Evidence of the circulation of pandemic influenza (H1N1) 2009 with D222D/G/N/S hemagglutinin polymorphisms during the first wave of the 2009 influenza pandemic.

BACKGROUND: The hemagglutinin HA1 D222G substitution may be associated with adverse outcomes in pandemic influenza A (H1N1) 2009 infections by enhancing the binding capacity of α2-3 sialyl receptors to pandemic influenza (H1N1) 2009 viruses. OBJECTIVES: To investigate the emergence of the D222G mutation and other polymorphisms at this position during the first southern hemisphere pandemic wave in 2009. STUDY DESIGN: A total of 127 samples that were nucleic acid test positive for pandemic influenza (H1N1) 2009 virus were subjected to a sequence-based genotypic assessment of viral populations. Specimens showing polymorphisms at position 222 were further cloned to characterise the viral quasispecies. RESULTS: A high proportion of intensive care unit (ICU) admissions (20%) and outpatients with mild symptoms (11.3%) carried polymorphisms of D/G/N/S at position 222 in hemagglutinin. Viral quasispecies derived from the upper and lower respiratory tract (URT and LRT) in ICU patients showed comparable levels of 222G populations. CONCLUSION: The detection of 222G quasispecies present in the URT in both ICU and outpatient groups suggest ready transmission of these variants, and its frequent detection (and clusters) in outpatients imply local community transmission.

Detection of the rapid emergence of the H275Y mutation associated with oseltamivir resistance in severe pandemic influenza virus A/H1N1 09 infections.

In 2009 a new swine-origin influenza virus A/H1N1 (A/H1N1 09) emerged, causing the century's first pandemic. Most isolates of the new A/H1N1 09 virus are susceptible to neuraminidase inhibitors, but the H275Y mutation in the neuraminidase gene region associated with high-level oseltamivir resistance has been detected. Using rolling circle amplification (RCA) technology, 96 A/H1N1 09-specific RT-PCR positive clinical samples collected from 80 oseltamivir-treated and untreated patients were screened for the presence of the H275Y mutation. Samples positive for 275Y mutation by RCA were cloned and sequenced for confirmation. From 25 patients who had been treated with oseltamivir and remained A/H1N1 09 RT-PCR positive, we identified three (12%) individuals with the H275Y mutation: one immuno-suppressed adult, one immuno-competent adult and one child. Samples collected at multiple time points from the two adults showed a switch from wild-type oseltamivir-sensitive 275H to oseltamivir-resistant 275Y population after 9 days of treatment. The child had the 275Y mutation detected after being persistently A/H1N1 09 RT-PCR positive while receiving oseltamivir treatment. Resistance was not detected in 17 pre-treatment samples and 54 A/H1N1 09 RT-PCR positive outpatients. RCA demonstrates the rapid emergence of the H275Y resistance mutation in individuals with severe A/H1N1 09 infection receiving neuraminidase inhibitors. Rapid detection of oseltamivir resistance in severe infection is essential for patients to receive maximum therapeutic benefit. In the light of emerging resistance, close monitoring and understanding of the nature and dynamics of resistance mutations in newly emerging strains should be a priority.

Emergence and spread of oseltamivir-resistant A(H1N1) influenza viruses in Oceania, South East Asia and South Africa.

