Replacement of neuraminidase inhibitor‐susceptible influenza A(H1N1) with resistant phenotype in 2008 and circulation of susceptible influenza A and B viruses during 2009‐2013, South Africa

Background Data on the susceptibility of influenza viruses from South Africa to neuraminidase inhibitors (NAIs) are scarce, and no extensive analysis was done. Objectives We aimed to determine oseltamivir and zanamivir susceptibility of influenza A and B virus neuraminidases (NAs), 2007‐2013, South Africa. Patients/Methods We enrolled participants through national influenza‐like illness surveillance, 2007‐2013. Influenza diagnosis was by virus isolation and quantitative polymerase chain reaction (qPCR). Drug susceptibility was determined by chemiluminescence‐based NA‐STAR/NA‐XTD assay. Sanger sequencing was used to determine molecular markers of NAI resistance. Results Forty percent (6341/15 985) of participants were positive for influenza viruses using virus isolation (2007‐2009) and qPCR (2009‐2013) methods. A total of 1236/6341 (19.5%) virus isolates were generated of which 307/1236 (25%) were tested for drug susceptibility. During 2007‐2008, the median 50% inhibitory concentration (IC50) of oseltamivir for seasonal influenza A(H1N1) increased from of 0.08 nmol/L (range 0.01‐3.60) in 2007 to 73 nmol/L (range 1.56‐305 nmol/L) in 2008. Influenza A isolates from 2009 to 2013 were susceptible to oseltamivir [A(H3N2) median IC50 = 0.05 nmol/L (range 0.01‐0.08); A(H1N1)pdm09 = 0.11 nmol/L (range 0.01‐0.78)] and zanamivir [A(H3N2) median IC50 = 0.56 nmol/L (range 0.47‐0.66); A(H1N1)pdm09 = 0.35 nmol/L (range 0.27‐0.533)]. Influenza B viruses were susceptible to both NAIs. NAI resistance‐associated substitutions H275Y, E119V, and R150K (N1 numbering) were not detected in influenza A viruses that circulated in 2009‐2013. Conclusions We confirm replacement of NAI susceptible by resistant phenotype influenza A(H1N1) in 2008. Influenza A and B viruses (2009‐2013) remained susceptible to NAIs; therefore, these drugs are useful for treating influenza‐infected patients.


| INTRODUC TI ON
Annually, influenza virus infections account for an estimated 3-5 million cases globally, with 250 000-500 000 deaths. 1  Administration to treat seasonal influenza. 4,5 Oseltamivir is the most widely used due to ease of oral administration. Data from Australasia and South-East Asia showed that influenza A viruses from 1998 through 2002 were overall more susceptible to the NAIs, oseltamivir, and zanamivir, than influenza B viruses. 6 However, in 2008, oseltamivir resistance was reported at a frequency of 90% globally for seasonal influenza A(H1N1) viruses and was associated with histidine to tyrosine mutation at position 275 (H275Y, N1 numbering) of the NA. 7,8 During the 2008 influenza season, oseltamivir-resistant influenza A(H1N1) viruses were also isolated from South African patients with influenza-like illness. These influenza A(H1N1) virus isolates (n = 49) had the H275Y substitution and were confirmed to be phenotypically resistant to inhibition by oseltamivir. 9 Neuraminidase inhibitor resistance associated with the H275Y mutation was reported at a frequency of 3% (169/5152) in influenza A(H1N1)pdm09 virus isolates received at the World Health Organization (WHO) collaborating centers (CCs) from various geographic regions, 2013-2014. 7,10 Global NAI susceptibility surveillance data from WHO-CCs for 2013-2014 include less than 3% African data. 10 Both oseltamivir and zanamivir are licenced in South Africa.
Zanamivir is available since the early 2000s and oseltamivir since 2006. 11,12 Zanamivir is approved for treatment of children aged 7 years and older, whereas oseltamivir can be given to individuals of all ages. 13,14 Although generally thought not to be widely prescribed, limited reports are available on the use of NAIs in South Africa. Benefit of oseltamivir for both treatment of and prophylaxis against influenza-associated respiratory illness in South African infants with low birthweight was reported. 15 Influenza A(H1N1) pdm09 H275Y-resistant phenotype viruses were reported in 1 of 54 (2%) of patients following 5-day standard dose oseltamivir treatment. 16 We aimed to determine oseltamivir and zanamivir susceptibility of influenza A and B virus NAs, 2007-2013, South Africa, and to investigate amino acid polymorphisms in NA.

