Background
Since its identification in April 2009 in the USA and Mexico, the 2009 pandemic influenza A (H1N1) virus has caused significant morbidity and mortality around the world [
1]. Most illnesses prove acute and self-limited, highest attack rates are concentrated among children and young adults [
2]. Mechanisms of person-to-person transmission of the 2009 H1N1 influenza virus appear similar to those of seasonal influenza [
2]. In light of medical knowledge derived from past experience with seasonal influenza, health care workers (HCWs), especially those taking care of sick children, run substantial risk of acquiring influenza [
3,
4]. To prevent transmission of 2009 H1N1 influenza in healthcare settings, US Centers for Disease Control and Prevention recommend that health facilities take preventive measures like elimination of potential exposure, engineering control, administrative control, personal protective equipment (PPE), and vaccination [
5]. Prevalence of 2009 H1N1 infections and efficacy of these preventive strategies among HCWs remain unclear.
Taiwan, a sub-tropical East Asia country with a population of 23 million, experienced two epidemic waves of 2009 pandemic influenza A (H1N1) [
6]. The first began in early July and ended in late September 2009. A second began in October or November 2009, then significantly declined after December, perhaps due to mass vaccination [
7]. We initiated a prospective cohort study, using serial blood samples to determine the seroprevalence of antibodies against 2009 influenza A (H1N1) among HCWs before, during, and after the 2009-2010 influenza seasons in Taiwan. The study targeted the seroepidemiology of 2009 pandemic influenza A (H1N1) in HCWs, along with efficacy of personal protective equipment and vaccination in prevention of transmission among HCWs in a children's hospital.
Methods
Design
In early August of 2009, we initiated a prospective cohort study in which HCWs in a children's hospital were recruited and followed up on until the late stage of the pandemic in early March, 2010. Three serial serum samples were collected from each participant. A baseline sample was collected in early August 2009, a time frame which more or less coincided with the early phase of the 2009 influenza A (H1N1) epidemic in Taiwan. The second sample was collected in late October 2009, around four weeks after the first epidemic peak and just before implementation of the monovalent 2009 pandemic influenza A (H1N1) vaccination program. The third sample was collected in early March 2010, about four weeks after the second epidemic wave had subsided. Hemagglutination inhibition (HAI) assay determined antibody levels for 2009 pandemic influenza A (H1N1).
A questionnaire collected information on demographic data, history of influenza-like illnesses (ILI), history of influenza vaccination, and PPE (mainly surgical masks) usage. An ILI was defined as fever higher than 38°C and a cough and/or sore throat in the absence of a known cause other than influenza. Participants were asked to actively report all recent onset ILI or other acute respiratory illnesses, such as rhinorrhea, nasal congestion, sore throat, or cough. The date of each illness episode was defined as the earliest symptom onset date or sickness absenteeism if onset dates were unavailable. Whether such episodes met the ILI criteria was judged by one of the investigators. Once ILI was diagnosed, throat swabs for viral isolation and real-time polymerase chain reaction (PCR) for 2009 pandemic influenza A (H1N1) virus were immediately performed to confirm the diagnosis. Acute respiratory episodes which did not meet criteria of ILI were categorized as acute respiratory illness. Self-reported level of personal protective equipment and hand hygiene adherence was rated by a five-point Likert scale [
8]. Adherence was classified as optimal if the response was "always" or "often."
Setting and study subjects
The National Taiwan University Hospital (NTUH) in Taipei is a major tertiary referral medical center containing 2,600 beds and providing medical care to about 7,000 outpatients daily. Staff members (aged 20-60 years) at the children's hospital (NTUCH, part of NTUH), which has 460 pediatric beds, were recruited through word-of-mouth referral. They worked on a daily basis from August 2009 through March 2010. They were divided into two groups based on risk of contracting influenza A (H1N1) infection. The high-risk group included staff that would come into direct contact with 2009 pandemic influenza A (H1N1) patients or their respiratory samples. This group mainly consisted of pediatricians, nurses, and medical technicians who took care of patients or handled their clinical respiratory samples in the pediatric emergency, out-patient, or laboratory departments. The low-risk group included staff members (mainly nurses and laboratory technicians) whose daily work requires no direct contact with ILI patients or clinical specimens.
During the study period, all ILI patients were tested for influenza A and B viruses by rapid test (QuickVue Influenza Test) for early identification. Droplet precautions like isolation, cohorting of patients, and use of surgical masks for both patients with ILI and HCWs were routinely implemented as per the policy set by the Taiwan Centers for Disease Control. Certain HCWs used N95 masks by personal preference when in direct contact with suspected or confirmed 2009 pandemic influenza A (H1N1) patients. NTUCH offers annual trivalent, inactivated influenza vaccines and supplied monovalent inactivated 2009 influenza A (H1N1) vaccines [
9,
10] free of charge to hospital staff in 2009. Monovalent H1N1 vaccines are highly recommended but not compulsory for HCWs. Vaccine campaigns and infection control training courses were implemented during the 2009 influenza A (H1N1) pandemic.