The neuraminidase inhibitors (NAIs) are an effective class of antiviral drugs for the treatment of influenza A and B infections. Until recently, only a low prevalence of NAI resistance (<1%) had been detected in circulating viruses. However, surveillance in Europe in late 2007 revealed significant numbers of A(H1N1) influenza strains with a H274Y neuraminidase mutation that were highly resistant to the NAI oseltamivir. We examined 264 A(H1N1) viruses collected in 2008 from South Africa, Oceania and SE Asia for their susceptibility to NAIs oseltamivir, zanamivir and peramivir in a fluorescence-based neuraminidase inhibition assay. Viruses with reduced oseltamivir susceptibility were further analysed by pyrosequencing assay. The frequency of the oseltamivir-resistant H274Y mutant increased significantly after May 2008, resulting in an overall proportion of 64% (168/264) resistance among A(H1N1) strains, although this subtype represented only 11.6% of all isolates received during 2008. H274Y mutant viruses demonstrated on average a 1466-fold reduction in oseltamivir susceptibility and 527-fold reduction in peramivir sensitivity compared to wild-type A(H1N1) viruses. The mutation had no impact on zanamivir susceptibility. Ongoing surveillance is essential to monitor how these strains may spread or persist in the future and to evaluate the effectiveness of treatments against them.

Nosocomial and community transmission of measles virus genotype D8 imported by a returning traveller from Nepal.

Measles is uncommon in Australia due to effective national vaccination strategies. In mid-2003, a cluster of nine cases of measles occurred in western Sydney. The index case was a 29-year-old traveller recently returned from Nepal. The case presented to hospital and transmitted the disease to two others in the Emergency Department. Further cases resulted from both community and nosocomial transmission. The median age of cases was 24 years, with three cases in children aged under four years. Only one person had a documented history of measles vaccination, a child who had received one dose of vaccine overseas. One case was a 2-month-old infant whose mother was immune and two cases were hospital staff members. Molecular analysis of measles virus isolates from four cases revealed the same D8 genotype, a strain previously identified in Nepal. Staff vaccination strategies implemented as a result of the outbreak were poorly patronised despite nosocomial transmission. As diseases such as measles become rare it is important to thoroughly investigate any outbreaks, and to maintain a high index of suspicion of measles, particularly in travellers presenting with a rash having returned from measles-endemic areas. Genetic analysis is important in tracing the origins of an outbreak, and to confirm relatedness between cases. The highly infectious nature of measles virus also underscores the need for appropriate infection control in minimising risk of nosocomial transmission. Such policies are of increasing importance with the emergence of novel viruses or the threat of pandemic influenza.

Challenges for the laboratory before and during an influenza pandemic.

Laboratory tests that reliably confirm infection with a novel influenza strain are a major component of pandemic planning. Combined nose and throat swabs are the most practical respiratory tract sample to safely obtain from patients. As nucleic acid tests are sensitive, specific and rapid, they will be the diagnostic test of choice during a pandemic. Virus isolation (in laboratories with Physical Containment level 3 facilities) is required for characterisation of the pandemic strain and vaccine development. Antiviral resistance testing may be required if antiviral drugs are used extensively to help control a pandemic. Diagnostic strategies will vary throughout the various pandemic phases.

Antiviral prophylaxis in the management of an influenza outbreak in an aged care facility.

Influenza in persons aged > or = 65 years is associated with an increased risk of severe complications. Residents in aged care facilities have a higher proportion of chronic illnesses and within closed settings there is an increased risk of transmission. In July 2002, a 50 bed aged care facility reported an influenza-like illness (ILI) among residents and staff despite over 90 per cent influenza vaccine coverage among residents. A total of 17 of 49 residents and 9 of 43 staff met the case definition for ILI with onset on or after 26 June 2002. Seven people required hospitalisation and two died. Nasopharyngeal swabs were collected from symptomatic residents and staff, and influenza A was detected in six residents and two staff. Five unimmunised residents and 33 unimmunised staff were offered influenza vaccine and all residents and staff were offered oseltamivir prophylaxis of 75mg daily for 10 days. Subsequently, of 41 residents tested, seven demonstrated a fourfold or greater rise in antibody titres specific to H3N2 yet reported no symptoms. All seven had been immunised at least eight weeks previously, and had taken oseltamivir prophylaxis. This outbreak was characterised by a high attack rate of ILI in a well-immunised community. The ability to access rapid diagnostic testing enabled the prompt initiation of antiviral prophylaxis, which may have a role in controlling influenza in this setting.