Inuenza outbreak among students in Ghana: a report from three time points

Background: Inuenza viruses are known to be responsible for a number of respiratory disease outbreaks worldwide. While there exists documented information on inuenza which allows for effective strategic preparedness activities in industrialized countries, the same cannot be said of developing and underdeveloped countries. It therefore behoves on developing countries to document information on circulating strains of inuenza. Such information will enable prioritization of prevention and more targeted preparedness strategies. In this paper, we report on three different outbreaks of Inuenza A among Ghanaian students in December 2017, May 2019 and December 2019 in the Ashanti region of Ghana. Methods: Throat or nasal samples were collected from all students who presented with signs and symptoms of u with ocked swabs, stored in RNAlater and transported to the laboratories of Kumasi Centre for Collaborative Research (KCCR) at ambient temperature. Following viral RNA extraction, detection of viral nucleic acid of both inuenza A and its subtypes including H1N1 and H3N2 was done by real-time reverse transcription Polymerase Chain Reaction (RT-PCR). Results: A total of 112 samples were taken. An overall Inuenza A prevalence of 51.8% (95% CI = 42.1-61.3) was recorded during the three outbreak time points. Of these, 11 (19.0%) were conrmed to be H1N1 (pdm09) and 47 (81.0%) were H3N2 strains. All the 11 H1N1 (pdm09) were identied during the rst outbreak. The H3N2 strains were identied during the second and third outbreaks in May 2019 and December 2019 respectively. Our results show that inuenza A virus activity was higher during the months of November and December compared to May. Age, contact with infected subjects, temperature, fever within the last 24 hours and headaches showed signicant variations (p-value <0.01) with respect to inuenza A. Wald’s test revealed that subjects presenting with headaches had about 5 times odds (95% CI = 1.4-17.7) for inuenza A infection compared to subjects without headache (p = 0.014). Conclusion: Our study provides epidemiological information to better understand the incidence and burden of inuenza to allow for prioritisation of prevention and control strategies. Such information would aid in controlling and preparing for future inuenza epidemics.


Introduction
In uenza viruses are implicated in highly infectious respiratory disease outbreaks worldwide [1]. The success of in uenza viruses in causing seasonal outbreaks year in and year out and eventually pandemics is dependent on the unique ability of the viruses to undergo frequent changes in their surface antigen [2]. As a result, immunity resulting from infection by one in uenza virus does not fully protect against antigenic or genetic variants of the same subtype. Annual global in uenza epidemics result in an estimated 3 to 5 million cases of severe respiratory illness and up to about 650,000 deaths [3].
The viruses attack all age groups with an estimated attack rate between 5%-30% [4]. Although the elderly stand the highest risk of in uenza associated mortalities, about 99% of mortalities resulting from in uenza are recorded in children, especially those in developing countries [5]. The burden of in uenza in developing tropical and sub-tropical countries is likely to be underestimated. A systematic review of 30 years of seasonal in uenza epidemiology in these regions revealed that in uenza accounts for 10% of all outpatient visits and about 6.5% of hospitalizations [6]. However, for most of these developing countries, reports on in uenza were considered insu cient to allow for prioritization of prevention, control and preparedness strategies [6]. It is therefore imperative for research laboratories to regularly report data gathered on in uenza to aid in planning prevention and control.
In Ghana, reports on in uenza viruses have been hospital-based or targeted at at-risk populations. For example, our group has reported on prevalence of 5.7% and 1.7% among outpatients [7] and in-patients [8] with acute respiratory infection respectively. Further, our study on respiratory pathogens among Ghanaian pilgrims returning from Mecca had 1.4% being positive for in uenza viruses [9]. Other studies focused on In uenza A H1N1 (pdm2009) and H3N2 reporting rates of 57% and 26% respectively [10]. Prior to this report, In uenza viruses had not been implicated in an epidemic or pandemic situation. In December 2017, the Kumasi Academy Senior High School (KUMACA) in the Ashanti region of Ghana was the site of an H1N1 (pdm09) epidemic. This led to the loss of four (4) lives [11]. The Ministry of Health in Ghana previously reported an attack rate of 14/1,000 population with a case fatality rate of 9.1% [12].
Similarly, in May 2019, there was an outbreak of H3N2 among medical students on a clinical rotation at the Komfo Anokye Teaching Hospital (KATH), the second largest referral hospital in Ghana. Another in uenza A outbreak was recorded for KUMACA in December 2019. In this study, we report on samples collected, laboratory diagnosis and results generated during these three independent outbreaks. This report will provide up to date information on currently circulating in uenza strains in Ghana and the importance of local laboratory involvement in such situations.

