Content uploaded by Virginia Hope
Author content
All content in this area was uploaded by Virginia Hope on Jan 13, 2015
Content may be subject to copyright.
Risk Factors and Immunity in a Nationally Representative
Population following the 2009 Influenza A(H1N1)
Pandemic
Don Bandaranayake
1
, Q. Sue Huang
1
*, Ange Bissielo
1
, Tim Wood
1
, Graham Mackereth
2
, Michael G.
Baker
3
, Richard Beasley
4
, Stewart Reid
5
, Sally Roberts
6
, Virginia Hope
1
, on behalf of the 2009 H1N1
serosurvey investigation team
"
1Institute of Environmental Science and Research, National Centre for Biosecurity and Infectious Disease, Upper Hutt, New Zealand, 2Ministry of Health, Wellington, New
Zealand, 3Wellington School of Medicine, University of Otago, Wellington, New Zealand, 4Medical Research Institute of New Zealand, Wellington Hospital, Wellington,
New Zealand, 5Ropata Medical Centre, Lower Hutt, New Zealand, 6Department of Microbiology, Auckland District Health Board, Auckland, New Zealand
Abstract
Background:
Understanding immunity, incidence and risk factors of the 2009 influenza A(H1N1) pandemic (2009 H1N1)
through a national seroprevalence study is necessary for informing public health interventions and disease modelling.
Methods and Findings:
We collected 1687 serum samples and individual risk factor data between November-2009 to
March-2010, three months after the end of the 2009 H1N1 wave in New Zealand. Participants were randomly sampled from
selected general practices countrywide and hospitals in the Auckland region. Baseline immunity was measured from 521
sera collected during 2004 to April-2009. Haemagglutination inhibition (HI) antibody titres of $1:40 against 2009 H1N1 were
considered seroprotective as well as seropositive. The overall community seroprevalence was 26.7% (CI:22.6–29.4). The
seroprevalence varied across age and ethnicity. Children aged 5–19 years had the highest seroprevalence (46.7%;CI:38.3–
55.0), a significant increase from the baseline (14%;CI:7.2–20.8). Older adults aged $60 had no significant difference in
seroprevalence between the serosurvey (24.8%;CI:18.7–30.9) and baseline (22.6%;CI:15.3–30.0). Pacific peoples had the
highest seroprevalence (49.5%;CI:35.1–64.0). There was no significant difference in seroprevalence between both primary
(29.6%;CI:22.6–36.5) and secondary healthcare workers (25.3%;CI:20.8–29.8) and community participants. No significant
regional variation was observed. Multivariate analysis indicated age as the most important risk factor followed by ethnicity.
Previous seasonal influenza vaccination was associated with higher HI titres. Approximately 45.2% of seropositive
individuals reported no symptoms.
Conclusions:
Based on age and ethnicity standardisation to the New Zealand Population, about 29.5% of New Zealanders
had antibody titers at a level consistent with immunity to 2009 H1N1. Around 18.3% of New Zealanders were infected with
the virus during the first wave including about one child in every three. Older people were protected due to pre-existing
immunity. Age was the most important factor associated with infection followed by ethnicity. Healthcare workers did not
appear to have an increased risk of infection compared with the general population.
Citation: Bandaranayake D, Huang QS, Bissielo A, Wood T, Mackereth G, et al. (2010) Risk Factors and Immunity in a Nationally Representative Population
following the 2009 Influenza A(H1N1) Pandemic. PLoS ONE 5(10): e13211. doi:10.1371/journal.pone.0013211
Editor: Benjamin J. Cowling, The University of Hong Kong, Hong Kong
Received July 7, 2010; Accepted September 3, 2010; Published October 14, 2010
Copyright: ß2010 Bandaranayake et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the New Zealand Ministry of Health. The funders had no role in study design, data collection and analysis, decision to publish,
or preparation of the manuscript.
Competing Interests: All authors have declared that they had (1) No financial support for the submitted work other than the fund from the New Zealand
Ministry of Health; (2) No financial relationships with commercial entities that might have an interest in the submitted work; (3) No authors have spouses, partners,
or children with relationships with commercial entities that might have an interest in the submitted work. (4) No authors have financial interests that may be
relevant to the submitted work.
* E-mail: sue.huang@esr.cri.nz
"
The members of the 2009 H1N1 serosurvey investigation team are listed in the Acknowledgments section.
Introduction
The detection of the 2009 influenza A (H1N1) pandemic (2009
H1N1) virus in the United States and Mexico in April 2009,
followed by widespread infection worldwide, prompted the World
Health Organization (WHO) to declare the first pandemic in 41
years [1,2,3]. Non-seasonal influenza (capable of being transmitted
between human beings) became a notifiable and quarantineable
disease in New Zealand on 30 April 2009. From 1 April to 31
December 2009, a total of 3211 confirmed cases of 2009 H1N1
had been notified, including 1122 hospitalisations and 35 deaths
[4]. Highest notification rates were seen in the under one year age
group, and high notification and hospitalisation rates were seen
among Pacific Peoples and Maori ethnic groups.
Estimating the true number of pandemic influenza cases in New
Zealand from clinical surveillance is not possible as the vast
majority of asymptomatic and mild symptomatic cases did not seek
medical attention. Various models have been utilised to estimate
PLoS ONE | www.plosone.org 1 October 2010 | Volume 5 | Issue 10 | e13211
the progress of the first wave of the pandemic but these have had
to depend on imprecise assumptions as many key variables are
unknown [5].
