Molecular biomarkers for chronological age in animal ecology.

The chronological age of an individual animal predicts many of its biological characteristics, and these in turn influence population-level ecological processes. Animal age information can therefore be valuable in ecological research, but many species have no external features that allow age to be reliably determined. Molecular age biomarkers provide a potential solution to this problem. Research in this area of molecular ecology has so far focused on a limited range of age biomarkers. The most commonly tested molecular age biomarker is change in average telomere length, which predicts age well in a small number of species and tissues, but performs poorly in many other situations. Epigenetic regulation of gene expression has recently been shown to cause age-related modifications to DNA and to cause changes in abundance of several RNA types throughout animal lifespans. Age biomarkers based on these epigenetic changes, and other new DNA-based assays, have already been applied to model organisms, humans and a limited number of wild animals. There is clear potential to apply these marker types more widely in ecological studies. For many species, these new approaches will produce age estimates where this was previously impractical. They will also enable age information to be gathered in cross-sectional studies and expand the range of demographic characteristics that can be quantified with molecular methods. We describe the range of molecular age biomarkers that have been investigated to date and suggest approaches for developing the newer marker types as age assays in nonmodel animal species.

High-throughput real-time PCR and melt curve analysis for sexing Southern Ocean seabirds using fecal samples.

Sex identification of birds is of great interest in ecological studies, however this can be very difficult in many species because their external features are almost monomorphic between the sexes. Molecular methodology has simplified this process but limitations still occur with widely accepted methods using polymerase chain reaction and gel electrophoresis, especially when applied to degraded DNA. Real-time polymerase chain reaction assays are emerging as a more efficient, sensitive, and higher throughput means of identification, but there are very few techniques validated using fecal samples and small target sizes. We present a real-time melt curve analysis assay targeting a small region of the CHD-1 gene allowing for high-throughput, sensitive, specific, and easy-to-interpret sexing results for a variety of Southern Ocean seabirds using fecal and tissue samples.

Human rhinovirus C in adult haematopoietic stem cell transplant recipients with respiratory illness.

BACKGROUND: A previously unidentified species of human rhinovirus, HRV-C, was described in 2006 in association with lower respiratory tract infection (LRTI). Features of infection in immunosuppressed adults are poorly characterised. OBJECTIVES: This study aims to determine the epidemiology of HRV-C in haematopoietic stem cell transplant (HSCT) recipients in a single centre. STUDY DESIGN: A prospective cohort study of all HSCT recipients admitted to Westmead Hospital, Westmead, Australia from 1 July 2005 to 30 September 2007 was undertaken. Nose/throat samples were collected from all patients at the time of admission and patients developing pre-defined symptoms and/or signs of respiratory infection during the admission. Samples were processed and tested for rhinoviruses and 14 other respiratory viruses using nucleic acid-based methods, immunofluorescence and culture. HRV genotyping was performed by sequencing a region of the rhinovirus 5' untranslated region (UTR). Clinical data on each episode were collected prospectively. RESULTS: HRVs were identified in 24 episodes: 8% of 299 episodes of clinically- defined respiratory infections and 39% of 61 episodes in which respiratory viruses were detected. HRV-C was most frequent (HRV-C: nine, HRV-A: eight and HRV-B: two). Seven episodes of HRV-C, five with pneumonia, occurred within 100 days of HSCT. Co-pathogens were frequent. CONCLUSIONS: The newly described HRV-C was the most common rhinovirus group detected in HSCT recipients with respiratory infection, with co-pathogens being frequent. Further research is required to understand the activity and pathogenicity of this virus in HSCT recipients.

Community-wide, contemporaneous circulation of a broad spectrum of human rhinoviruses in healthy Australian preschool-aged children during a 12-month period.

Human rhinovirus (HRV) replication triggers exacerbation of asthma and causes most acute respiratory illnesses (ARIs), which may manifest as influenza-like illness. The recent assignment of 60 previously unknown HRV types to a third HRV species, Human rhinovirus C, raised questions about the prevalence of these picornavirus types in the community, the extent of HRV diversity at a single site, and whether the HRVs have an equally diverse clinical impact on their hosts. We quantified HRV diversity, and there was no clinical impact attributable to HRV species and genotypes among a community population of preschool-aged children with ARI who provided respiratory samples during 2003. All HRV species were represented among 138 children with ARI, and 74 distinct HRV types were cocirculating. Fever accompanied 32.8% of HRV-positive ARI cases. HRVs were less likely than DNA viruses to be codetected with another virus, suggesting virus interference at the community level, demonstrated by the inverse correlation between influenza virus detection and HRV detection.

Usefulness of published PCR primers in detecting human rhinovirus infection.

We conducted a preliminary comparison of the relative sensitivity of a cross-section of published human rhinovirus (HRV)-specific PCR primer pairs, varying the oligonucleotides and annealing temperature. None of the pairs could detect all HRVs in 2 panels of genotyped clinical specimens; >1 PCR is required for accurate description of HRV epidemiology.

Influenza surveillance in Australia: we need to do more than count.