Materials And Methods
The outbreak setting/study area The three outbreaks were centered around three focal areas within the Kumasi Metropolitan Area (KMA). These were KUMACA, Kwame Nkrumah University of Science and Technology (KNUST) and KATH.
Outbreak one occurred in December 2017 at KUMACA, outbreak two was in May 2019 at KATH and outbreak three at KUMACA in December 2019.
KUMACA is a senior high school in the Asokore Mampong Municipality of the Ashanti Region. The school has a student population of about 3000, of which close to 60% are males. The school has both day and boarding facilities. The boarding students are accommodated in 8 houses namely Prempeh, Sadler, Boakye Dankwa (BoDank), Yaa Achiaa, Akua Nyarko, Jubilee, Nana Boakye Ansah Debrah (NABOAD) and New house. KNUST is a tertiary institution with about 55,000 student size. The KNUST hospital is the university's main health care facility within which is a clinic designated for only students. The hospital, though situated on the university campus, serves the general community surrounding the school. Students from KUMACA are usually referred to the University Hospital, KNUST due to its close proximity to the school. KATH is a tertiary healthcare facility which serves as a teaching hospital for the School of Medicine and Dentistry of KNUST. The facility receives referrals from 12 out of the 16 administrative regions of Ghana and has a staff size of about 4000 working in 15 different directorates (13 clinical and 2 non-clinical). The hospital is bordered by communities such as Bantama, Adum, Kejetia and Nhyiaeso. Medical students on clinical rotation are housed in hostels within the hospital environment and in homes in the surrounding neighbourhoods. Figure 1 shows the study areas and the different hospitals where the patients were admitted or visited.

Inclusion criteria
All students who presented with signs and symptoms of u were assessed by a Physician and included in the study.

Sample collection
During the three outbreaks, respiratory samples (throat or nasal swabs) for 112 patients/students were received at the Kumasi Centre for Collaborative Research (KCCR) laboratories at the Kwame Nkrumah University of Science and Technology (KNUST) for testing. The samples were collected with ocked swabs (Copan Group, Brescia, Italy) and stored in 500ml RNAlater (Qiagen, Hilden, Germany) in 1.5ml tubes (Eppendorf, Regensburg, Germany) and immediately transported to the KCCR laboratories. Some samples were accompanied by case report forms (CRFs). The CRFs examined mainly socio-demographic characteristics, history with animal exposure and treatment outcome of suspected students/patients. All samples taken at the University Hospital, KNUST and the Komfo Anokye Teaching Hospital (KATH) were transported at ambient temperature to the KCCR and tested at its laboratories. External quality control testing of selected samples was performed by Institute of Virology in Charité, Berlin during the rst outbreak in December, 2017. For subsequent outbreaks, viral aetiology testing was locally con rmed by KCCR laboratories.

Viral RNA extraction and testing
Extraction of viral RNA from the samples was done using the Qiagen viral RNA mini kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. Samples were extracted individually with an input volume of 140μl and eluted in 60µl of elution buffer.