A serological measure of the population immunity profile in a
community provides a truer picture of infection during the first
wave, and allows for evidence-based decisions on interventions
during future waves. A direct measure of neutralising antibodies to
2009 H1N1 before and after the first wave provides the cumulative
incidence estimates of asymptomatic and symptomatic infections
in a population, which could inform modelling initiatives for
predicting subsequent pandemic waves [6]. Investigation of the
potential risk factors of infection by analysis of information on
host, environmental, behavioural and health service utilization
factors obtained by a questionnaire would help guide public health
interventions.
This report describes the first large nationally representative
seroprevalence study from the southern hemisphere where 2009
H1N1 coincided with seasonal influenza infections. Immunity
levels were measured in representative community participants
and healthcare workers after the first wave of 2009 H1N1. The
cumulative incidence of 2009 H1N1 was estimated by measuring
neutralising antibodies to 2009 H1N1 using pre-pandemic
(baseline) and post-pandemic serum samples. The risk factors for
2009 H1N1 were also analyzed by using information collected
from questionnaires.
Methods
Ethics Statement
Ethics approval (MEC/09/09/106) was obtained from the
Multiregional Ethics Committee of the New Zealand Ministry of
Health. Written informed consent was obtained from all
participants.
Study design and population
Both community and healthcare worker studies involved a
multi-stage random cross-sectional design and a questionnaire
evaluating demographics and potential risk factors.
Community study. The study population consisted of the
registered patients enrolled in the selected general practitioner
(GP) clinics and were individuals residing in New Zealand before,
during and after the first wave of the pandemic. Random samples
of patients stratified by age and ethnicity were obtained from the
study population during the period November 2009 to March
2010. Serological results from these samples reflected immunity
acquired during the first wave from April to September 2009 as
well as any pre-existing immunity.
The first stage of the cross-sectional study was a purposive
cluster sample of general practices, followed by a stratified random
sample of registered patients. The study included 14 GP clinics
across the country. The study localities were selected in
predetermined areas based on observed incidence during the
pandemic as high, medium and low, as well as the ethnic
distribution. Practices already participating in the on-going
national sentinel surveillance system for influenza were preferred.
Within each practice, registered patients were stratified by age
group and ethnicity. Five age groups were categorised as 1 to 4, 5
to 19, 20 to 39, 40 to 59, and $60. Ethnicity was recorded
according to New Zealand Census categories, but for analytical
purposes was divided into three categories as Maori, Pacific
Peoples and Other.
Within each stratum, simple random sampling was performed
to select sufficient numbers of participants. Taking into account
stratification, a minimum sample size of 1500 participants was
required, at design prevalence of 20% and confidence level (CI) of
95%, to maintain +/210% acceptable margin error of the
estimate.
Following random selection from GP registers with purposive
sampling of GP practices to meet strata requirements, telephone
contact with a participant was made and a questionnaire was
administered to collect exposure and risk factor information. The
questionnaire included information on the participant’s demo-
graphics, history of influenza-like illness (ILI) and other acute
illnesses, contact with ILI patients, general health status,
vaccination history, and living conditions. Information sheets,
consent forms and blood sample request forms were made
available to the participants. The expected low response from
minority ethnic groups and very young children, was counteracted
by systematic recruitment during consultations in three GP clinics
and the use of finger-prick samples respectively. A 5 ml venous or
finger-prick blood sample was collected and transported to the
WHO National Influenza Centre (NIC) at Institute of Environ-
mental Science and Research (ESR) for haemmagglutination
inhibition testing. In total, 1156 participants were enroled in the
study. Nine participants did not return the questionnaires and thus
were excluded from this analysis. This gave an overall response
rate of 76% (1147/1500).
Healthcare worker study. The study population included
secondary healthcare workers (HCWs) located in Auckland and
Middlemore hospitals and primary HCWs from the 14 GP
practices included in the community study. HCWs were divided
into three categories as medical, nursing, and other staff (including
allied health and support staff). A simple random sampling
procedure was performed to select sufficient numbers of
participants. In total 171 primary HCWs and 369 secondary
HCWs were enroled during January to March 2010.
Baseline study. The baseline immunity to 2009 H1N1 was
measured from 521 serum samples taken before 22-April 2009
from individuals aged 1 to 98 years. 184 sera, collected from
children aged 1–19 years during 2004–2005, were obtained from
ESR’s Invasive Pathogen Laboratory for the purpose of the
meningococcal seroprevalence study. 337 sera, collected from
adults aged $20 years during 2004 to 22-April 2009, mainly for
the serological testing for arboviruses and polioviruses, were
obtained from ESR’s Clinical Virology Reference Laboratory.
These were residual samples submitted to the laboratories for
diagnostic testing or antibody screening. Only information about
age, sex, sample collection date and collecting laboratory was
available for these samples.
Laboratory Method
Antibodies against 2009 H1N1 were detected by using
haemmagglutination inhibition (HI) assay, according to standard
methods [7,8]. The HI assay used 1.0% guinea pig erythrocytes. A
reference antigen, pandemic influenza A/California/7/2009 virus
propagated in embryonated chicken eggs, was provided by
WHOCC-Melbourne. Serial two-fold dilutions of serum were
tested beginning with a 1:10 dilution and a final dilution of 1:1280.
