Overview of influenza subtype distribution during May 1 through July 28, 2009, from the 2683 samples received for testing during period of enhanced surveillance in Singapore. This shows the percentage of all samples received each day at the Molecular Diagnosis Centre at National University Hospital, Singapore, that were positive for influenza.
Tang JW, Tambyah PA, Lai FYL, Lee HK, Lee CK, Loh TP, Chiu L, Koay ES. Differing Symptom Patterns in Early Pandemic vs Seasonal Influenza Infections. Arch Intern Med. 2010;170(10):861-867. doi:10.1001/archinternmed.2010.108
Singapore is a tropical country with a temperature range of 23°C to 35°C and relative humidity of 48% to 100% throughout the year. Influenza incidence peaks in June through July and November through January, though influenza cases can be detected throughout the year.
Between May 1 and July 28, 2009, a novel dual-gene diagnostic polymerase chain reaction assay targeting the hemagglutinin (HA) and nucleoprotein (NP) genes of the new influenza A(H1N1/2009) virus was specifically designed for enhanced influenza surveillance using nasopharyngeal swabs collected from symptomatic patients (including their close contacts) and returning travelers returning from influenza A(H1N1/2009)–affected areas, presenting to affiliated primary care clinics as well as the main hospital emergency department.
From the week of June 16 to June 23, 2009, this pandemic influenza A(H1N1/2009) displaced and then replaced the seasonal influenzas (H3N2, H1N1, and B). Of 2683 samples tested during this 12-week surveillance period, 742 (27.6%) were positive for any influenza virus using this assay, with 547 cases of A(H1N1/2009) (20.4%), 167 cases of A(H3N2) (6.2%), 14 cases of A(H1N1) (0.5%), and 12 cases of influenza B (0.4%). Results of multivariate analysis showed that age (P < .001), fever (P < .001), cough (P < .001), sore throat (P = .002), rhinorrhea (P = .001), and dyspnea (P < .001) were significantly different among these groups.
From this large prospective study, there was a lower incidence of fever and dyspnea in patients with pandemic influenza A(H1N1/2009) infection. Similar to reports from elsewhere, it was also found that this pandemic virus tends to infect younger people, though with fewer symptoms, on average, than seasonal influenza. Early pandemic influenza A(H1N1/2009) infections appeared to be slightly milder than seasonal influenza as indicated by different symptom patterns in the presentation of more than 500 cases of influenza A(H1N1/2009) during April through July to a large teaching hospital in Singapore.
Various aspects of the new pandemic influenza A(H1N1/2009) virus have been described, including its putative origins from swine influenza viruses1,2 and its emergence in several regions worldwide, including Mexico,3 the United States,4 Europe,5- 8 Australia,9 and Thailand.10 More detailed clinical presentations of this novel virus have been reported mainly from Mexico3 and the United States,4,11,12 with 1 report from Japan.13 Other countries are presumably documenting their cumulative cases in a similar way.
However, as yet, there have been no large series of clinical cases of influenza A(H1N1/2009) described in geographical zones with a tropical climate, with high temperatures and relative humidity, where the survival of the influenza virus may be least optimal.14- 16 More data on influenza incidence in tropical countries have been called for,17 particularly since, at least in Singapore, there have been suggestions of high mortality rates in previous influenza pandemics.18
There is also the observation that in many settings, pandemic influenza A(H1N1/2009) has displaced seasonal influenza virus infection. This is reflected in the recent recommendation of the World Health Organization (WHO) for the Southern hemisphere vaccination for 2010 to include an antigen for pandemic influenza A(H1N1/2009).19 From published analyses of previous influenza pandemics, this phenomenon is well known.20 However, with current rapid diagnostic technologies, it is now possible to follow such virological events prospectively in real-time.
