Molecular diagnosis of respiratory viruses.

The increasing availability of nucleic acid amplification tests since the 1980s has revolutionised our understanding of the pathogenesis, epidemiology, clinical and laboratory aspects of known and novel viral respiratory pathogens. High-throughput, multiplex polymerase chain reaction is the most commonly used qualitative detection method, but utilisation of newer techniques such as next-generation sequencing will become more common following significant cost reductions. Rapid and readily accessible isothermal amplification platforms have also allowed molecular diagnostics to be used in a 'point-of-care' format.

Detection of influenza A and B with the Alere ™ i Influenza A & B: a novel isothermal nucleic acid amplification assay.

Background: Rapid influenza diagnostic tests (RIDTs) have an important role in clinical decision-making; however, the performances of currently available assays vary widely.

Objectives: We evaluated the performance of the Alere(™) i Influenza A&B (Alere(™) iNAT), a rapid isothermal nucleic acid amplification assay that has recently received FDA clearance, for the detection of influenza A and B viruses during the Australian influenza season of 2013. Results were compared to two other RIDTs tested in parallel; Quidel Sofia(®) Influenza A+B fluorescent immunoassay (FIA) and Alere(™) BinaxNOW(®) Influenza A & B immunochromatographic (ICT) assay.

Evaluation of the Sofia Influenza A + B fluorescent immunoassay for the rapid diagnosis of influenza A and B.

Rapid influenza diagnostic tests (RIDTs) can facilitate the appropriate prescription of antivirals for influenza, obviate the need for unnecessary testing and antibacterial agents and allow the implementation of infection control measures. However, the reported sensitivities and specificities of different RIDTs vary widely in clinical settings, as does assay ability to distinguish between influenza types and subtypes. To evaluate the performance of the Sofia Influenza A + B fluorescent immunoassay (FIA) for the detection of influenza A and B during the 2013 Southern Hemisphere influenza season, a total of 209 consecutive respiratory tract swabs from adult patients with an influenza-like illness were tested by both Sofia Influenza A + B and an in-house real-time, reverse transcription-polymerase chain reaction (RT-PCR) assay.

Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia.

In March 2010, an outbreak of low pathogenicity avian influenza A (H10N7) occurred on a chicken farm in Australia. After processing clinically normal birds from the farm, 7 abattoir workers reported conjunctivitis and minor upper respiratory tract symptoms. Influenza virus A subtype H10 infection was detected in 2 workers.

Epstein-Barr virus genotypes and strains in central nervous system demyelinating disease and Epstein-Barr virus-related illnesses in Australia.

Objectives: To identify Epstein-Barr virus (EBV) genotypes and strains in samples from individuals with and without a first diagnosis of central nervous system (CNS) demyelinating disease (a possible precursor to multiple sclerosis) and patients with EBV-associated diseases in Australia.

Methods: Samples from 55 EBV DNA and serology positive subjects including individuals with (n = 17) and without (n = 21) a first clinical diagnosis of CNS demyelination and patients with EBV-related diseases (n = 17) were examined. EBV genotype and strain were identified by sequence mutations within the Epstein-Barr nuclear antigen-2 region (EBNA-2) using DNA sequence analysis.

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.

Comparison of the incidence of influenza in relation to climate factors during 2000-2007 in five countries.

Relatively few international comparisons of the incidence of influenza related to climate parameters have been performed, particularly in the Eastern hemisphere. In this study, the incidence of influenza and climate data such as temperature, relative humidity, and rainfall, from cities at different latitudes with contrasting climates: Singapore, Hong Kong (China), Ulaanbaatar (Mongolia), Vancouver (Canada), and three Australian cities (Brisbane, Melbourne and Sydney) were examined to determine whether there was any overall relationship between the incidence of influenza and climate. Applying time-series analyses to the more comprehensive datasets, it was found that relative humidity was associated with the incidence of influenza A in Singapore, Hong Kong, Brisbane, and Vancouver.

Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye.

Background: Reactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases.

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.

Molecular identification and analysis of nonserotypeable human enteroviruses.

Conventional approaches to characterizing human enteroviruses (HEVs) are based on viral isolation and neutralization. Molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) and nucleotide sequencing of the entire or partial VP1 gene. A modified RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient way to characterize common and nonserotypeable (by neutralization) HEVs.

Comparison of a rapid antigen test with nucleic acid testing during cocirculation of pandemic influenza A/H1N1 2009 and seasonal influenza A/H3N2.

The rapid diagnosis of influenza is critical in optimizing clinical management. Rapid antigen tests have decreased sensitivity in detecting pandemic influenza A/H1N1 2009 virus compared to seasonal influenza A subtypes (53.4% versus 74.

Outbreak of human metapneumovirus infection in a residential aged care facility.

Summer outbreaks of respiratory illness in residential aged care facilities are uncommonly reported in New South Wales. A respiratory illness outbreak in an aged care facility during January 2008 prompted a response to contain the outbreak by implementing infection control measures, including cohorting of symptomatic residents, cohorting nursing care, closure to new admissions and the use of personal protective equipment by staff. In addition, respiratory tract specimens were collected to determine the causative agent.

Identification of 20 common human enterovirus serotypes by use of a reverse transcription-PCR-based reverse line blot hybridization assay.

The more than 100 human enterovirus (HEV) serotypes can also be classified into four species, HEV-A to -D, based on phylogenetic analysis of multiple gene regions. Current molecular typing methods depend largely on reverse transcription-PCR (RT-PCR) amplification and nucleotide sequencing of the entire or 3' half of the VP1 gene. An RT-PCR-based reverse line blot (RLB) hybridization assay was developed as a rapid and efficient approach to characterize common HEVs.

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.

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.

Segregation of human immunodeficiency virus type 1 subtypes by risk factor in Australia.

The aim of this study was to determine which human immunodeficiency virus type 1 (HIV-1) subtypes were circulating in Australia and to correlate the subtypes with risk factors associated with the acquisition of HIV-1 infection. DNA was extracted from peripheral blood mononuclear cells, and HIV-1 env genes were amplified and subtyped using heteroduplex mobility analysis, with selected samples sequenced and phylogenetic analysis performed. The HIV-1 env subtypes were determined for 141 samples, of which 40 were from female patients and 101 were from male patients; 13 samples were from children.