Suitable control serum samples were included in all assays,
including post-infection ferret serum samples raised against the A/
Auckland/1/2009 strain and a known human sample with a
known HI titre as a positive control. All human sera samples were
treated with receptor destroying enzyme (Vibrio Cholera Neur-
aminidase) and guinea-pig erythrocytes to inactivate non-specific
inhibitors of viral haemagglutination. The antibody level was
measured as the titre of heamagglutination inhibition. The
reciprocal of the highest dilution causing complete haemaggluti-
nation inhibition of erythrocytes by the 2009 H1N1 virus was used
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 2 October 2010 | Volume 5 | Issue 10 | e13211
as a measure of the antibody level to the pandemic virus. It has
been shown that susceptibility to influenza virus infection is
inversely related to the initial titre of serum HI antibody. HI
antibody titres of $1:40 are correlated with a reduction of 50% of
the risk of contracting an influenza infection or disease
[9,10,11,12]. Thus, in this study, an HI titre of 1:40 is used as
the threshold of seroprotection as well as seropositivity. The
proportion of individuals with HI titres of $1:40 against 2009
H1N1 in the serosurvey is referred to as seroprevalence.
Geometric mean titres (GMTs) were estimated by assigning a
value of 1:10 for titres of 1:10 or lower and a value of 1:1280 for
titres of 1:1280 or higher.
Data analysis and statistics
For the community seroprevalence study, individuals have had
different probabilities of being selected for the sample, due to
stratification and unequal allocation. Therefore, stratified and
weighted analysis was performed to account for the study design.
Rao-Scott Chi-squares test, which is a design-adjusted version of
the Pearson chi-square test, was used to test the significance of the
estimates at p value equal to 0.05.
Descriptive analysis. We performed descriptive analysis for
the categorical and numerical data using PROC SURVEYFREQ
and PROC SURVEYMEANS of SAS 9.1 version (SAS Institute
Inc., Box 8000, Cary, NC), respectively. These procedures allow
incorporating the sample design by specifying the age-ethnicity
strata and sampling weights.
Multivariable analysis. The main hypothesis being tested in
this analysis was whether age group or ethnicity affected the
likelihood of 2009 H1N1 immunity. The final model included age
group, ethnic group, sex, vaccination history, chronic illness,
reported damp housing, and study area as explanatory variables,
with 2009 H1N1 seroprotective result as the outcome.
Multivariable survey logistic regression was the method of choice
since the outcome was binary (1 = evidence of infection to 2009
H1N1, 0 = no evidence). This analysis included 820 of the 1147
participants, who had complete information for age, ethnicity,
serology results, and for selected risk factors.
Univariable screening analysis for inclusion was done at P#0.2.
Variables associated with seropositive test at P#0.2 were then
included into a multivariable survey logistic regression model.
Pearson correlation was performed to assess the correlation
between risk factors. If factors were significantly correlated, then
only one of these was selected for the model. Variables were
allowed to remain in the model if statistically significant at P,0.05
using stepwise selection, with seropositive status (0/1) as the
dependent variable. Potential confounders such as housing
condition and seasonal vaccination history were forced into the
model. Interaction terms were constructed from main effect
variables and tested for significance. The final model included age
group, ethnic group and sex, vaccination history, chronic illness,
and reported damp housing as independent variables. Since damp
housing correlated with those also reporting cold or musty housing
conditions, we used the former in our model. Statistical analyses
were performed using SAS version 9.1 (SAS Institute Inc., Box
8000, Cary, NC).
Results
Characteristics of the sample
Table 1 shows demographis characteristics of the samples
collected for the community, healthcare workers and baseline
study.
Seroprevalence in study populations
We analyzed serology results for the 1147 community
participants, 532 healthcare workers, and 521 baseline samples
(Table 2). The overall community seroprevalence was 26.7%
(CI:23.4–29.9). Seroprevalence varied across age groups. School
aged children (5–19 years) had the highest seroprevalence
(46.6%;CI:38.3–54.9), a significant increase from the baseline
(14%;CI:7.2–20.8). Older adults aged $60 had no significant
difference in seroprevalence between the serosurvey
(24.8%;CI:18.7–30.9) and baseline (22.6%;CI:15.3–30.0). Pacific
Peoples had significantly higher seroprevalence (49.5%;CI:34.9–
64.2) than Maori and Other (Europeans etc) ethnic groups. There
were no statistically significant difference in seroprevalence by sex
or by study areas. The seroprevalence of primary HCWs
(29.3%;CI:22.4–36.3) and secondary HCWs (25.3%;CI:20.8–
29.8) showed no significant difference from the community
participants aged 20–59 years (21%;CI:16.5–25.7). There was no
difference in seroprevalence among doctors (29.9%;CI:21.9–37.9),
nurses (27.5%;CI:21.3–33.7) and support staff (25.0%;CI:18.7–
31.3). Based on age and ethnicity standardization to the national
population, an estimated 29.5% of New Zealanders (1.3 million)
had immunity to 2009 H1N1. Based on the questionnaire survey
approximately 45.2% (CI:38.0–52.4) of seropositive individuals
had had no symptoms. This percentage did not change to any
appreciable extent following different calculations where the
seropositive individuals with pre-existing immunity were excluded
and those with symptoms due to pathogens other than 2009 H1N1
were included.
Determinants of immunity
Responses to the questionnaires from community participants
were analyzed to identify deteminants of immunity. These
included a range of host, environmental, behavioural and health
service utilization factors (Table S1). Most factors showed no
association with increased immunity. Large household size (.4
people) was associated with higher immunity (Crude
OR,1.6;CI:1.11–2.31, p = 0.011) compared to small household
size (1–4 people). However, after the age adjustment, this effect
was no longer present (Adjusted OR,1.36;CI:0.92–2.03,
p = 0.126).
