Incidence and seroprevalence of seasonal influenza a viruses in Egypt: Results of a community‐based cohort study

Abstract Background H1N1 and H3N2 influenza A viruses circulate in people as seasonal influenza viruses. Data on influenza infection rates and circulation in demographic subpopulations in Egypt are limited. In this study, we aimed to determine the incidence and seroprevalence of seasonal influenza A virus infections in a cohort of rural Egyptians between 2017 and 2020. Methods A total of 2383 subjects were enrolled from 390 households in five study sites in Northern Egypt. Informed consents were obtained. Sera were collected from participants on an annual basis (Baseline: 2016–2017, Follow up 1: 2017–2018, Follow up 2: 2018–2019, and Follow up 3: 2019–2020) to determine seroprevalence of antibodies against H1N1 and H3N2 viruses by hemagglutination inhibition assay and to estimate incidence based on seroconversion. Results Seropositivity against H1N1 was over 40% and over 60% against H3N2. The high seroprevalence was due to natural infection because participants were mostly unvaccinated. Seropositive participants were younger than seronegative participants indicating that the infection rate is higher in children. Incidence of both viruses ranged from 4% to 28% depending on study year. The incidence and seroprevalence of H3N2 and H1N1 infections at Follow up 1, 2, and 3 showed an increase at Follow up 2 observed for all age categories corresponding to season 2018–2019, at which the vaccine efficacy was the lowest worldwide compared with preceding and following seasons. Conclusions This cohort study provided estimates of influenza A infection rates among rural Egyptians. We recommend updating influenza vaccination programs to focus on such populations.


| INTRODUCTION
H1N1 and H3N2 subtypes of influenza A viruses that emerged in 2009 and 1968, respectively, currently circulate in people as seasonal influenza viruses and cause illness, hospitalization, and death worldwide every year. Seasonal influenza is an acute respiratory infection causing mild to severe illness. Symptoms include fever, cough, headache, muscle and joint pain, severe malaise, sore throat, and a runny nose. 1 Hospitalization and death occur mainly among high-risk groups including children and the elderly. According to the World Health Organization, seasonal influenza epidemics cause three to five million cases of severe illness and between 250,000 and 500,000 deaths annually. 1 While the epidemiology of seasonal influenza is well defined in developed countries, less is known about the epidemiology of influenza A in the developing world, particularly in the Middle East and North Africa region. It is estimated that 99% of deaths in children under 5 years of age with influenza-virus associated lower respiratory tract infections are in developing countries. 2

A study conducted in
Saudi Arabia showed that 15% of suspected influenza cases captured by the surveillance system over a period of 7 years (2010-2016) tested positive for H1N1 and needed hospitalization out of which 10% needed admission into intensive care unit. 3 Another study from Saudi Arabia examining influenza A(H1N1)pdm09 epidemiology in the Eastern Province between April 2015 and February 2016 showed that younger people have a greater risk of influenza A(H1N1)pdm09 infection than older people. 4 In a study conducted to estimate the burden of seasonal influenza in Tunisia, the incidence rate of influenzaassociated influenza-like illness (ILI) was 12.6% in the 2014-2015 season. Among positive patients, 39.2% were of A(H1N1)pdm2009 subtype and 15.5% of H3N2 subtype. 5

| Cohort study design
Details of the study design and protocol have been previously published. 12

| Serological testing
Blood specimens were collected in vacuum tubes containing clotting agents. Clotted blood was kept on ice and delivered to the laboratory on the same day, where it was stored at 4 C. On the following day, serum was separated from cells by centrifugation for 5 min at 1000Âg and then aliquoted and frozen at À20 C until use. Seasonal influenza A/Brisbane/10/07(H3N2) and pandemic A/California/04/09(H1N1) viruses were used to determine seroprevalence of antibodies against both viruses by hemagglutination inhibition (HI) assay, using 0.5% turkey red blood cells (RBCs). Sera were treated 1:3 with Receptor-Destroying Enzyme (RDE; Denka Seiken, Tokyo, Japan), incubated overnight at 37 C then inactivated at 56 C for 30 min. Inactivated sera were hemadsorbed by 5% packed turkey RBCs for 1 h at 4 C.
The hemadsorbed sera were separated by centrifugation at 1000Âg for 5 min, adjusted to 1:40 with phosphate buffered saline (PBS), diluted in two-fold dilutions, and incubated with an equal volume of 4 hemagglutination units per 25 μl of virus. Virus-sera mix was incubated for 30 min at room temperature. A 0.5% turkey RBCs solution was applied to all dilutions. Hemagglutination inhibition was scored after 30 min at room temperature. HI positivity was considered at end point titer of ≥1:40.

| Incidence calculation
A subject with a fourfold increase in antibody titer against each subtype in the consecutive sample was considered to be infected with that subtype during the time between the samples were obtained.

| Statistical analysis
The chi-square test was used to compare categorical variables. The McNemar test was used to compare seroprevalence and incidence accounting for repeated measurements. The SPSS version 24 (IBM, Armonk, NY, USA) was used. A p value <0.05 was considered statistically significant.

| RESULTS
The demographic distribution and health data of the study participants are shown in Table 1. The majority of participants were adults older than 18 years (58%) while children were 42%. The age range of the participants was 2 to 104 years old, and the mean age of the subjects was 26.73 years with standard deviation of 18.48 years.
Females constituted 55% of the study population. More than half of the participants were those with elementary and intermediate education representing 52.2%, followed by uneducated individuals (34.3%), and secondary or university educated individuals (13.5%).
Almost half of the subjects were single, and the rest were either married, divorced, or widowed. Students constituted 32.8%, housewives 29.2%, toddlers 14%, and the rest were either professionals, skilled laborers, or unemployed. Most of the participants did not suffer from chronic diseases.
Seroprevalence of antibodies against H3N2 and H1N1 among study participants during the period from 2017 to 2020 is shown in    The annual rate of seasonal influenza in a study in Egypt was estimated to be 20%-30% in children and 5%-10% in adults. 9 Our study provides comparable numbers and shows the annual variation in incidence and seroprevalence. The incidence and seroprevalence of H3N2 This study has a number of limitations. The seroprevalence is likely underestimated as collection of samples was not done after or during the season only but was spread over the year. Moreover, incidence is underestimated due to the potential underestimation of seroprevalence. The findings of this study may not be generalizable to the general population as it was restricted to rural areas.
In conclusion, this cohort study provided a better estimate of influenza A infection rates than regular SARI surveillance as clinical surveillance may miss milder infections that do not meet a traditional ILI surveillance profile and thus can underestimate the true burden of influenza. The seroprevalence of influenza A was high due to natural infection because participants were mostly unvaccinated. We recommend updating the influenza vaccination program to include exposed individuals in high-risk categories. writing-review and editing.

PATIENT CONSENT STATEMENT
Written informed consent was obtained from all subjects over 18 years old, written assent was obtained for children between 14 and 17 years old, parental written consent was obtained for all participants less than 18 years old.

DATA AVAILABILITY STATEMENT
Data are available in the manuscript.