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The incidence of enterovirus D68 (EV-D68) in New South Wales, Australia, is unknown. As part of a state-wide surveillance program, enterovirus positive diagnostic specimens were assessed from patients presenting to hospitals with respiratory and meningitis syndromes from August 2018 to November 2019. Diagnostic enterovirus positive samples were collected from 339 patients and re-extracted followed by targeted PCR across the whole EV-D68 genome (7.4 kb). Obtained amplicons (n=208) were sequenced using Illumina sequencing technology and the phylogenetic relationships analysed relative to EV-D68 Fermon strain. We identified EV-D68 in 31 patients, both children (n=27) and adults (n=4). Phylogenetically, the majority (n=30) were from subclade B3, the same as that causing outbreaks of EV-D68 across the USA and Europe during 2018. These data strengthen the importance of having an active enterovirus surveillance network.

Human cytomegalovirus congenital infection is the leading non-genetic cause of fetal malformation in developed countries. There are currently no safe antivirals for use during pregnancy. Placental trophoblast cells specifically secrete exosomes containing miRNA from the chromosome 19 miRNA cluster (C19MC) which confer viral resistance to recipient cells. We show the highly expressed C19MC miRNA miR-517a-3p inhibits HCMV replication and viral protein expression in both fibroblast and trophoblast cell cultures (71.6% and 50.4% inhibition of HCMV DNA at 7 days post infection respectively; p 0.05). This naturally occurring molecule has potential for opening-up antiviral therapeutic strategies for pregnancy.

The current public health emergency surrounding the COVID-19 pandemic, that is the illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in thousands of cases in Australia since 25 January 2020 when the first case was diagnosed. This emerging virus presents particular hazards to researchers and laboratory staff in a clinical setting, highlighted by rapid and widespread global transmission. Based on the epidemiological and clinical data that have become available in mid-2020, we propose the interim classification of SARS-CoV-2 as a Risk Group 3 organism is reasonable, and discuss establishing Biosafety Level 3 (BSL-3) regulations accordingly. Despite its global spread, the reported mortality rate of SARS-CoV-2 ranging from 0.13% to 6.22% is considerably less than that of other Risk Group 4 agents including Ebola and Marburg viruses with fatality rates as high as 90%. In addition, studies have demonstrated that approximately 86% of patients presenting with severe courses of the disease are aged 70 years or above, with the presence of comorbid conditions such as cardiovascular and respiratory system diseases in the majority of all fatal cases. In contrary to recent discussions surrounding the protective and administrative measures needed in a laboratory, the emerging evidence surrounding mortality rate, distinct demographics of severe infections, and the presence of underlying diseases does not justify the categorisation of SARS-CoV-2 as a Risk Group 4 organism. This article summarises biosafety precautions, control measures and appropriate physical containment facilities required to minimise the risk of laboratory-acquired infections with SARS-CoV-2.

Purpose Neurological complications of influenza cause significant disease in children. Central nervous system inflammation, the presumed mechanism of influenza-associated encephalopathy, is difficult to detect. Characteristics of children presenting with severe neurological complications of influenza, and potential biomarkers of influenza-associated encephalopathy are described. Methods A multi-center, retrospective case-series of children with influenza and neurological complications during 2017 was performed. Enrolled cases met criteria for influenza-associated encephalopathy or had status epilepticus. Functional outcome at discharge was compared between groups using the Modified Rankin Scale (mRS). Results There were 22 children with influenza studied of whom 11/22 had encephalopathy and 11/22 had status epilepticus. Only one child had a documented influenza immunization. The biomarker CSF neopterin was tested in 10/11 children with encephalopathy and was elevated in 8/10. MRI was performed in all children with encephalopathy and was abnormal in 8 (73%). Treatment of children with encephalopathy was with corticosteroids or intravenous immunoglobulin in 9/11 (82%). In all cases oseltamivir use was low (59%) while admission to the intensive care unit was frequent (14/22, 66%). Clinical outcome at discharge was moderate to severe disability (mRS score > 2) in the majority of children with encephalopathy (7/11, 64%), including one child who died. Children with status epilepticus recovered to near-baseline function in all cases. Conclusion Raised CSF neopterin was present in most cases of encephalopathy, and along with diffusion restriction on MRI, is a useful diagnostic biomarker. Lack of seasonal influenza vaccination represents a missed opportunity to prevent illness in children, including severe neurological disease., Highlights • Elevated CSF neopterin was common in cases of IAE. • Short term outcome for IAE was poor, particularly in those with MRI diffusion restriction. • Seasonal influenza vaccination is important for all children.