Detection of in uenza viruses
All samples were tested for both in uenza A and its subtypes including H1N1pdm09 and H3N2. Detection of viral nucleic acid was done by real-time reverse transcription Polymerase Chain Reaction (RT-PCR).
Primers and probes used for targeting the viruses and cycling conditions for the PCR run were as previously described for In uenza A and In uenza A subtypes H1 and H3 [10] using the Invitrogen SuperScript III Platinum OneStep buffer system (ThermoFisher Scienti c, MA, USA). Testing was performed on a CFX96 Bio-Rad real-time PCR platform with CFX software version 1.6 (Bio-Rad, Singapore). Results of the real time RT-PCR assays were determined by analyzing the cycle threshold (Ct) values of the various samples generated by the CFX software. In all tests, samples with Ct values less than 38 were considered positive and included in the nal analysis. All PCR runs were validated by the inclusion of positive and negative controls.

Data management
Data were double-entered and aggregated into a Microsoft Excel le (version 2019) and cleaned.
Statistical analysis was done using R statistical software (version 3.5.1, 2018). Descriptive analysis was performed to determine the frequency and percentages of categorical variables. The association between in uenza A detection and other variables was analysed using Fisher's exact test or Chi-square test where appropriate. Variables that were signi cant were entered into a logistic regression model. Estimates were expressed as Odds Ratio (OR) and 95% Wald's Con dence interval. A statistically signi cant result was considered when p-value was ≤0.05.

Results
Socio-demographic and clinical presentations of the study participants Samples were received for 133 patients during the three outbreaks. However, case report forms were received for 112 (84.2%) and therefore used in the statistical analysis. The average age of all subjects was 19.2±3.7 years. More than half (62, 55.3%) of the students were below 20 years. The percentage of females was slightly higher than that of males (51.8% vs. 48.2%). No history of animal contact or exposure to swine was reported by the subjects, however, contact with suspected or con rmed infected people was reported (44, 38.6%). Table 1 gives descriptive information on the study participants and study variables. Epidemiological picture during the three time points For the three outbreaks that occurred, twenty (20) and 40 samples were collected during outbreak 1 and 2 respectively, while 52 samples were collected during outbreak 3.
The majority (77.6%) of in uenza positive cases were recorded between November to mid-December.
Accordingly, outbreaks 1 and 3 which occurred in December had higher proportion of in uenza A positive cases than the second outbreak in May. Figure 2 shows the daily distribution of cases during the three outbreak periods.

Distribution of in uenza by site
The majority of the participants were recruited from KUMACA (57, 50.9%) followed by KATH (40,  Of the 58 subjects who tested positive for in uenza A, 10 individuals who were exposed to infection with in uenza A were younger (