Singapore is a tropical country with a temperature range of 23°C to 35°C and relative humidity of 48% to 100% throughout the year. Influenza incidence peaks in June through July and November through January, though influenza cases can be detected throughout the year.21,22 The first case of pandemic influenza A(H1N1/2009) was detected in a traveler from New York on May 17, 2009. The initial clinical features of the first 10 cases have been reported.23 Enhanced surveillance for influenza was put in place nationwide from the end of April 2009 when the WHO announced that it had raised its global pandemic alert level and Singapore correspondingly raised its national pandemic alert level.24 There have not been any reports of the clinical and/or epidemiological features of the widespread locally acquired community cases from Singapore following the initial imported cases.
We describe herein the comparative symptomatology of pandemic, seasonal, and noninfluenza cases that were diagnosed using a novel dual-gene diagnostic polymerase chain reaction (PCR) assay during this enhanced surveillance period in Singapore (May-July 2009) and examine the phenomenon of viral strain displacement and replacement among these influenza viruses in the cohort of patients attending our large teaching hospital, the National University Hospital (NUH), Singapore, and its related primary care clinics.
Samples collected for virological testing consisted of nasopharyngeal “flocked” swabs collected into 3 mL of standard virus transport medium (Copan Diagnostics, Corona, California), using standard techniques as comprehensively described elsewhere.25 These newer flocked swabs (Copan Diagnostics) were used because they have been shown to increase the yield of such respiratory sampling.26 These samples were obtained from patients and their close contacts who presented to NUH during May 1 through July 28, 2009, as well as those attending hospital outpatient clinics, the hospital emergency medicine department, and affiliated primary care clinics (both government and private), as well as hospital in-patients with influenza-like symptoms and staff or patients with compatible contact or travel histories. No demographic restriction was imposed for the purpose of this study. Samples were tested for influenza virus at the Molecular Diagnosis Centre, NUH (MDC/NUH). All these diagnostic and surveillance respiratory samples collected from these patients during this period of heightened surveillance (May through July 2009) were transported to the MDC/NUH laboratory within a few hours of sampling.
Most of the patients included in this analysis either manifested at least one of the following symptoms: fever, cough, sore throat, “runny nose” (rhinorrhea), “muscle pain” (myalgia), “shortness of breath” (dyspnea), diarrhea, and vomiting. The few asymptomatic patients who were tested were usually those returning from currently endemic areas (eg, staff attending overseas events or on vacation) or were asymptomatic contacts of confirmed or suspected cases of influenza A(H1N1/2009).
Clinicians obtaining samples from these patients completed a standardized data collection form (available from authors on request) that was based on the WHO data collection form as stipulated in their International Health Regulations.27 These data were collected with the physician asking the questions and completing the questionnaire with the patient being present. Symptoms presenting in the previous 5 to 7 days were considered relevant. Fever was defined conventionally as being a body temperature higher than 37.5°C and was measured using ear thermometers by the health care staff at patient presentation or admission.
Samples and data received from patients with appropriately completed symptom surveillance forms were included in this study. For the purposes of data analysis, these patients were also stratified by age (0-5, 6-18, 19-35, 36-50, and ≥51 years) and sex.
Note that during the study period, Singapore transitioned from a “containment” to a “mitigation” strategy on July 8, 2009, and from this time on, sampling was mainly targeted at symptomatic individuals for the purpose of deciding on the appropriate treatment (although there were a limited number of cases that still underwent isolation and contact tracing and then follow-up of potentially vulnerable or infected contacts). Despite the transition from containment to mitigation, enhanced surveillance continued at selected primary care clinics with at least 10 to 20 samples a day randomly obtained from patients presenting with influenza-like illness for A(H1N1/2009) virological testing. During this mitigation stage (ie, after July 8, 2009), with community-wide transmission occurring, the travel history was no longer considered as useful and was not included in the data analysis.
Briefly, after RNA extraction, all samples were initially screened by a sensitive, multiplex, real-time reverse–transcriptase PCR (RT-PCR) assay designed in-house to detect all influenza A viruses of human, avian, and swine origin and influenza B virus. For samples with detected influenza A, further seasonal influenza A subtyping real-time RT-PCR assays (to human H1 or H3 viruses) ensued, and, if yet untypeable, by an in-house, specifically designed A(H1N1/2009) dual-gene (HA and NP genes) real-time RT-PCR assay to confirm the presence of the pandemic influenza A(H1N1/2009) virus (further details of these assays are available from the authors on request).