BACKGROUND: A cornerstone of Australia’s ability to control COVID-19 has been effective border control, using an extensive supervised quarantine program. However, a rapid recrudescence in COVID-19 cases was observed in the state of Victoria in June 2020. Here, we describe the genomic findings that located the source of this second wave as a breach in supervised hotel quarantine and demonstrate the successful elimination of COVID-19 for a second time in Australia. METHODS: Genome sequencing was performed on all available SARS-CoV-2-positive samples in Victoria and integrated genomic and epidemiological investigation undertaken. RESULTS: At 31st January 2021, 20,451 COVID-19 cases were reported in Victoria; samples were sequenced from 75% of cases (15,431/20,451). Genomics revealed 98% (10,426/10,646) of locally-acquired cases during the second wave were derived from a single incursion from hotel quarantine, with the outbreak strain rapidly detected in other Australian states and territories. Phylodynamic analyses indicated an epidemic growth rate comparable to emerging variants, such as B.1.1.7 in the United Kingdom. Strict public health interventions resulted in the elimination of the outbreak strain by 29th October 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. CONCLUSIONS: Rapid escalation of clonal outbreaks can occur from even a single breach of control practices, as revealed through our genomic ‘enhanced outbreak-detection' system. The subsequent elimination and rapid control of new SARS-CoV-2 incursions reinforce that decisive public health responses to emergent cases are effective even with high epidemic growth rates, and “elimination” should be favored in settings where this is achievable. FUNDING STATEMENT: The Microbiological Diagnostic Unit Public Health Laboratory (MDU PHL) and the Victorian Infectious Diseases Reference Laboratory (VIDRL) at The Doherty Institute are funded by the Victorian Government. This work was supported by the National Health and Medical Research Council, Australia (NHMRC); Partnership Grant (APP1149991), Investigator Grant to BPH (APP1196103), Investigator Grant to DAW (APP1174555), Research Fellowship to TPS (APP1105525), MRFF COVID-19 Genomics Grant (MRF9200006). DECLARATION OF INTERESTS: None to declare. ETHICS APPROVAL STATEMENT: Data were collected in accordance with the Victorian Public Health and Wellbeing Act 2008. Ethical approval was received from the University of Melbourne Human Research Ethics Committee (study number 1954615.3).

The first laboratory confirmed case of Coronavirus disease 2019 (COVID-19) in Australia was in Victoria on 25 January 2020 in a man returning from Wuhan city, Hubei province, the People's Republic of China. This was followed by three cases in New South Wales the following day. The Australian Government activated the Australian Health Sector Emergency Response Plan for Novel Coronavirus on 27 February 2020 in anticipation of a pandemic. Subsequently, the World Health Organization declared COVID-19 to be a Public Health Emergency of International Concern followed by a pandemic on 30 January 2020 and 11 March 2020, respectively. Laboratory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is key in identifying infected persons to guide timely public health actions of contact tracing and patient isolation to limit transmission of infection. This article aims to provide a comprehensive overview of current laboratory diagnostic methods for SARS-CoV-2, including nucleic acid testing, serology, rapid antigen detection and antibody tests, virus isolation and whole genome sequencing. The relative advantages and disadvantages of the different diagnostic tests are presented, as well as their value in different clinical, infection control and public health contexts. We also describe the challenges in the provision of SARS-CoV-2 diagnostics in Australia, a country with a relatively low COVID-19 incidence in the first pandemic wave but in which prevalence could rapidly change.

Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication.DYRK expression was investigated in AD169- and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR.HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3-fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p 0.05) indicating these cellular kinases are required during HCMV placental replication.HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

Australian national surveillance data was used to assess recognition, sequelae, and antiviral therapy for congenital cytomegalovirus (CMV) cases.Data from congenital CMV cases reported through the Australian Paediatric Surveillance Unit born January 1999 to December 2016 were described and Chi-square tests used to characterise trends and associations in case reporting, maternal CMV serology testing, and antiviral therapy. Descriptive analyses for hearing loss and developmental delay were reported for cases born ≥2004, following introduction of universal neonatal hearing screening.There were 302 congenital CMV cases (214 symptomatic, 88 asymptomatic). Congenital CMV was suspected in 70.6% by 30 days of age, with no differences across birth cohorts. Maternal CMV serology testing was associated with maternal illness during pregnancy but not birth cohort. There was increasing antiviral use for symptomatic cases, being used in 14% born 1999-2004, 19.6% born 2005-2010, and 44.4% born 2011-2016 (p 0.001). For those born ≥2004, hearing loss was reported in 42.1% of symptomatic and 26.6% of asymptomatic cases; while developmental delay was reported in 16.9% of symptomatic and 1.3% of asymptomatic cases.There appears to be under-reporting and under-recognition of congenital CMV despite increasing use of antiviral therapy. Universal newborn CMV screening should be considered to facilitate follow-up of affected children and targeted linkage into hearing and developmental services, and to provide population-level infant CMV epidemiology to support research and evaluation of antiviral and adjunctive therapies.

Human cytomegalovirus (HCMV) is a major human pathogen with seropositivity rates in the adult population ranging between 40% and 95%. HCMV infection is associated with severe pathology, such as life-threatening courses of infection in immunocompromised individuals and neonates. Current standard therapy with valganciclovir has the disadvantage of adverse side effects and viral drug resistance. A novel anti-HCMV drug, letermovir, has been approved recently, so that improved therapy options are available. Nevertheless, even more so far unexploited classes of compounds and molecular modes of action will be required for a next generation of antiherpesviral treatment strategies. In this study, we focused on the analysis of the antiviral potency of a novel class of compounds, i.e. pyrrolopyridine analogs, and identified both hit compounds and their target protein candidates. In essence, we provide novel evidence as follows: (i) screening hit SC88941 is highly active in inhibiting HCMV replication in primary human fibroblasts with an EC50 value of 0.20 ± 0.01 μM in the absence of cytotoxicity, (ii) inhibition occurs at the early-late stage of viral protein production and shows reinforcing effects upon LMV cotreatment, (iii) among the viruses analyzed, antiviral activity was most pronounced against β-herpesviruses (HCMV, HHV-6A) and intermediate against adenovirus (HAdV-2), (iv) induction of SC88941 resistance was not detectable, thus differed from the induction of ganciclovir resistance, (v) a linker-coupled model compound was used for mass spectrometry-based target identification, thus yielding several drug-binding target proteins and (vi) a first confocal imaging approach used for addressing intracellular effects of SC88941 indicated qualitative and quantitative alteration of viral protein expression and localization. Thus, our findings suggest a multifaceted pattern of compound-target binding in connection with an unusual mode of action, opening up further opportunities of antiviral drug development.

Preterm birth is a common cause of adverse neonatal and childhood outcomes. It is commonly associated with infection of the maternal-fetal interface. The relationship between periodontitis and preterm labour is controversial.Control placental tissues from uncomplicated term births were compared with those from spontaneous preterm births for incidence of common periodontal bacteria. A chi-square analysis was used to compare the populations, with significance determined at p=0.05.The study group comprised 29 control women who had an uncomplicated term birth, 25 delivered by caesarean section and 4 vaginal deliveries, and 36 women with a spontaneous preterm labour and subsequent delivery at less than 34 weeks gestation. There were significant (p=0.05) differences between the preterm and term groups maternal age with 28.7 compared to 32.0 years old respectively. There was no significant (p=0.05) differences between the groups fetal risk factors or co-morbidities, except the preterm group had a significantly higher (p=0.05) rate of premature rupture of membrane (PROM). There were significantly (p=0.01) more Fusobacterium spp. in the placentas from term births than preterm births.This study found that the common periodontal pathogen, Fusobacterium spp., is not detected more in placentas from preterm birth and may potentially be lower, possibly resulting from bacterial ecological factors in term placentas.

