Specimens were collected from subjects with influenza-like illnesses as part of the sentinel surveillance system for Influenza-like Illness (ILI) conducted in Dakar since 1996 and as requested by the Ministry of Health in order to decipher the etiology of febrile illnesses in children seeking care in primary healthcare centers in Dakar.
An ILI case was identified as an outpatient presenting with sudden onset of fever (≥38°C) and cough or sore throat, accompanied or not by myalgia, prostration, headache or malaise, with the onset of symptoms occurring within the previous 3 days. A standardized questionnaire was used to collect demographic and clinical information from the enrolled patients. Throat and/or nasopharyngeal swabs were taken from patients within 48–72 hours from onset of symptoms and then tested for virus isolation and/or molecular detection with standard protocols validated by WHO. When both throat and nasal swabs were available, the two samples were collected in the same tube to increase sensitivity of testing. Aliquots of samples were also stored at -80°C for further analysis.
For virus isolation, the clinical materials obtained from patients were inoculated on Madin Darby Canine Kidney (MDCK) cell lines as described previously . Tissue culture fluid was harvested after observing inoculated MDCK cell line for cytopathic effect. Hemagglutination and Hemagglutination Inhibition (HAI) tests were performed (detection and typing of viruses) using specific antisera, as recommended by WHO standard protocols .
For molecular characterizations, ribonucleic acid (RNA) extraction was performed from 200 μl of each sample using the QIAamp Viral RNA kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions. Each RNA sample was eluted with 50 μl of nuclease-free water.
We used an RT-PCR/RFLP technical  to discriminate between oseltamivir-sensitive and oseltamivir-resistant influenza A(H1N1) isolates. RT-PCR primers target a 183-bp region of the NA gene, encompassing the codon prone to H274Y mutation (N1-770Fw: AGA TCG AAA AGG GGA AGG TTA CTA, and N1-881Rv: TCC CTG CAT ACA CAC ATC ACT). Briefly, 6 μl of each RT-PCR product were digested with BspHI (New England Biolabs®) in a final volume of 20 μl, the restriction site for this enzyme (TCATGA/AGTACT palindrome) covering the codon 274 (H: CAT/Y: TAT). Thus, BspHI should cut only amplicons without mutation, i.e. amplicons from oseltamivir-sensitive A(H1N1) viruses (predicted size of the major digest product: 147 bp). Digested and undigested amplicons were co-electrophoresed on 2% agarose with appropriated molecular weight markers (100 bp ladder, New England Biolabs®). Oseltamivir-sensitive isolates from the 2007 Influenza season served as positive controls for BspHI digestion. Gels were stained with ethidium bromide (0.5 μg/ml).
The matrix M2 gene was amplified and sequenced in order to look for mutations conferring resistance to adamantanes.
Selected viruses were phenotypically (resistance to NAI), antigenically, and genetically characterized by the WHO Collaborating Centre for Influenza at the National Institute for Medical Research (NIMR) in London, UK. The influenza virus neuraminidase (NA) activity of the isolates and sensitivity to neuraminidase inhibitors (NI) was measured by an enzyme assay using a fluorogenic substrate i.e MUNANA (2’ 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate) with known NA inhibitor-resistant viruses as controls. HA and NA genes were sequenced using ABI Prism BigDye terminator cycle sequencing kits and an ABI-3730XL DNA analyser. Phylogenetic analysis of the neuraminidase whole gene sequence (1400 bp) and the haemagglutinin HA1 genes (1200 first bp) of H1N1 viruses were performed using MEGA version 5  for constructing Maximum Likelihood Tree using the Tamura-nei evolutionary model. Alignments of sequences were performed using the BioEdit Sequence alignment Editor .
The surveillance protocol was approved as less than minimal risk research by the Senegalese National Ethical Committee of the Ministry of Health, and written consent forms were not required. Throughout the study, the database was shared with the Epidemiology Department at the Senegalese Ministry of Health and Prevention for appropriate public health actions.