When sample numbers were high during the early phase of the pandemic, both the screening and the A(H1N1/2009)-specific assays were performed simultaneously as first-line tests, and the subtyping to either H1 or H3 seasonal influenza A viruses in cases that screened positive for influenza A, but were A(H1N1/2009) negative, was performed later.
Univariate comparison of demographic characteristics and presence of various symptoms among influenza subtypes were conducted with the χ2 test. Multivariate analysis was conducted using logistic regression with multinomial categories. All statistical analysis was performed using SPSS statistical software package version 17.0 (SPSS Inc, Chicago, Illinois). P < .05 was considered statistically significant.
A total of 2750 patient samples were received for influenza virus testing during the study period. There were 67 patients whose data surveillance forms were not adequately completed. These patients were excluded from analysis, resulting in 2683 valid distinct patients.
During the period under study (May 1, 2009, through July 28, 2009) at this hospital, a total of 547 cases of A(H1N1/2009) (20.4%), 167 cases of A(H3N2) (6.2%), 14 cases of A(H1N1) (0.5%), and 12 cases of influenza B (0.4%) were detected under the enhanced surveillance program, giving an influenza positivity rate of 27.6%, with the remainder (1943 [72.4%]) being noninfluenza cases. It became clear that the previous predominantly circulating seasonal influenza A(H3N2) virus was being rapidly replaced by the new pandemic influenza A(H1N1/2009) subtype (Figure). In Singapore, this transition occurred around the week of June 16 to 23, 2009, approximately a month after the first imported case was detected nationwide.
The most common presenting symptoms with the pandemic strain were cough (88.1%), fever (79.3%), sore throat (53.7%), and rhinorrhea (49.9%). For A(H3N2), the most common presenting symptoms were fever (88.0%), cough (81.4%), runny nose (55.7%), and sore throat (38.3%). For seasonal A(H1N1) and influenza B, the most common presenting symptoms were similar to A(H3N2) (Table 1). Very few of our patients, infected with either the pandemic or seasonal strain or even those who were negative for influenza, had gastrointestinal symptoms (ie, vomiting and/or diarrhea).
Because of the relatively few seasonal A(H1N1) and influenza B cases, they were grouped together with A(H3N2) as a “seasonal influenza” group for comparative analysis vs pandemic influenza A(H1N1/2009). Results from univariate analysis suggested that patterns of age (P < .001), fever (P < .001), cough (P < .001), rhinorrhea (P < .001), sore throat (P < .001), headache (P < .001), myalgia (P = .005), dyspnea (P < .001), and diarrhea (P = .03) were significantly different between the pandemic vs seasonal vs noninfluenza cases (Table 2).
The age distribution of those infected with pandemic vs seasonal influenza was also different. The seasonal influenza viruses appeared to affect all ages, with a higher proportion of those 5 years or younger, while the pandemic virus affected the children and young adults more, with very few elderly cases. This age pattern was statistically significantly different among the new pandemic, seasonal, and noninfluenza cases (P < .001) (Table 2). Furthermore, multivariate analysis showed that age (P < .001), fever (P < .001), cough (P < .001), sore throat (P = .002), rhinorrhea (P=.001), and dyspnea (P < .001) were significantly different among the 3 groups. The odds of being infected with A(H1N1/2009) were only 54.3% compared with seasonal influenza when a patient presented with fever. Similarly for patients presenting with runny nose and dyspnea, their odds of being infected with A(H1N1/2009) were significantly lower than seasonal influenza (Table 3). However, when patients developed cough or sore throat, their odds of being infected with A(H1N1/2009) compared with seasonal influenza were twice and 1.4 times as high, respectively, relative to when they did not develop these symptoms.