Background Congenital cytomegalovirus (CMV) is the most common non-genetic cause of sensorineural hearing loss. Currently, there are no universal CMV screening programs for newborns or routine CMV testing of neonates with hearing loss in Australia, or elsewhere. Objectives This study was undertaken to determine the prevalence of congenital CMV infection in infants with hearing loss identified using routine resources via the Australian universal neonatal hearing screening (UNHS) program. Study design Infants who failed UNHS, referred for audiological testing and found to have permanent hearing loss were screened for CMV via PCR of urine and saliva. Congenital CMV was diagnosed if CMV was detected in infants ≤30 days of age, or using retrospective testing on stored new born screening cards, retrospective testing, or using clinical criteria if >30 days of age. The cohort was analyzed for time of testing and prevalence of congenital CMV determined. Results The Audiology Department reviewed 1669 infants who failed UNHS between 2009 and 2016. Thirty percent (502/1669) had permanent hearing loss confirmed, of whom 336/502 were offered CMV testing. A definite (n = 11) or probable (n = 8) diagnosis of congenital CMV occurred in 19/323 (5.9%), of whom definite diagnoses were made in 4/19 on tests positive prior to 21 days of life, in 5/19 who were positive on neonatal blood screening card (NBSC) testing, in 2/19 who were positive on placental testing. In 8/19 probable diagnoses were made based on positive testing between ages 23–42 days and a consistent clinical syndrome in the absence of another cause for hearing loss after genetic and other testing. CMV testing mirrored the timing of audiological testing, with ∼40% completing audiology and CMV testing by 21 days, and 64% by 30 days. Conclusion This program, utilizing existing clinical services identified probable congenital CMV in ∼6% of a large cohort failing UNHS with permanent hearing loss, of whom more than half were definite diagnoses. No additional assets were required to those already existing in this tertiary referral pediatric centre, whilst providing useful and timely data for clinical and audiological management.

Congenital cytomegalovirus is the most frequent, yet under-recognised, infectious cause of newborn malformation in developed countries. Despite its clinical and public health importance, questions remain regarding the best diagnostic methods for identifying maternal and neonatal infection, and regarding optimal prevention and therapeutic strategies for infected mothers and neonates. The absence of guidelines impairs global efforts to decrease the effect of congenital cytomegalovirus. Data in the literature suggest that congenital cytomegalovirus infection remains a research priority, but data are yet to be translated into clinical practice. An informal International Congenital Cytomegalovirus Recommendations Group was convened in 2015 to address these questions and to provide recommendations for prevention, diagnosis, and treatment. On the basis of consensus discussions and a review of the literature, we do not support universal screening of mothers and the routine use of cytomegalovirus immunoglobulin for prophylaxis or treatment of infected mothers. However, treatment guidelines for infected neonates were recommended. Consideration must be given to universal neonatal screening for cytomegalovirus to facilitate early detection and intervention for sensorineural hearing loss and developmental delay, where appropriate. The group agreed that education and prevention strategies for mothers were beneficial, and that recommendations will need continual updating as further data become available.