Laboratory-confirmed influenza is a nationally notifiable disease in Australia. According to notification data, Queensland has experienced more severe influenza seasons than other states and territories. However, this method ignores available denominator data: the number of laboratory tests performed. We propose that negative results of laboratory tests for influenza should be made notifiable, alongside laboratory-confirmed disease, and used to calculate the proportion of positive test results in real-time. Using data from the public health pathology services of three Australian states - Queensland Health laboratories, the Victorian Infectious Diseases Reference Laboratory and Western Australia's PathWest - for 2004 to 2008, we show that incorporating laboratory-negative test data into national surveillance data would add to and improve our understanding of influenza epidemiology.

A simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses.

BACKGROUND: Laboratories often have difficulties obtaining positive control material for polymerase chain reaction (PCR) diagnosis of rare or emerging viruses. This is particularly problematic during outbreaks caused by emerging infectious diseases, when delays can impede the public health response. OBJECTIVES: The aim of this study was to develop a simple approach for preparing real-time PCR positive reaction controls for rare or emerging viruses. STUDY DESIGN: We describe a universal method for preparing PCR positive reaction controls (Uni-Control), which uses two synthetic control oligonucleotides and irrelevant viral nucleic acid as an initiator template. In this study, we prepared Uni-Controls for novel influenza A(H1N1) and human metapneumovirus (HMPV) RT-PCR assays. Parainfluenza type 2 virus RNA and equine herpes virus DNA were used as initiator templates. RESULTS: Using the Uni-Controls, characteristic sigmoidal real-time PCR amplification curves were observed in the influenza A(H1N1) and HMPV RT-PCR assays. Comparable cycle threshold values were observed in both assays when using the same concentration of the initiator template. CONCLUSIONS: The Uni-Control method for preparing real-time PCR positive reaction controls provides an interim measure by which real-time PCR assays can be rapidly introduced for rare or emerging viruses in the absence of wild-type control material. The system is versatile and we propose can readily be adapted to almost any viral template.

Early evidence for direct and indirect effects of the infant rotavirus vaccine program in Queensland.

OBJECTIVE: To assess the impact of introducing a publicly funded infant rotavirus vaccination program on disease notifications and on laboratory testing and results. DESIGN AND SETTING: Retrospective analysis of routinely collected data (rotavirus notifications [2006-2008] and laboratory rotavirus testing data from Queensland Health laboratories [2000-2008]) to monitor rotavirus trends before and after the introduction of a publicly funded infant rotavirus vaccination program in Queensland in July 2007. MAIN OUTCOME MEASURES: Age group-specific rotavirus notification trends; number of rotavirus tests performed and the proportion positive. RESULTS: In the less than 2 years age group, rotavirus notifications declined by 53% (2007) and 65% (2008); the number of laboratory tests performed declined by 3% (2007) and 15% (2008); and the proportion of tests positive declined by 45% (2007) and 43% (2008) compared with data collected before introduction of the vaccination program. An indirect effect of infant vaccination was seen: notifications and the proportion of tests positive for rotavirus declined in older age groups as well. CONCLUSIONS: The publicly funded rotavirus vaccination program in Queensland is having an early impact, direct and indirect, on rotavirus disease as assessed using routinely collected data. Further observational studies are required to assess vaccine effectiveness. Parents and immunisation providers should ensure that all Australian children receive the recommended rotavirus vaccine doses in the required timeframe.

Detection of novel influenza A(H1N1) virus by real-time RT-PCR.

Accurate and rapid diagnosis of novel influenza A(H1N1) infection is critical for minimising further spread through timely implementation of antiviral treatment and other public health based measures. In this study we developed two TaqMan-based reverse transcription PCR (RT-PCR) methods for the detection of novel influenza A(H1N1) virus targeting the haemagglutinin and neuraminidase genes. The assays were validated using 152 clinical respiratory samples, including 61 Influenza A positive samples, collected in Queenland, Australia during the years 2008 to 2009 and a further 12 seasonal H1N1 and H3N2 influenza A isolates collected from years 2000 to 2002. A wildtype swine H1N1 isolate was also tested. RNA from an influenza A(H1N1) virus isolate (Auckland, 2009) was used as a positive control. Overall, the results showed that the RT-PCR methods were suitable for sensitive and specific detection of novel influenza A(H1N1) RNA in human samples.

A Novel Duplex Real-Time Reverse-Transcription PCR Assay for the Detection of Influenza A and the Novel Influenza A(H1N1) Strain.

Timely implementation of antiviral treatment and other public health based responses are dependent on accurate and rapid diagnosis of the novel pandemic influenza A(H1N1) strain. In this study we developed a duplex real-time PCR (RT-PCR) (dFLU-TM) assay for the simultaneous detection of a broad range of influenza A subtypes and specific detection of the novel H1N1 2009 pandemic strain. The assay was compared to the combined results of two previously described monoplex RT-PCR assays using 183 clinical samples and 10 seasonal influenza A isolates. Overall, the results showed that the dFLU-TM RT-PCR method is suitable for detection of influenza A, including the novel H1N1 pandemic strain, in clinical samples.