Individuals with previous seasonal influenza vaccination showed
higher geometric mean titres than those without vaccinations,
particularly in children aged 1–4 and 5–19 years (Figure 1).
Multivariate analysis was performed to test whether age group
and ethnicity affected 2009 H1N1 immunity level. Table 3 shows
the ouputs from the multivariable survey logistic model. Younger
age groups were associated with an increased likelihood of
immunity. The likelihood of 2009 H1N1 immunity among the
age group 5–19 and age group 1–4, respectively, was 5.3 (CI:3.2–
8.7 p,0.001) times and 3.5 (CI:2.0–6.2 p = 0.029) times higher
compared with that of age group 40–59 (the reference group). The
likelihood of 2009 H1N1 immunity was 2.2 (CI:1.5–3.4 p,0.001)
times higher in the Pacific People compared with that of the
‘‘Other’’ ethnic group (the reference group). These results have
confirmed the findings in the descriptive analysis as shown by the
ethnicity effect (p = 0.009). Participants with previous seasonal
influenza vaccinations were 1.8 times (p = 0.002) more likely to
have HI titers of $1:40 compared with those who had never been
vaccinated.
The difference in the proportion of seroprotective individuals
from the baseline and the serosurvey of 2009 H1N1 was
considered as a proxy measure of the cumulative incidence of
infection due to the pandemic virus [13]. Figure 2 showed
proportions of the baseline and serosurvey samples equal to or
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 3 October 2010 | Volume 5 | Issue 10 | e13211
above each titre level for age groups of 1–4, 5–19, 20–59 and $60
years. Children in 1–4 and 5–19 years showed significant
differences between the baseline and serosurvey at almost every
titre level while older adults had little difference at any titre level.
Infection and health impact of the 2009 H1N1 pandemic
The difference in the proportion of individuals with HI titre of
$1:40 was compared between the baseline and serosurvey samples
among different age groups. Based on age and ethnicity
standardization to the national population, an estimated 18.3%
of New Zealanders were infected with 2009 H1N1. Our
population cumulative incidence estimates (781,867 cases) are
substantially higher than the case estimates from various clinical
surveillance data [4,14]. Based on the questionnaire survey, a
substantial proportion of seropositive individuals (45.2%;CI:38.0–
52.4) did not report any symptoms while 54.8% (CI:47.6–62.0) of
seropositive individuals reported at least one symptom. This gives
an estimated total of 428,463 symptomatic cases. Taking
symptomatic case and infected case estimates from the serosurvey
and the known number of deaths (35), the case fatality ratio was
8.2 per 100,000 (0.008%, 35/428,463) of symptomatic cases and
4.5 per 100,000 (0.004%, 35/781,867) of infected cases. Using
hospital admission as an indicator of severity and the known
number of admissions (1122), the hospitalization ratio was 262 per
100,000 (0.262%, 1122/428,463) of symptomatic cases and 144
per 100,000 (0.144%, 1122/781,867) of infected cases.
Discussion
To our knowledge, this is the first nationally representative
serological study from a temperate southern hemisphere country.
It provides useful information on the population immunity profile
in New Zealand and new insights into the epidemiology of the
pandemic virus infection during the first wave. This study used a
simple and replicable design which produced adequate response
rates while minimizing the in-built bias inherent in other
seroprevalence studies utilizing non-random samples. Further-
more, this survey allows analysis of information on potential
contributing factors to 2009 H1N1 infection, which will inform
future public health interventions.
The highest proportion of individuals with protective immunity
and pandemic virus infection was found in school aged children
(5–19 years) at 46.7% with a significant increase of 32.7% from the
baseline immunity of 14.0%. Our study showed a higher infection
rate with the pandemic virus in the school age children. This
finding accords with the notion that school age children constitute
the main conduit for spread of influenza, probably due to
generally higher levels of contact in school. In this respect our
results were very similar to the findings reported from several other
developed countries [13,15,16].
A high proportion (22.6%) of older adults aged $60 years had
cross-reactive antibodies against 2009 H1N1 before the first wave.
Older adults could acquire immunity to 2009 H1N1 virus, as a
Table 1. Sample demographics for the community, healthcare worker, and baseline study.
Community Study Healthcare workers Baseline
Demography
Number of
samples Percent (%)
Number of
samples Percent (%) Number of samples Percent (%)
Age group (years)
1 to 4 152 13.2 84 16.1
5 to 19 209 18.1 100 19.2
20 to 39 221 19.2 238 44.2 106 20.4
40 to 59 258 22.4 250 46.4 107 20.5
60 and over 314 27.2 51 9.5 124 23.8
Ethnic group
Maori 184 15.9 24 4.6
Pacific 171 14.8 18 3.4 Not Available
Other 801* 69.3 485 92.0
Gender
Female 640 55.6 436 80.7 176 52.4
Male 511 44.4 104 19.3 160 47.6
Study area
Auckland 269 23.3 423 78.3
Waikato 107 9.3
Bay of Plenty 122 10.6 18 3.3
MidCentral 113 9.8 Not Available
Wellington 370 32.0 78 14.4
Canterbury 109 9.4
Otago 66 5.7 21 3.9
Overall 1156 540 521
*Including 457 Europeans only.
doi:10.1371/journal.pone.0013211.t001
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 4 October 2010 | Volume 5 | Issue 10 | e13211
result of previous exposure to a 1918-like A(H1N1) virus circulating
during 1918–1957, or a lifetime of exposure to influenza A, which
has resulted in broad heterotypic immunity [12,17,18,19,20]. This
pre-existing immunity is consistent with clinical surveillance
reported in New Zealand where pandemic cases were concentrated
in younger age groups [14,21]. Older adults (24.8%) had HI titers of
$1:40 in the serosurvey with little increase from the baseline and no
increase in GMT. However, we only assessed neutralizing antibody
against 2009 H1N1 haemagglutinin in this study. It is possible that
heterotypic immunity to influenza from antibody against the
neuraminidase or cellular responses to highly conserved viral
epitopes might have also contributed to the apparent protective
effect in older adults [22]. Further study on the effect of heterotypic
immunity on age-specific populations is needed.