Background Before pandemic H1N1 vaccines were available, the potential benefit of existing seasonal trivalent inactivated influenza vaccines (IIV3s) against influenza due to the 2009 pandemic H1N1 influenza strain was investigated, with conflicting results. This study assessed the efficacy of seasonal IIV3s against influenza due to 2008 and 2009 seasonal influenza strains and against the 2009 pandemic H1N1 strain. Methods This observer-blind, randomized, placebo-controlled study enrolled adults aged 18–64 years during 2008 and 2009 in Australia and New Zealand. Participants were randomized 2:1 to receive IIV3 or placebo. The primary objective was to demonstrate the efficacy of IIV3 against laboratory-confirmed influenza. Participants reporting an influenza-like illness during the period from 14 days after vaccination until 30 November of each study year were tested for influenza by real-time reverse transcription polymerase chain reaction. Results Over a study period of 2 years, 15,044 participants were enrolled (mean age ± standard deviation: 35.5 ± 14.7 years; 54.4% female). Vaccine efficacy of the 2008 and 2009 IIV3s against influenza due to any strain was 42% (95% confidence interval [CI]: 30%, 52%), whereas vaccine efficacy against influenza due to the vaccine-matched strains was 60% (95% CI: 44%, 72%). Vaccine efficacy of the 2009 IIV3 against influenza due to the 2009 pandemic H1N1 strain was 38% (95% CI: 19%, 53%). No vaccine-related deaths or serious adverse events were reported. Solicited local and systemic adverse events were more frequent in IIV3 recipients than placebo recipients (local: IIV3 74.6% vs placebo 20.4%, p

Background Early detection of bacterial pathogens in the lower airway is an important part of managing CF. This study aimed to assess the diagnostic accuracy of oropharyngeal suction (OPS) samples in obtaining airway bacterial cultures in young children with cystic fibrosis (CF), and the level of child distress caused by obtaining OPS samples. Methods Young children with CF undergoing broncho-alveolar lavage (BAL) as part of concurrent research or routine annual surveillance were studied. OPS was performed by stimulating a cough and suctioning the back of the oropharynx in the awake child to replicate clinical practice. BAL of the right upper, middle and lingula lobes was then performed. Samples were sent for standard bacterial culture. The child's distress during OPS was rated using the Groningen Distress Scale (1=calm, 2=timid/nervous, 3=serious distress but still under control, 4=serious distress with loss of control, 5=panic). Results There were 65 paired samples obtained from 39 children (21 boys, mean age on day of first sampling was 34.1months, SD 19.1months). For Pseudomonas aeruginosa , specificity, sensitivity, NPV and PPV with 95% CI were 98% (87–99), 75% (20–96), 98% (91–98) and 60% (15–93%) respectively. In all age groups combined, median level of distress was 3 (IQR 2–4), with distress highest in 2 and 3year olds, with a median of 4 (IQR 3–4). Conclusion OPS has diagnostic utility in determining the absence of organisms in the lower airway, with specificity for P.aeruginosa detection of 98%. However, a positive OPS result is not necessarily a good indicator of lower airway infection. Distress levels were high during OPS, mostly in 2 and 3year olds.

Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.

Background: Quality assurance (QA) of testing is important for our ability to be certain results we provide to patients are accurate and definitive Testing agents of biosecurity concern includes Security Sensitive Biological Agents (SSBA) such as Tier 1 agents (anthrax, Ebolavirus, influenza, HPAI, SARS, smallpox, plague) and Tier 2 agents (swine fever, botulism, Tularaemia, yellow fever, etc) Test QA is limited by need for containment at BSL3 and BSL4 level, difficulties accessing reagents due to serious public health consequences of organism release, and increasing regulation Methods in providing QA: The Biosecurity Quality Assurance Program (BSQAP) was formed after 2008 when the SSBA Regulatory Scheme for control of select agents was established The program addresses the unique problems of SSBA by utilising novel methods for QA, including subgenomic non-infectious molecular targets, online e-learning, and recently whole genome sequencing (WGS) programs assessing new technologies Discussion(s): Emerging technologies (multiplex Biofire systems, WGS) and continuing emergence of novel threats (Ebolavirus, HPAI) mean laboratories need rapid, accurate QA support Tests introduced nationally by reference laboratories in response to emergent threats often lack prior QA, experience, and appropriate reagents Addressing these systematically through regulatory preparedness (the 'PHLN' module, early governmental engagement), novel methodologies (above), high level engagement (Commonwealth, WHO, AFP) and expertise has assisted successful public health responses Acknowledgements: BSQAP program members Joanna Gray, Torsten Theis, Katherine Lau, Peter Santosa, RCPQAP Tony Badrick, Commonwealth Government References 1 Lau KA, Theis T, Gray J, et al Ebola preparedness: diagnosis improvement using rapid approaches for proficiency testing J Clin Microbiol 2016;55: 783-90 2 Theis T, Lau KA, Gray J, et al Proficiency testing for the detection of Middle East respiratory syndrome-coronavirus demonstrates global capacity to detect MERS-CoV J Med Virol 2018;90: 1827-33 Copyright © 2020