Determining 2009 pandemic influenza A (H1N1) antibody
Hemagglutination inhibition (HAI) assay determined antibody levels against 2009 influenza A/California/7/2009 A (H1N1). Drawn blood was centrifuged to separate serum, which was immediately frozen and stored at -80°C for later use. The HAI assay was performed by one experienced technician according to standard protocol: 50 uL of serially diluted (2-fold serially diluted, starting from 10-fold dilution) serum was incubated with equal volume of A/California/07/2009 influenza virus containing 8 HA units for 30 minutes at room temperature. Then 50 uL 0.75% suspension of chicken red blood cells were added to the mixture and incubated for another 30 minutes. Extent of hemagglutination was visually inspected and the highest dilution capable of agglutinating red blood cells determined. All samples were tested in duplicate, titers expressed as the reciprocal of highest dilution of serum where hemagglutination was prevented. A titer was defined as seroprotective with a HAI antibody titer > = 40, a titer representing the level at which approximately 50% of individuals will be protected [
11]. Seroconversion was defined as having a four-fold or greater increase in HAI antibody titers between any successive paired sera. The diagnosis of 2009 influenza A (H1N1) infection was made based on positive viral culture, real time polymerase chain reaction from a throat swab, or seroconversion detected in blood samples.
Definition of 2009 pandemic influenza A (H1N1) infection
Laboratory diagnosis of H1N1 relied on either virological or serological tests. Subjects with positive virological tests, either virus isolation or real time PCR from throat swabs, were defined as virologically confirmed 2009 pandemic influenza A (H1N1) infections. Subjects having seroconversions were defined as having serological evidence of 2009 pandemic influenza A (H1N1) infection. Subjects with any of the above were defined as having laboratory evidence of H1N1 infection.
Epidemiology of 2009 pandemic influenza A (H1N1) in Taiwan
In April 2009, the Taiwan Center for Disease Control added 2009 pandemic influenza A (H1N1) infection to its list of reportable diseases [
12]. Throat swab samples from suspected cases were sent to reference laboratories for confirmation. A figure was plotted to show the number of confirmed cases of 2009 pandemic influenza A (H1N1) infection from May 2009 to April 2010 in Taiwan. ILI episodes and laboratory-confirmed 2009 pandemic influenza A (H1N1) infections in our study subjects were also compared against influenza epidemic activity in Taiwan in the same figure by date.
Statistical analysis
Geometric mean titers (GMT) were calculated for HAI titers. Value of 5 was assigned for HAI titers below 10 and 1280 for titers of 1280 or higher while calculating GMT. Comparisons of paired samples used Wilcoxon-signed rank tests, and Mann-Whitney tests were used to compare two independent samples. Fisher's exact test compared proportions; Chi-square tests were used for independent cases, two-tailed p values below 0.05 are considered statistically significant. We performed univariate and multivariate logistic regression using these variables alongside baseline titer, age, sex, 2009 pandemic influenza A vaccine status, high-risk group, ILI history, 2009 pandemic influenza infection history, optimal surgical mask usage, and hand hygiene status to gauge their contribution to seroconversion and 2009 pandemic influenza A (H1N1) infection rates. Odds ratios (ORs) with asymptotic Wald 95% confidence intervals (CIs) and two-tailed p values are plotted with statistical significance set at 0.05. Multivariate analysis entailed stepwise logistic regression, wherein variables that did not improve model fit at p < 0.10 were discarded. Data analyses used the Statistical Package for Social Sciences (SPSS version 16.0).
Ethics
The study was approved by the Institutional Review Board of National Taiwan University Hospital. Written informed consent was obtained from all participants.
Discussion
Since the worldwide pandemic of 2009 influenza A (H1N1) virus, studies on the risk of infection for healthcare workers have burgeoned: e.g., a prospective seroepidemiologic cohort study done in Singapore, 6.6% (35/531) on HCWs showing seroconversion during the pandemic [
13]. Another cross-sectional study in Hong Kong revealed 12% of 599 HCWs had antibody titers that were seroprotective against the virus after the first wave of the pandemic [
14]. In one study from Australia, 19.9% of 231 frontline HCWs had positive antibody titer against the virus [
15]. Yet studies on the prevalence and seroepidemiology of 2009 pandemic influenza A (H1N1) in HCWs taking care of children remain limited. By showing seroconversion, our longitudinal follow-up cohort study further pinpointed 14.4% (13/90) of Taiwan's unvaccinated HCWs as infected during the pandemic. Even with virological evidence, only 16.7% (15/90) of all unvaccinated HCWs had laboratory evidence of infection in this study. The majority of HCWs in Taiwan remained susceptible.