An overall low proportion of children and adults (1–59 years) had
cross-reactive antibodies to 2009 H1N1 in the baseline samples,
Table 2. 2009 H1N1 seroprevalence in the community, healthcare workers, and baseline samples.
Sero-survey No. Tested No. Sero Positive (Titre
.
40) Seroprevalence P-value for group
(95% CI)
Overall* 1147 347 26.7 (23.4–29.9)
Age group (years)
1
,0.001
1 to 4 148 55 29.5 (21.0–38.0)
5 to 19 206 102 46.7 (38.3–55.0)
20 to 39 221 61 22.2 (15.6–28.9)
40 to 59 258 56 20.2 (14.0–26.5)
60 and over 314 73 24.8 (18.7–30.9)
Ethnic group
2
0.001
Maori 181 62 36.3 (28.0–44.6)
Pacific 167 73 49.5 (35.1–64.0)
Other 799 212 25.9 (22.4–29.4)
Sex* 0.94
Female 636 194 26.5 (22.2–30.9)
Male 506 152 26.8 (21.8–31.8)
Study area* 0.36
Auckland 262 82 23.6 (16.3–30.8)
Waikato 107 22 20.0 (10.2–29.7)
Bay of Plenty 122 38 27.7 (18.5–36.9)
MidCentral 113 36 26.4 (16.8–36.0)
Wellington 369 117 30.2 (24.5–36.0)
Christchurch 109 32 19.4 (11.1–27.7)
Otago 65 20 29.4 (16.8–41.9)
Healthcare workers
Primary 169 50 29.6 (22.6–36.5)
Secondary 363 92 25.3 (20.8–29.8)
Occupation .0.05
Doctor 127 38 29.9 (21.9–37.9)
Nurse 200 55 27.5 (21.3–33.7)
Other 184 46 25.0 (18.7–31.3)
Baseline immunity
Overall 521 62 11.9 (9.1–14.7)
Age group (Years) ,0.001
1 to 4 84 5 6.0 (0.9–11.0)
5 to 19 100 14 14.0 (7.2–20.8)
20 to 39 106 8 7.5 (2.5–12.6)
40 to 59 107 7 6.5 (2.0–11.1)
60 and over 124 28 22.6 (15.3–30.0)
P-value calculated using the Rao-Scott chi-square test.
1
Ethnicity-adjusted estimates for the study population.
2
Age-adjusted estimates for the study population.
*Age- and ethnicity-adjusted overall estimate.
doi:10.1371/journal.pone.0013211.t002
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 5 October 2010 | Volume 5 | Issue 10 | e13211
similar to the British and Victoria reports [13,23]. However, 6% of
children aged 0–4 years had HI titers of $1:40 in the baseline
samples, higher than 1.8% reported by the British study [13], but
not significantly different. This difference may reflect the varied
influenza exposure these young children experienced in the two
countries. Also, the relatively small numbers of opportunistic
diagnostic sera in the baseline samples were collected without
randomization and with no information on seasonal influenza
vaccination and other important determinants. This is one of the
limitations of this study regarding the representativeness of baseline
samples across all age groups. Random sampling of the population
for the baseline would have been ideal.
Figure 1. Effect of seasonal influenza vaccination on geometric mean titres by age groups in the community study.
doi:10.1371/journal.pone.0013211.g001
Table 3. Results from the multivariate survey logistic regression model for selected factors.
Risk factors Odds Ratio for antibody titer
$
1:40 Lower CI Higher CI P - value
Age group (years)
1 to 4 3.5 2 6.2 ,0.001
5 to 19 5.3 3.2 8.7 ,0.001
20 to 39 1.4 0.85 2.3 0.18
40 to 59 Reference - - -
60 and over 0.95 0.6 1.5 0.84
Ethnic group
Maori 1.4 0.95 2.2 0.09
Pacific 2.2 1.5 3.4 ,0.001
Other Reference - - -
Sex (male/female) 0.82 0.59 1.1 0.21
Any vaccination history (yes/no) 1.8 1.2 2.6 0.002
Prior chronic illness (yes/no) 1.2 0.81 1.7 0.41
Damp housing (yes/no) 1.1 0.83 1.4 0.62
doi:10.1371/journal.pone.0013211.t003
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 6 October 2010 | Volume 5 | Issue 10 | e13211
Pacific Peoples had the highest seroprevalence followed by those
of Maori origin. Pacific and Maori peoples also had much higher
hospitalization and intensive care unit admission rates compared
with European and other groups [4,14]. The younger population
age structure in Pacific and Maori peoples does not fully explain
these ethnic groups’ apparent susceptibility to 2009 H1N1. Other
contributing factors to these ethnic differences may include: higher
prevalence of the infection in Pacific and Maori peoples: higher
prevalence of co-morbidities (such as asthma and diabetes),
unfavorable environmental factors (such as household crowding
and poor quality housing), behavioral differences in responses to
influenza, differences in socio-cultural-economic status, differences
in health service utilization and increased genetic susceptibility
[24]. Further study on the contributing factors to ethnic differences
in the risk of 2009 H1N1 infection and severe disease is underway
in New Zealand.