Background The most recent documented Australian outbreak of enterovirus A71 (EV-A71) occurred in Sydney from 2012 to 2013. Over a four-month period more than 100 children presented to four paediatric hospitals with encephalitic presentations including fever and myoclonic jerks. The heterogeneous presentations included typical encephalomyelitis, and cardiopulmonary complications. Objectives To characterise the genomes of enterovirus strains circulating during the 2013 Sydney EV-A71 outbreak and determine their phylogeny, phylogeography and association between genome and clinical phenotype. Study design We performed an analysis of enterovirus (EV) positive specimens from children presenting to hospitals in the greater Sydney region of Australia during the 2013 outbreak. We amplified near full-length genomes of EV, and used next generation sequencing technology to sequence the virus. We used phylogenetic/phylogeographic analysis to characterize the outbreak viruses. Results We amplified and sequenced 23/63 (37 %) genomes, and identified the majority (61 %) as EV-A71. The EV-A71 sequences showed high level sequence homology to C4a genogroups of EV-A71 circulating in China and Vietnam during 2012-13. Phylogenetic analysis showed EV-A71 strains associated with more severe symptoms, including encephalitis or cardiopulmonary failure, grouped together more closely than those from patients with hand, foot and mouth disease. Amongst the non-EV-A71 sequences were five other EV subtypes (representing enterovirus subtypes A and B), reflecting the diversity of EV co-circulation within the community. Conclusions This is the first Australian study investigating the near full-length genome of EV strains identified during a known outbreak of EV-A71. EV-A71 sequences were very similar to strains circulating in Asia during the same time period. Whole genome sequencing offers additional information over routine diagnostic testing such as characterisation of emerging recombinant strains and inform vaccine design.

Introduction Chlamydia retesting 3 months after treatment is recommended to detect reinfections, but retesting rates are typically low. The purpose of this study is to determine if the addition of a postal home collection kit to a short message service (SMS) reminder at 3 months increases the percentage of patients retested for chlamydia at 1–4 months, compared to SMS alone. Design In this unblinded randomized controlled trial, participants were randomized 1:1 to intervention (home arm) or control (clinic arm) status. Setting/participants Participants included 200 each of women, heterosexual men, and men who have sex with men diagnosed and treated for chlamydia at sexual health services. Intervention Three months after chlamydia diagnosis, home arm participants received an SMS reminder and postal home collection kit (women, vaginal swab; heterosexual men, Copan UriSwab; men who have sex with men, UriSwab and rectal swab). Main outcome measures The main outcome measures were the percentage of participants retested at 1–4 months after chlamydia diagnosis and the percentage in each arm with repeat positive tests, by risk group and overall, analyzed by intention to treat. Data were collected from 2011 to 2013 and analyzed in 2014. Results The percentage retested within 1–4 months of chlamydia diagnosis was significantly higher in home versus clinic arm participants among women (64% [66/103] vs 39% [38/97], p p =0.002); men who have sex with men (62% [61/98] vs 44% [45/102], p =0.010); and overall (61% [184/302] vs 39% [117/298], p p =0.021) and overall (10% [31/302] vs 4% [12/298], p =0.006). Conclusions The addition of a postal home collection kit to routine SMS reminders resulted in substantial improvements in chlamydia retesting rates in all three risk groups and detection of more repeat positive tests, compared with SMS alone. Extending the intervention to other primary care settings with low retesting rates should be considered.

