higher among small households, individuals with managerial occupations, those living in owner-occupied accommodations, and those who had lived abroad during childhood (table 3). Gender was not associated with vaccination, and ethnicity proved significant only in the regression model when the non-White categories were broken down further (a significantly higher proportion of South Asian reported vaccination).


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TABLE 3. Factors independently associated with hepatitis A virus seropositivity among nonvaccinees and with hepatitis A virus vaccination among all individuals* in a multivariable logistic regression model, England and Wales, 2001–2002
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To our knowledge, this was only the second population-based survey of immunity to viral infections using postal oral fluid collection to investigate the presence of antiviral antibodies and the first to collect extensive demographic and social risk factor information in relation to antibody prevalence data. This is not possible through the current serosurveillance network in the United Kingdom that greatly limits the inferences to be made concerning the distribution of immunity and transmission of a viral infection in the population (5).

This novel surveillance approach appeared logistically feasible with instructions being well followed, resulting in the return of over 5,000 viable oral fluid samples and questionnaires. The questionnaire allowed detailed risk factor information to be collected and was well tolerated by participants (<1 percent of samples were returned without a questionnaire). The feasibility of the general practice-based sampling frame was encouraging, as it allowed access to individual adults and children, unlike other sampling frames such as the electoral roll (21). The sampling of individuals, rather than households or families, gives greater power in a risk factor analysis as members of the latter groups are more likely to have the same antibody and risk factor status.

The novel oral fluid approach also had cost advantages over a traditional serologic survey. There was no requirement for specially trained personnel to be employed to collect serum samples, and therefore there were no travel costs for either participants or researchers. A serology-based risk factor study is also more expensive in terms of equipment (Vacutainer (Becton, Dickinson and Company, Franklin Lakes, New Jersey), syringe, rubber gloves, spot plaster, and antiseptic cream in children cost about $4.00 per person compared with a $1.14 oral fluid swab).

The overall response rate of 40 percent, which was lower in young adult males and ethnic minorities, introduces the possibility of bias in the overall antibody prevalence estimates and limits the generalizability of the results. As the ethnic minority population (with a distinct HAV profile) in England and Wales is small in comparison with that in the United States (<9 percent of the population), the underrepresentation of these groups in the sample makes little difference to the overall prevalence estimate. However, as this study indicated an increased risk of HAV seropositivity among ethnic minority groups, additional targeted studies are now needed to better understand the epidemiology of HAV within these groups.

Despite these limitations, this survey indicated a low prevalence of immunity to HAV in England and Wales. The overall prevalence was 18.9 percent (20.5 percent when adjusted for ethnic minority underrepresentation), with a peak estimate in adults aged 25–44 years of only 26 percent. These results were very similar to those of a 1996 seroprevalence survey (figure 1), which used anonymous residual sera (in all but those aged 20–24 years, although the poor response in our survey in that group could mean that age sample was not representative). However, the availability of data on self-reported HAV vaccination status in the oral fluid survey made the contribution of vaccination to the HAV seroprevalence profile clear. If self-reported vaccinees were eliminated from the analyses, the overall seroprevalence dropped to 9.2 percent, indicating a very low incidence of natural infection in England and Wales.

The inactivated HAV vaccine is not a routine vaccination in England and Wales. It is recommended for travelers to moderate or high endemic areas, as well as for some additional risk groups including hemophiliacs and homosexual men. The use of human normal immunoglobulin, which confers short-term immunity, is no longer recommended for routine prophylaxis and is seldom used. Although HAV vaccination proved the most important risk factor in determining HAV serostatus, it remained the self-reported variable most prone to misclassification (30 percent of respondents were unsure of their status). A significant proportion of self-reported vaccinees were not seropositive to HAV (48 percent), although some discrepancies may be explained through incomplete vaccine doses and confusion between human normal immunoglobulin and the inactivated vaccine. However, the strength of the association between vaccination and HAV seropositivity and the large difference in the percentage immune who were vaccinated and nonvaccinated (table 3) support the quality of the data and reinforce the importance of being able to collect information on vaccination status. Future studies to validate self-reported vaccination status should be encouraged as vaccination data appear increasingly important for the interpretation of HAV seroprevalence data.

The availability of extensive demographic and social data allowed the identification of some unexpected associations. HAV has traditionally been thought of as a food-borne infection and one linked to poor sanitation (22). Poor sanitation is in turn associated with poor living conditions and social deprivation. However, this survey reported the higher social classes, represented through managerial occupations, as having a higher prevalence of seropositivity. This association held only among vaccinees, emphasizing changes in the relative influence of natural infection and vaccination on the epidemiology of HAV in England and Wales that could also apply to many other developed countries. Individuals in managerial occupations and owning property are more able to travel on holiday or business and are therefore more likely to be vaccinated.

The use of oral fluid allowed the collection of comprehensive risk factor data at a population level. This proved particularly important for HAV with the identification of two separate risk factor profiles relating to natural infection and vaccination. With the number of antiviral vaccines increasing and changing social conditions (e.g., hygiene), it is becoming increasingly important to collect risk factor data to be able to interpret basic virus-specific antibody prevalence profiles.


    ACKNOWLEDGMENTS
 
This survey was funded by the Public Health Laboratory Service, now part of the Health Protection Agency.

The authors would like to thank Dr. Madge Vickers and Jeanett Martin of the Medical Research Council for allowing access to the GPRF and for helpful comments in the initial stages of study design. The authors are very grateful to Nicky Fasey for her tireless efforts in practice recruitment and to Eddie Matthews for resolving the many computer problems that arose. Sample collection would also not have been possible without the extensive time given by the 40 practice nurses. Finally, the authors thank Dr. John Parry and Tamara McDonald for their patience and expertise in testing many thousands of oral fluid samples.


    NOTES
 
Correspondence to Dr. Marianne Morris-Cunnington, GlaxoSmithKline, New Frontiers Park South, 3rd Avenue, Harlow, Essex CM19, United Kingdom (e-mail: marianne.c.cunnington{at}gsk.com). Back


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 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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