The seroprevalence among primary and secondary healthcare
workers did not differ significantly compared with that of the
general population. In the Taiwanese study, the front-line hospital
workers (20% seroprevalence with mean ages of 36.9610.6) is
significantly higher than the the general population (less than 3%
with mean ages of 52.0612.6 years), which may reflect a higher
contact risk [25]. In this context it is likely that the standard
infection control measures routinely used in New Zealand HCWs
provide adequate protection even though such measures do not
include pre-exposure antiviral prophylaxis. In addition, there was
no significant difference in seroprevalence between doctors, nurses
and support staff. Further study with individualised information
Figure 2. Proportions of the baseline and serosurvey samples equal to or above each titre level for the age groups of 1–4 (a), 5–19
(b), 20–59 (c) and
$
60 years (d).
doi:10.1371/journal.pone.0013211.g002
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 7 October 2010 | Volume 5 | Issue 10 | e13211
regarding risk exposures and personal protective measures is
needed.
The difference in the proportion of individuals with HI titre of
$1:40 between the baseline and serosurvey among different age
groups, was considered an appropriate proxy measure of the
cumulative incidence of infection due to 2009 H1N1 [13]. There
are some limitations associated with this measure. Firstly,
cumulative incidence estimates required comparison of the
proportion of neutralizing antibodies against 2009 H1N1 before
and after the pandemic, which reduces the precision of the
estimate for a given sample. Secondly, this measure may lead to an
underestimate of infection by 2009 H1N1 because the threshold
(HI titre of 1:40) may underestimate the true proportion of
individuals who were infected. Thirdly, this measure assumes that
all age groups respond to the pandemic virus in the same way
immunologically. This is a simplified assumption for a complex
host immunological response that may vary across age groups.
Our findings suggested that 2009 H1N1 triggered different
response in titers of neutralizing antibodies in different age groups.
Lastly, this measure may underestimate cumulative incidence for
individuals who were infected with 2009 H1N1 but never
developed HI antibodies. Further studies are needed to define a
serological marker of infection specific to 2009 H1N1 that do not
detect cross-reactive antibodies to other seasonal influenza
A(H1N1) viruses.
The high proportion of seropositive individuals that did not
report illness gives an indication of a relatively ‘silent’ spread of the
disease in any naive population. While asymptomatic individuals
may be less infective, their role in the spread of 2009 H1N1 cannot
be discounted. This finding has important implications for public
health policy measures that were instituted at ports of entry and
educational institutions during the first wave of the pandemic. It
underscores the need for vigilance both at the community and
individual levels to reduce the spread of disease. Basic hygiene
measures such as regular hand-washing become important
whether or not one has a conspicuous ILI.
Our serosurvey showed that previous seasonal influenza
vaccination was associated with higher HI titers against 2009
H1N1, similar to the findings in other reports [22] [26]. Hancock
et al analyzed stored-serum samples from trials of seasonal
trivalent inactivated vaccines predating the 2009 pandemic and
showed the presence of cross-reactive antibodies to 2009 H1N1 in
adults and very little response in children [22]. The same study
showed that vaccination with the seasonal vaccine resulted in a
doubling in titers of these cross-reactive antibodies to 2009 H1N1.
Interestingly, our study also showed that participants with any
previous seasonal influenza vaccination were about twice (1.8
times) more likely to have HI titers of $1:40 against 2009 H1N1
compared with those who have never been vaccinated. Two
possible mechanisms can be used to explain this observation.
Firstly, it was assumed the participants with or without seasonal
influenza vaccines had a similar probability to be infected with the
pandemic virus. Compared to the non-vaccinated participants, the
vaccinated participants would respond with higher HI titres when
infected with the pandemic virus because they had been primed
with seasonal influenza vaccines previously. Secondly, recipients of
seasonal influenza vaccines may have had an increased risk of
contracting 2009 H1N1 compared to non-recipients. This
confounding would result in higher HI titres in the vaccinated
participants than in the non-vaccinated ones. Several studies have
examined the effectiveness of seasonal influenza vaccine against
2009 H1N1. The case-control study from Australia [27], the case-
cohort study from USA [28] and the outbreak investigation in
New York City [29] did not support a significant effect of 2008-09
trivalent influenza vaccine in either decreasing or increasing the
risk for 2009 H1N1 illness. In addition, investigators from Mexico
conducted a hospital-based case-control study and reported a
vaccine effectiveness of 73% (CI = 34% to 89%) from the 2008-09
trivalent inactivated vaccine against 2009 H1N1 illness [30].
Conversely, a series of five studies conducted in four Canadian
provinces reportedly found that receipt of seasonal 2008-09
influenza vaccine was associated with a 1.5- to 2-fold greater risk
for medically attended 2009 H1N1 illness [31]. Further research is
needed to evaluate the effects of seasonal influenza vaccination on
infection with 2009 H1N1.
Vaccination strategies include targeting people at risk of adverse
health outcomes and boosting population immunity to prevent
transmission. Our findings can help public health authorities to
make evidence-based decisions on vaccination strategies and
priority listing for 2009 H1N1 and future pandemics. For
example, while children 5–19 years who played an important
role in the community transmission of infection are now largely
protected against 2009 H1N1, they could be a high priority for
pandemic influenza vaccination in the event of another novel
pandemic strain.
Supporting Information
Table S1 Univariate and age-adjusted analysis for selected
health determinants of immunity in community participants.