| Study specimens
Respiratory specimens collected included primarily nose and throat swabs collected at the time of diagnosis of the acute respiratory illness episode prior to the initiation of treatment. All upper respiratory tract specimens from patients enrolled from 2007 to 2013 (Figure 1) were collected in viral transport medium (Highveld Biological, Johannesburg, South Africa) or universal transport medium (Copan, Murrieta, CA, USA) as previously described. 18 Briefly, MDCK cells were seeded in shell vials and maintained with Dulbecco's modified Eagle's medium (DMEM, Lonza, Allendale, NJ, USA) containing 4.5 g/L glucose, with l-glutamine, 10% fetal bovine serum, 2 mmol/L l-glutamine, and antibiotics. After inoculation with 250 microliter clinical specimen for 2 hours on washed serum-free cell monolayers, the cultures were incubated with DMEM serum media at 37ºC and harvested 24-73 hours later. Culture supernatant fluids from influenza virus cultures were stored at −70°C. 22,23 Influenza virus isolates (2007-2009) were subtyped serologically by hemagglutination inhibition (HAI) and subtype-specific antisera. [23][24][25] Influenza viruses were detected by qRT-PCR, and influenza A positives were subtyped using a qRT-PCR assay from Centers for Disease Control and Prevention (CDC, Atlanta, GA, USA). 18,20,21,26 From 2009 to 2013, influenza-positive specimens with qPCR with cycle threshold (C t ) values <30 were selected for virus isolation.
In addition, we selected specimens for influenza virus isolation at the beginning, middle, and end of the South African influenza season (typically May through September).

| The NA-STAR and NA-XTD chemiluminescent assay
The NA-STAR and NA-XTD chemiluminescent assays (Applied Biosystems, Foster City, CA, USA) were used to determine the susceptibility of NA from influenza A and B virus isolates to oseltamivir and zanamivir. Virus culture supernatant fluid aliquots were retrieved and pre-treated with 10% Triton X-100 solution to inactivate virus. 27 The NA inhibition assays were performed following the manufacturers' protocols as described. 28 Briefly, virus isolates were titrated to determine the signal-to-noise ratio of 40 as NA concentration for use in the inhibition assay. The final NAI concentrations ranging from 0.028 to 550 nanomolar (nmol/L) and the appropriate virus dilution (signal:noise = 40 or a minimum concentration of NA

| Detection of drug resistance mutations and drift in influenza virus neuraminidase
The NA genes of influenza A and B strains from 2009 to 2013 were amplified and sequenced from original clinical specimens.
Briefly, the universal influenza A and B primers (uni12 and Buni11) were used for complementary DNA (cDNA) preparation followed by nested PCR. 30,31 Sequencing was performed using the BigDye

| D ISCUSS I ON
In this study, we confirm change in circulation of oseltamivir-  34  40  44  48  106  200  223  241  247  248  260  264  270  275  299  321  351  369  382  386  388  394 407 432 Limitations to our study were as follows: This study provides baseline data on influenza A and B viruses with oseltamivir-susceptible phenotypes in South Africa.
Guidelines for the prevention and treatment of influenza in South Africa are published electronically. 52 However, although oral oseltamivir and inhaled zanamivir are available in South Africa, they are not routinely prescribed. 14,53 This is also observed for hospitalized patients enrolled through our pneumonia surveillance program in public hospitals (C. Cohen, unpublished data). Oseltamivir is prescribed more frequently by private healthcare practitioners, but no data are available on the extent of use in South Africa.
Given the very low frequency of resistance reported globally, the use of NAIs to treat patients at high risk of developing severe illness should be promoted.

CO N FLI C T O F I NTE R E S T
The authors do not declare financial or personal conflict of interest related to this study.

D I SCL A I M ER
The findings and conclusions in this report do not necessar-