This study has presented and analyzed the patterns of symptoms according to influenza virus subtypes in patients presenting with influenza-like illness to the NUH in Singapore. The study covered a period of heightened influenza surveillance, triggered by the emergence of the pandemic influenza A(H1N1/2009) virus during May 1 through July 28, 2009, and is to our knowledge the largest clinical report on laboratory-confirmed influenza cases from tropical Asia.
Several studies have described the symptom pattern of infections by the novel pandemic influenza A(H1N1/2009) virus. In a comparison of the same symptoms as those that have been described in this study, one of the earliest reports from Mexico found that of 18 patients with confirmed pandemic influenza A(H1N1/2009) infection, the most common symptoms were fever (100%), cough (100%), and dyspnea (100%), followed by myalgia (44%), rhinorrhea (28%), headache (22%), and diarrhea (22%).3 In a much larger cohort from the United States, of 642 patients with confirmed A(H1N1/2009) infection, the most common symptom was fever (94%) and then cough (92%), sore throat (66%), diarrhea (25%), and vomiting (25%).4 A report on 49 patients admitted with A(H1N1/2009) infection from Japan described fever being the most common symptom (87.7%) and then cough (79.1%), sore throat (71.4%), myalgia (55.1%), headache (52%), diarrhea (14.2%), and vomiting (12.2%).13 Another early study from the United States (from California only), based on a smaller number patients (n = 30) infected with A(H1N1/2009), again reported fever and cough being the most common symptoms (97% and 77%, respectively), but with a larger proportion complaining of vomiting (46%).11 In contrast, from this study (Table 1), the Singapore cohort of 547 A(H1N1/2009)-infected patients, had similar proportions with cough (88%), fever (79%), sore throat (54%), and rhinorrhea (50%) but fewer reporting headache (21%), vomiting (1.1%), and diarrhea (0.7%). Interestingly, dyspnea, which was reported in 0.5% of our patients and 100% of the early Mexican patients, was not reported in these US or Japanese studies.
However, none of these previous studies compared symptom patterns between patients presenting with seasonal (H3N2, H1N1, and B) and pandemic A(H1N1/2009) influenza infections. In this study, it can be seen from Table 2 and Table 3 that there were significant differences between the symptom patterns of seasonal and pandemic influenza when fever, cough, sore throat, rhinorrhea, and dyspnea were compared. These results also showed that the age ranges of patients infected with seasonal vs pandemic influenza were significantly different. In this Singaporean population, the pandemic influenza A(H1N1/2009) infected a lower age group of patients compared with seasonal influenza, with the majority of A(H1N1/2009) infections occurring in the younger age range of 9 to 32 years (median age, 19 years). This is comparable to other reports from Germany (mean age, 23 years; median age, 18 years [n = 190]),7 the Netherlands (median age, 18 years for indigenous and 21 years for imported cases [n = 51 and n = 64, respectively]),8 Australia (median age, 21 years; range, 2-63 years [n = 223]),9 the United States (median age, 20 years; range, 3 months–81 years [n = 642])4 and Mexico (median age, 38 years; range, 9 months–61 years [n = 18]).3 With very few 0- to 5-year-old children presenting in this cohort, it is difficult to compare their symptom patterns with similar studies elsewhere, especially since these other studies do not specifically present symptoms for this particular age range alone.3,4,13
It is becoming more generally accepted that this tendency for pandemic influenza to cause symptomatic infection in younger patients is probably owing to the older patients (age, >60 years) having some cross-reacting immunity that had arisen from their longer lifetime history of previous exposure to influenza A(H1N1) viruses that may have been more similar to the pandemic influenza A(H1N1/2009) strain than the current seasonal influenza A(H1N1) virus.28 This paradoxically may account for the lower overall influenza A(H1N1/2009)–related pneumonia mortality observed in the United States despite the markedly higher incidence of influenza A(H1N1/2009) identified in laboratory surveillance of influenza-like illnesses detected by the US Centers for Disease Control and Prevention (CDC),28 ie, this may be the first influenza pandemic to have a lower mortality rate than seasonal influenza.