Found at: doi:10.1371/journal.pone.0013211.s001 (0.03 MB
DOC)
Acknowledgments
The members of the 2009 H1N1 serosurvey investigation team are as
follows: WHO National Influenza Centre, Institute of Environmental Science and
Research: J. Ralston, W. Gunn, J. Bocacao, M. Peacey, B. Adlam, S. Walker,
S. Paine, A. McLaughlin, D. Martin, A. Glennie; Ropata Medical Centre:A.
Cunliffe, M. Day, T. Bettany; Newlands Medical Centre: T. McKenzie, W.
Horo-Gregory; Upper Hutt Health Centre: L. Kljakovic, M. Mackle, J. Yee;
Porirua Union and Community Health Service: J. Ward; Ora Toa Cannons Creek
Medical Centre: D. Stevens, K. Thornbury; Hamilton East Medical Centre:H.
Mullins, L. Alexander; Northcare Medical Centre: S. Ryan, D. Astwood;
Kopeopeo Health Centre: M. Dohrman, N. Baker, S. Isbister; Christchurch South
Health Centre: S. Carson, J. Faimaid, M. Bayliss; Mornington Medical Centre:L.
Medlin J. Bain; Mairangi Medical Centre: A. Thomson, K. Bannister, M.
Duncan; East Tamaki Healthcare at Bairds Rd: R. Patel, O. Khalil, S.
Sivakumar, K. Patel; East Tamaki Healthcare at Glen Innes: D. Chee, S.
Nadarajah, M. Patel; Spring Vale Medical Centre: S. Cantillon, J. Cantillon;
Stewart St. Surgery: A. Corser,T. Cook, L. Fordyce; Auckland District Health
Board (DHB): D. Williamson; Counties Manukau DHB: S. Taylor, E. Best;
Public Health Unit in Whanganui DHB: P. O’Connor; Public Health Unit in Bay
of Plenty DHB: P. Shoemack; Public Health Unit in Public Health South DHB:J.
Holmes, M. Poore; Ministry of Health: M. Jacobs; WHO Collaborating Centre in
Melbourne:A. Kelso, I. Barr, R. Shaw, C. Durrant.
We would like to thank the participating general practitioners and their
staff, Auckland Hospital and Middlemore Hospital staff and Medical
Officers of Health involved in seroprevalence study for their time and
cooperation. We would also like to acknowledge the WHO National
Influenza Centre at ESR for the provision of laboratory testing and results;
ESR’s Invasive Pathogen Laboratory for providing baseline sera samples
for children aged 1–19 years; ESR’s epidemiology group for the data
analysis. A special thanks to the WHO Collaborating Centre in Melbourne
for providing the pandemic influenza A/California/7/2009 viral antigen
for this serosurvey.
Author Contributions
Conceived and designed the experiments: DB QSH AB MGB RB SR SR
VH. Performed the experiments: QSH SR SR. Analyzed the data: DB
QSH AB TW GM MGB RB. Contributed reagents/materials/analysis
tools: DB QSH AB TW. Wrote the paper: DB QSH AB TW GM MGB
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 8 October 2010 | Volume 5 | Issue 10 | e13211
RB VH. Designed questionnaires for the community and healthcare
workers: MB. Managed the laboratory testing: QSH. Proved the concept of
the community study in Ropata Medical Centre: S. Reid. Managed the
data and sample collection for secondary healthcare workers: S. Roberts.
Read, contributed to, and approved the final version of the manuscript:
QSH DB AB TW GM MGB RB S. Reid S. Roberts VH.
References
1. CDC (2009) Update: swine influenza A (H1N1) infections–California and Texas,
April 2009. MMWR Morb Mortal Wkly Rep 58: 435–437.
2. WHO Swine Influenza - Update 3. Available: http://wwwwhoint/csr/don/
2009_04_27/en/indexhtml Accessed on 22 January 2010.
3. Chan M (2009) Swine influenza [statement by WHO Director-General Dr
Margaret Chan]. April 25, 2009. Available: http://wwwwhoint/mediacentre/
news/statements/2009/h1n1_20090425/en/indexhtml Accessed 3 May, 2010.
4. Lopez L, Huang QS Influenza in New Zealand in 2009. Available: http://
wwwsurvesrcrinz/virology/influenza_annual_reportphp Accessed on 3 May,
2010.
5. Paine S, Mercer G, Kelly P, Bandaranayake D, Baker M, et al. (2010)
Transmissibility of 2009 pandemic influenza A(H1N1) in New Zealand: effective
reproduction number and influence of age, ethnicity and importations. Euro
Surveill 15.
6. Lipsitch M, Hayden FG, Cowling BJ, Leung GM (2009) How to maintain
surveillance for novel influenza A H1N1 when there are too many cases to
count. The Lancet 374: 1209–1211.
7. Kendal A, Pereira M, Skehel J (1982) Concepts and procedures for laboratory-
based influenza surveillance. Atlanta: Centers for Disease Control.
8. Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, et al. (1999)
Detection of antibody to avian influenza A (H5N1) virus in human serum by
using a combination of serologic assays. J Clin Microbiol 37: 937–943.
9. Hobson D, Curry RL, Beare AS, Ward-Gardner A (1972) The role of serum
haemagglutination-inhibiting antibody in protection against challenge infection
with influenza A2 and B viruses. J Hyg (Lond) 70: 767–777.
10. Potter CW, Oxford JS (1979) Determinants of immunity to influenza infection in
man. Br Med Bull 35: 69–75.