Introduction Congenital human cytomegalovirus (HCMV) infection is a major public health problem due to severe sequelae in the fetus and newborns. Currently, due to their toxicity anti-CMV treatments cannot be administered to pregnant women. We thus developed an ex vivo model of 1st trimester placental CMV infection to observe the route of infection across the placenta and to test the efficacy of various new drugs targeting different stages of viral cycle. Methods After validation of the viability of floating villi explants by ELISA β-HCG, the kinetics of placental infection were determined by immunochemistry and qPCR in this ex vivo model. Antiviral susceptibility was determined in vitro using focus reduction assay and by qPCR in the ex vivo model. Results The ex vivo model showed viral infection in trophoblasts and mesenchymal space of floating villi. In vitro, antiviral combinations of maribavir with baicalein or artesunate inhibited viral infection by more than 90%. On the other hand, in ex vivo model, infection was reduced by 40% in presence of maribavir and artesunate. The synergistic effect observed in vitro was not observed ex vivo. Discussion This model allowed us to understand the CMV spread in 1st trimester floating villi better and to analyze the anti-CMV efficacy and toxicity of new drugs that could be administered to pregnant women, either alone or in combination. Conclusions Such an ex vivo model could be applied to other viruses such as rubella or parvovirus B19 and in new drug development.

Norovirus is the leading cause of acute gastroenteritis with most infections caused by GII.4 variants. To understand the evolutionary processes that contribute to the emergence of GII.4 variants, we examined the molecular epidemiology of norovirus-associated acute gastroenteritis in Australia and New Zealand from 893 outbreaks between 2009 and 2012. Throughout the study GII.4 New Orleans 2009 was predominant; however, during 2012 it was replaced by an emergent GII.4 variant, Sydney 2012. An evolutionary analysis of capsid gene sequences was performed to determine the origins and selective pressures driving the emergence of these recently circulating GII.4 variants. This revealed that both New Orleans 2009 and Sydney 2012 share a common ancestor with GII.4 Apeldoorn 2007. Furthermore, pre-epidemic ancestral variants of each virus were identified up to two years before their pandemic emergence. Adaptive changes at known blockade epitopes in the viral capsid were also identified that likely contributed to their emergence.

Summary Introduction Accurate diagnosis of hepatitis B virus (HBV) infection is essential for infection control, treatment and screening of potential blood, organ and tissue donors. We assessed the sensitivity of the HBsAg and HBcAb as screening assays alone and in combination for detecting HBV infection in a series of Australian patients. The performance of the Architect (Abbott Diagnostics, Germany) and the Elecsys (Roche Diagnostics, Germany) platforms were assessed for detection of HBcAb. Methods There were 2778 blood samples assessed using the COBAS Ampliprep/TaqMan test for HBV DNA, of which 331 sera had concurrent HBV serology testing. This allowed determination of the correlation between HBV DNA and different serological markers. Of the 331 sera, 260 had sufficient residual volume to be retested for HBcAb using both Elecsys and the Architect assays. Results Of the 331 patients, one (0.3%) was negative by the Architect Anti-HBc II assay, in the presence of HBV DNA and positive HBsAg, consistent with recent infection. Positive HBcAb in the absence of HBV DNA was found in 67 of 331 (20.2%) patients. Of these, 18 of 67 had isolated HBcAb with negative results on all other tests, with 12 of 18 (3.6%) demonstrating low HBcAb signals on chemiluminscent microparticle assay. No cases of detectable HBV DNA in the presence of negative serology were found. When the HBcAb was used as a marker for past exposure or chronic HBV infection, the Architect Anti-HBc II assay demonstrated sensitivity and specificity of 98% and 79.9%, respectively, compared to 90% and 78.9%, respectively, for the Elecsys Anti-HBc assay. The combination of the Architect Anti-HBc II and HBsAg assays, as per conventional solid organ donor and recipient screening protocols, had 90% specificity and 100% sensitivity for determining HBV infection. Conclusion This study shows that the use of combined HBsAg and HBcAb is sensitive and reliable for screening and predicting HBV nucleic acid test (NAT) positivity, whereas HBcAb alone missed an acute infection in this study population. There were no significant differences detectable between the Architect and the Elecsys HBcAb assays ( p = 0.001), suggesting laboratories should assess individual assays in the local population before use as screening tests.