Previous studies had described differences in symptom patterns with different influenza subtypes. For example, an earlier Japanese study compared the symptomatology of seasonal influenzas (H1N1, H3N2, and B) and found slightly higher fevers in patients with influenza A(H3N2) and more gastrointestinal symptoms in patients with influenza B.29 In addition, Khiabanian et al30 found that both seasonal influenza A(H1N1) and the recently emerged pandemic influenza A(H1N1/2009) viruses tended to infect younger individuals, which may be the result of some partial cross-immunity in the more elderly population who were alive during the previous circulation of influenza A(H1N1) viruses prior to 1957.28
Recently, Presanis et al31 used a mathematical model that combined different pandemic influenza clinical data sets. Their analysis suggested that the current pandemic influenza A(H1N1/2009) virus causes milder disease than seasonal influenza. The analysis from the present study, which compared symptom patterns between pandemic and seasonal influenza cases, also supports this conclusion.
The numbers of laboratory-confirmed seasonal (subtypes A/H3N2, A/H1N1, and B) and pandemic A(H1N1/2009) influenza-positive samples, stratified by age, over this period have also been presented. This demonstrated the rapid displacement, then replacement, of the seasonal influenza viruses by the novel pandemic A(H1N1/2009) influenza strain, a pattern that had been seen in previous pandemics.20 Other surveillance data, publicly available at the surveillance Web sites of other regions (United States,32 Canada,33 Europe,34 Japan,35 and New Zealand36) were also retrieved and plotted in the same way for comparison purposes (data not shown). Together, these results demonstrated a sequential wave of influenza A(H1N1/2009) dominance from the Western to the Eastern hemisphere, with a roughly similar rate of displacement and/or replacement of the local seasonal circulating influenza subtypes in these countries. This trend has become more obvious now, with both Southern then Northern hemisphere countries having experienced the continued spread of the novel pandemic virus during their annual influenza seasons. The reasons for this rapid displacement, then replacement, of current circulating seasonal influenza subtypes are unclear, particularly since antibodies to the pandemic A(H1N1/2009) virus do not cross-react (and therefore do not offer any cross-immunity) to the seasonal influenza A(H3N2) and A(H1N1) viruses.28
This observation also raises an interesting question. Epidemiological evidence of the past influenza pandemics from 1918 (H1N1), 1957 (H2N2), and 1968 (H3N2) had revealed that after the introduction of each new pandemic subtype, the previous pandemic subtype eventually disappeared from general circulation. The recently circulating seasonal A(H1N1) virus was in fact believed to be the result of an accidental release in 1977,20 and if not for this unfortunate event, only the A(H3N2) virus, present since the 1968 influenza pandemic, would be circulating, prior to the emergence of the pandemic influenza A(H1N1/2009) virus.
Given that the immune clearance and resulting immunity of influenza viruses depend mainly on the hemagglutinin (HA) gene, it is unclear why these different subtypes do not continue to circulate and infect new susceptible persons. There is relatively little serological cross-reacting immunity between H1, H2, and H3 subtypes, which is why the annual WHO recommendations for the seasonal influenza vaccine contains separate H1 and H3 HA antigen components.19 Despite studies examining the underlying mechanism for antigenic drift in seasonal influenza viruses,20 these have been mainly theoretical and still do not adequately address the reasons why antigenically different pandemic influenza subtypes cannot naturally cocirculate.
There are several limitations to this study. First, the symptom data relied completely on the requesting physicians to accurately complete the influenza surveillance forms, in addition to their performing the normal clerking for each patient. In busy departments at busy times, the data on such surveillance forms may not be completely accurate owing to physician fatigue or simply a lack of time. Unfortunately, during these early stages of the emerging pandemic, body mass index measurements were not part of the routine surveillance questionnaire collected for these patients (which is also not mentioned in the WHO International Health Regulations health questions27), though it is now thought to be an important factor that may affect clinical outcomes with A(H1N1/2009) infections.37,38
Second, in this patient cohort, there is a sample bias toward those who experienced symptoms with their infection. There may well be a considerable proportion of influenza A(H1N1/2009)–infected but asymptomatic individuals in the community who do not present to health care services. Therefore, these data can only be interpreted as representative of those mostly symptomatic patients presenting to this health care service during this early phase of the pandemic. Over time, as the general population becomes more familiar with this new virus, it may well be that (as with seasonal influenza) those who are only mildly symptomatic may no longer present to health care services.