11. de Jong JC, Palache AM, Beyer WE, Rimmelzwaan GF, Boon AC, et al. (2003)
Haemagglutination-inhibiting antibody to influenza virus. Dev Biol (Basel) 115:
63–73.
12. Potter CW, Jennings R, Nicholson K, Tyrrell DA, Dickinson KG (1977)
Immunity to attenuated influenza virus WR L 105 infection induced by
heterologous, inactivated influenza A virus vaccines. J Hyg (Lond) 79: 321–332.
13. Miller E, Hoschler K, Hardelid P, Stanford E, Andrews N, et al. (2010)
Incidence of 2009 pandemic influenza A H1N1 infection in England: a cross-
sectional serological study. Lancet DOI:10.1016/S0140-6736(09). pp 62126–
62127.
14. Baker MG, Wilson N, Huang QS, Paine S, Lopez L, et al. (2009) Pandemic
influenza A(H1N1)v in New Zealand: the experience from April to August 2009.
Euro Surveill 14.
15. Zimmer SM, Crevar CJ, Carter DM, Stark JH, Giles BM, et al. (2010)
Seroprevalence following the second wave of Pandemic 2009 H1N1 influenza in
Pittsburgh, PA, USA. PLoS One 5: e11601.
16. Ross T, Zimmer S, Burke D, Crevar C, Carter D, et al. (2010) Seroprevalence
Following the Second Wave of Pandemic 2009 H1N1 Influenza. PLoS Curr
Influenza: RRN1148.
17. Greenberg SB, Couch RB, Kasel JA (1974) An outbreak of an influenza type A
variant in a closed population: The effect of homologous and hete rologous
antibody on infection and illness. Am J Epidemiol 100: 209–215.
18. Larson HE, Tyrrell DA, Bowker CH, Potter CW, Schild GC (1978) Immunity to
challenge in volunteers vaccinated with an inactivated current or earlier strain of
influenza A(H3N2). J Hyg (Lond) 80: 243–248.
19. Pereira MS, Chakraverty P, Schild GC, Colem an MT, Dowdle WR (1972)
Prevalence of antibody to current influenza viruses and effect of vaccination on
antibody response. Br Med J 4: 701–703.
20. Potter CW, Jennings R, Phair JP, Clarke A, Stuart-Harris CH (1977) Dose-
response relationship after immunization of volunteers with a new, surface-
antigen-adsorbed influenza virus vaccine. J Infect Dis 135: 423–431.
21. Huang QS, Bandaranayake D, Lopez LD, Pirie R, Peacey M, et al. (2009)
Surveillance for the 2009 pandemic influenza A (H1N1) virus and seasonal
influenza viruses - New Zealand, 2009. MMWR Morb Mortal Wkly Rep 58:
918–921.
22. Hancock K, Veguilla V, Lu X, Zhong W, Butler EN, et al. (2009) Cross-reactive
antibody responses to the 2009 pandemic H1N1 influenza virus. N Engl J Med
361: 1945–1952.
23. Grills N, Piers LS, Barr I, Vaughan LM, Lester R, et al. (2010) A lower than
expected adult Victorian community attack rate for pandemic (H1N1) 2009.
Aust N Z J Public Health 34: 228–231.
24. La Ruche G, Tarantola A, Barboza P, Vaillant L, Gueguen J, et al. (2009) The
2009 pandemic H1N1 influenza and indigenous populations of the Americas
and the Pacific. Euro Surveill 14.
25. Chan YJ, Lee CL, Hwang SJ, Fung CP, Wang FD, et al. (2009) Seroprevalence
of Antibodies to Pandemic (H1N1) 2009 Influenza Virus Among Hospital Staff
in a Medical Center in Taiwan. Journal of the Chinese Medical Association 73:
62–66.
26. Chen MI, Lee VJ, Lim WY, Barr IG, Lin RT, et al. (2010) 2009 influenza
A(H1N1) seroconversion rates and risk factors among distinct adult cohorts in
Singapore. JAMA 303: 1383–1391.
27. Kelly H, Grant K (2009) Interim analysis of pandemic influenza (H1N1) 2009 in
Australia: surveillance trends, age of infection and effectiveness of seasonal
vaccination. Euro Surveill 14.
28. Gargiullo P, Shay D, Katz J, Bramley A, Nowell M, et al. (2009) Effectiveness of
2008-09 trivalent influenza vaccine against 2009 pandemic influenza A (H1N1) -
United States, May-June 2009. MMWR Morb Mortal Wkly Rep 58:
1241–1245.
29. Iuliano AD, Reed C, Guh A, Desai M, Dee DL, et al. (2009) Notes from the
field: outbreak of 2009 pandemic influenza A (H1N1) virus at a large public
university in Delaware, April-May 2009. Clin Infect Dis 49: 1811–1820.
30. Garcia-Garcia L, Valdespino-Gomez JL, Lazcano-Ponce E, Jimenez-Corona A,
Higuera-Iglesias A, et al. (2009) Partial protection of se asonal trivalen t
inactivated vaccine against novel pandemic influenza A/H1N1 2009: case-
control study in Mexico City. Br Med J 339: b3928.
31. Skowronski DM, De Serres G, Cro wcroft NS, Janjua NZ, Boulianne N, et al.
(2010) Association between the 20 08-09 seasonal i nfluenza vaccin e and
pandemic H1N1 illness during Spring-Summer 2009: four observational studies
from Canada. PLoS Med 7: e1000258.
2009 H1N1 Seroprevalence
PLoS ONE | www.plosone.org 9 October 2010 | Volume 5 | Issue 10 | e13211