The psychosocial impact of the 2009 pandemic was great; HCWs were under tremendous stress taking care of infected patients because they are regarded high-risk for contracting the virus. Our study showed moderate overall infection rates among HCWs, surprisingly no higher than that of household contacts of an index case with pandemic influenza. Chao DY et al. calculated a 19% seroconversion rate during April-July and October-November 2009 among 147 household contacts (mean age: 39.34, range: 25-60) with schoolchildren in Taiwan [
6]. A report from Hong Kong claimed no sharp difference in incidence between clinical versus non-clinical staff among 1,158 confirmed 2009 pandemic influenza A (H1N1) infections in HCWs [
15]. Current preventive strategies like proper use of surgical masks, hand hygiene, and vaccination afford HCWs partial protection against influenza. A large-scale serological study from four distinct cohorts in Singapore, showed seroconversion rates even lower for HCWs (6.5%) than the general population (13.5%) or military personnel (29.4%) [
16]. One prospective sero-epidemiological cohort in Singapore also reported seroconversion among health care workers (11%) significantly lower than that in normal (44%) military personnel [
17]. A higher alertness and strict PPE compliance by HCWs account for the low seroconversion rates observed.
HCWs with diverse daily tasks actually differ in risk level for contracting H1N1 virus. Those in direct contact with ILI patients or their samples presumably face higher risk. Nurses working in 2009 pandemic influenza A (H1N1) isolation wards and coming in contact with infected colleagues showed independent occupational risk factors for seroconversion [
13]. Data confirmed high risk or direct contact with influenza patients and their respiratory samples as independent risk factors for seroconversion (Table
4), yet suboptimal surgical mask usage or hand hygiene as independent risk factors, obviously because high- or low-risk participants had good adherence to PPE usage (100% for high- and 70.2-87.7% for low-risk). Surgical masks were cited as effective as N95 respirators in preventing laboratory-confirmed influenza infections [
18,
19]. The World Health Organization recommends surgical masks for all patient care, other than N95 masks for aerosol generation procedures. Reinforcement of current strategies is justified.
Taiwan's first pandemic influenza A (H1N1) infection was confirmed in May and remained very rare until early July 2009. When we began this study in August, 4.7% of our subjects already had protective antibodies against the newly emerged virus; 6.0% had ILI prior to first sampling. One possible explanation is the virus spreading more widely and rapidly than previously thought. Our earlier study showed subjects vaccinated with seasonal influenza vaccines unlikely to have cross reactivity to this virus [
20]. Another possibility is that the 2009 influenza A (H1N1) virus shared antigen similarities with older influenza strains. Prevalence of cross-reactive antibodies to the 2009 influenza A (H1N1) virus in adults aged 18-60 were 0-13% from studies in the U.S., Australia, UK, and Finland [
21‐
24].
Improving the vaccination coverage rate among HCWs is one priority in combating future influenza epidemics. One US national health objective for 2010 was to achieve 60% HCW vaccination coverage. Overall vaccination rates of monovalent 2009 pandemic influenza A (H1N1) vaccines were about 70% for school age children and adolescents, 11% for adults, and 30% for preschoolers in Taiwan [
25]. Coverage rates for HCWs in the current study fell somewhere among 57.0% for high- versus only 12.3% for low-risk. Such disparity might emanate from lower perceived risk. Results suggest that as long as HCWs follow current infection control guidelines, risk of infection in any healthcare setting is quite low. Monovalent pandemic influenza A (H1N1) vaccine in this study has immunogenicity of 90% or higher in adolescents and adults [
9,
10]; data suggest rapid decline of vaccine-induced influenza antibodies. A mere 56.7% (34/60) of vaccinees have detectable seroprotective antibodies three to four months after vaccination.
Discrepancy arose between the number of subjects reporting ILI (n = 25) and those showing laboratory evidence of 2009 pandemic influenza A (H1N1) infection (n = 15). Good serological studies can normally identify asymptomatic infections and yield more infections than clinical ILI; data suggests self-reported ILI as lacking specificity, especially during a major influenza pandemic. Seasonal influenza H3N2 virus infections, plus other respiratory viruses like rhinovirus and adenovirus co-circulated in the early stage of 2009 pandemic influenza A (H1N1) in Taiwan. More than 86% of study subjects reported acute respiratory illness during this study (Table
2). People tend to be more sensitive and might exaggerate symptoms during a pandemic. Many common colds caused by other viruses might be reported as ILI.
Our current study shows several limitations. First, the number of participants was relatively small, all from one hospital. Receiving H1N1 vaccinations likewise decreased number of subjects eligible for certain analysis. Unexpectedly, low seroconversion rates in our subjects may distort identification of true risk factors. Finally, self-administered questionnaires can raise biased information on clinical symptoms.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
TYY made major contributions to sample collection, statistical analysis and manuscript preparation. CYL conceived and designed the study, participated in the analysis of the data and preparation of the manuscript. Y-TT carried out antibody determinations and data interpretation. LYC and LMH made substantial contributions to acquisition and analysis of data. All authors contributed to the writing of the manuscript and approved the final manuscript.