Third, although Singapore is an ethnically diverse population (approximately 77% Chinese, 14% Malays, 8% Indian, and 1% other races), the symptom patterns in patients belonging to each of these specific ethnic groups were not analyzed separately. Ethnic differences may not be significant, since other studies have not found any ethnic differences in presentation of influenza in other settings.4
Fourth, although Singapore has one of the world's highest incidences of diabetes mellitus and a rising incidence of obesity, this study did not obtain such clinical data or other risk factors, which have been shown to be potentially associated with more severe presentations of pandemic influenza A(H1N1/2009). In addition, the change in criteria for laboratory testing from widespread testing of mildly symptomatic individuals and contacts during the containment phase to a more targeted testing of patients with more clinically severe illness during the mitigation phase might have skewed our results.
This study summarizes and distinguishes the patterns of displacement and replacement and symptom presentations of pandemic influenza A(H1N1/2009) vs seasonal influenza viruses, observed during a unique time window (May 1–July 28, 2009) of the early influenza A(H1N1/2009) pandemic, as this novel virus gradually displaced and replaced the previously circulating seasonal influenza viruses in Singapore.
It is likely that these symptom patterns will continue to evolve and change as the novel pandemic influenza A(H1N1/2009) eventually predominates, globally, in the susceptible human population. However, this early window period in this first wave of the pandemic has provided an opportunity to compare the symptomatology of these different influenza viruses in this particular tropical environment and ethnically diverse population during this transitional period.
Intriguingly, the lower incidence of fever and dyspnea at presentation in our population seems to correlate with US CDC surveillance data, which does not show an increase in pneumonia and influenza mortality despite markedly higher incidence rates of influenza in nationwide surveillance in the United States.28 However, this is still a relatively small patient cohort compared with the global population infected with this novel virus. Hence, although there is some suggestion that these patients who attended our diagnostic services during this time—in the early phase of the influenza A(H1N1/2009) pandemic in Singapore—appear to be less symptomatic than those with seasonal influenza, this may change as the pandemic unfolds, especially as the sampling frequency and circumstances in which such samples are taken become more similar to that of annual seasonal influenza.
Correspondence: Julian Wei-Tze Tang, PhD, MRCP, MRCPath, Department of Laboratory Medicine, National University Hospital, 5 Lower Kent Ridge Rd, Singapore 119074 (email@example.com).
Accepted for Publication: November 25, 2009.
Author Contributions: All authors had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Tang. Acquisition of data: Tambyah, H. K. Lee, C. K. Lee, Loh, Chiu, and Koay. Analysis and interpretation of data: Tang and Lai. Drafting of the manuscript: Tang. Critical revision of the manuscript for important intellectual content: Tang, Tambyah, Lai, H. K. Lee, C. K. Lee, Loh, Chiu, and Koay. Statistical analysis: Lai. Obtained funding: Koay. Administrative, technical, and material support: Tambyah, H. K. Lee, C. K. Lee, Loh, Chiu, and Koay. Study supervision: Tang, Tambyah, Chiu, and Koay. Tambyah clinically managed the patients; Loh retrieved the surveillance data; H. K. Lee, C. K. Lee, and Loh designed and performed the influenza A(H1N1/2009)–specific PCR; and Chiu and Koay supervised the molecular testing.
Financial Disclosure: Dr Tambyah has received research support from Baxter and Adamas and honoraria from Novartis and has served on the board of the Asia Pacific Advisory Council of Influenza.
Funding/Support: Dr Koay is supported by the H1N1 Advance Urgent Funds (#R-179-000-045-720) from the Yong Loo Lin School of Medicine, National University of Singapore.