Discussion
The sudden emergence and detection of In uenza A H1N1 (pdm09) in KUMACA in the Ashanti region of Ghana in 2017 was totally unexpected. This tested the preparedness of local health response systems and exposed the weaknesses in these systems as it took close to two weeks and four deaths [11] to con rm aetiology. This revealed the need for strong local capacities for preparedness and response against in uenza and emphasised the importance of multisectoral collaboration in epidemic situations to establish aetiology in a timely manner to inform patient management strategies. The importance of a strong local capacity was manifested during the second and third outbreaks in May and December 2019 respectively where aetiology was con rmed within a day.
The different in uenza strains detected during the three outbreaks may be attributable to the frequent genetic shift of the surface antigens of the in uenza virus and the circulation of these viral subtypes in Ghana. This calls for a concerted effort by all stakeholders to make provisions and enhance preparedness for epidemics by the different strains of the virus at any point in time.
It is apparent from our results that in uenza A virus activity may be highest during the months of November and December compared to May. Even though such a rm observation can only be made after surveillance of respiratory viruses all year-round, these months (November and December) are the transition months from the minor rainy season to the dry season in Ghana with moderate intensity of warmth and low humidity. Low temperatures and humidity favour in uenza virus infectivity as has been demonstrated by a number of studies [13][14][15][16]. The virus keeps maximal infectivity at relatively low humidity while viral inactivation occurs at relatively high humidity and temperature. Consequently, the virus is rendered non-viable after being carried in respiratory droplets for relatively longer periods in high humid air. This moderate warmth and low humidity could explain the high in uenza activity during the months of November and December. The high humidity of the rainy season around the month of May potentially limits the extent of spread and viability of the virus resulting in the low in uenza A activity around that period.
The overall high prevalence of in uenza A observed under this study and at the different time points could be due to the relative activity of the in uenza virus all year round in tropical regions of Africa as reported by Radin et al after 5 years surveillance of in uenza activity in some African countries [17]. Prevalence rates for the rst (December 2017) H1N1 (pdm09) and the second (May 2019) H3N2 outbreaks conform to the ndings of a three year surveillance report on in uenza viruses in Ghana [10] and could represent the prevalence rate of these subtypes in Ghana. The high prevalence rate (71.2%) recorded during the December 2019 H3N2 period is characteristic of a temperate pattern of in uenza activity between November and January in North African countries and other temperate regions [17][18][19], and suggestive of the in uence of climatic conditions on the transmission of in uenza viruses [20]. This high prevalence also points to the endemicity of the virus subtype in Ghana and other West African states [17].
In our analyses, we found that in uenza A was more frequently detected in subjects < 20 years which is consistent with reports that suggest that in uenza A, especially H1N1 has a higher attack and relative mortality rates in people under 20 years [21,22]. The predominance among this group could be explained by the fact that there is a high level of physical contact and interaction among this group due to overcrowding of students as witnessed in many Ghanaian second cycle schools [23]. Consequently, when one is infected, there is a high rate of spread of the viral infection among the students [20]. This makes them e cient transmitters of the virus and could represent an at-risk group. This study could not establish contact with an infected person as a risk factor (OR=0.37) of in uenza A infection as evidenced by the large proportion of in uenza positive cases (70.2%) who had not had any prior physical contact with an infected person. This is because the virus could be transmitted through infectious droplets and aerosols and as such physical contact with an infected person might not be necessary for viral transmission especially in an epidemic situation [24]. This underlies the effect of overcrowding on the transmission of the virus through infectious droplets and aerosols.
During outbreaks of zoonotic origins, information on contact with animals or wildlife and/or consumption of their meat becomes important [25,26]. Our results indicated no history of animal contact. Even though our limited data may not warrant such a conclusion, an observed high prevalence of In uenza A H1N1 (pdm09) in pigs in Kumasi [27] provides hints of the possibility of human-to-swine or vice versa transmission. This underscores the need for systematic swine surveillance in Ghana considering the close contact between humans and livestock especially pigs which are now seen in many developing countries including those of sub-Saharan Africa [28][29][30].
Although many in uenza patients may not present with speci c signs and symptoms of illness, the acute onset of respiratory illness is typical for in uenza. This includes coughing, fever and headache [31].
Although the triad of headache, coughing, and pharyngitis has been found to constitute the major predictors of in uenza disease [32], our study found headache as the most important predictor of in uenza infection during the outbreaks, as those who presented with headache were ve times more susceptible to in uenza A infection. This contrasts the ndings of Monto et al, (2000) who found fever and coughing to be the most important predictors of in uenza disease [33].
Our data comes with some limitations. Key limitation is the lack of data for some of the cases who were recruited. This affects the sample size and statistical power of the study and consequently does not allow for generalisation of ndings. Also, information provided by this study does not depict possible seasonal variability as data was only taken during outbreaks and not a year-round surveillance.
In conclusion, our study provides epidemiological information to better understand the incidence and burden of in uenza to allow for prioritisation of prevention and control strategies. Information on the current circulating strains would aid in controlling and preparing for the next in uenza epidemic and this requires information on the seasonality of in uenza, which is sorely lacking. The recent in uenza outbreak experience is necessary to develop the next generation of strategic, operational, and tactical plans for any anticipated or unplanned outbreaks.