Despite greater than 99% of influenza A viruses circulating in the Asia-Pacific region being resistant to the adamantane antiviral drugs in 2011, the large majority of influenza A (>97%) and B strains (∼99%) remained susceptible to the neuraminidase inhibitors oseltamivir and zanamivir. However, compared to the first year of the 2009 pandemic, cases of oseltamivir-resistant A(H1N1)pdm09 viruses with the H275Y neuraminidase mutation increased in 2011, primarily due to an outbreak of oseltamivir-resistant viruses that occurred in Newcastle, as reported in Hurt et al. (2011c, 2012a), where the majority of the resistant viruses were from community patients not being treated with oseltamivir. A small number of influenza B viruses with reduced oseltamivir or zanamivir susceptibility were also detected. The increased detection of neuraminidase inhibitor resistant strains circulating in the community and the detection of novel variants with reduced susceptibility are reminders that monitoring of influenza viruses is important to ensure that antiviral treatment guidelines remain appropriate.

Background Health care workers' (HCW) compliance with infection control measures is influenced by organizational, environmental and individual factors. However, it is unknown whether HCWs' perceptions of transmission risk and protectiveness of infection control measures influences the uptake of infection control measures. Methods A questionnaire of perceptions and intentions to use infection control measures was completed by 74 HCWs at 2 hospitals. HCWs also indicated a 1-m transmission risk zone and their perceived transmission risk zone. Responses were used in logistic regression models to predict intended behaviors. Results Poor recognition of the importance of employing a 1-m transmission risk zone predicted intention not to don a mask in single rooms where patient contact was unexpected (adjusted odds ratio [AOR], 0.5; P = .032). When contact was expected, perceived protectiveness of pre-exposure prophylaxis (AOR, 7.9; P P = .023), and a minimum 1-m risk zone (AOR, 9.8; P = .022) predicted mask use. HCWs perceived transmission risk zones within 2.45 m from attending an adult and 1.12 m from attending pediatric patients. Conclusion Intention to use a facemask was poor for care in single rooms but improved if patient contact was expected and in multibed rooms. HCWs attending pediatric patients measured a smaller transmission risk zone than what is currently recommended under droplet precautions.

The drugs currently available for treatment of severe human cytomegalovirus (HCMV) infections suffer from many drawbacks, particularly toxicity, and potential teratogenicity contraindicating their use in target populations such as pregnant women. The emergence of drug-resistant strains is still a problem for disease management, particularly in immunosuppressed populations where antivirals are used for extended periods of time. The flavonoid family of drugs contains promising candidates as they have low toxicity and inhibit different targets to currently available antivirals. We report here that, unlike their chalcon homologs, four flavonoids (baicalein, quercetin, quercetagetin and naringenin) inhibit various stages of HCMV replication, the most active anti-HCMV compound being baicalein and the less active and less selective being quercetagetin. These drugs could provide potential inhibitors of virus replication alone or in combination, without increased toxicity.

Background Prompt and accurate laboratory diagnosis of measles is essential for case detection, outbreak management and ongoing surveillance in low incidence countries. Several disease markers are employed for diagnosis and are important to determine epidemiological and molecular characteristics for future control measures. Objectives To report different disease markers, genotypes and epidemiology of a measles outbreak in Australia, a low incidence country. Study design A retrospective descriptive study of the clinical and epidemiological features and laboratory diagnosis in 16 confirmed measles cases using measles serum IgM/IgG, antigen detection (IFA), viral RNA detection by real-time PCR and genotyping results for respiratory and urine specimens processed in one reference laboratory. Results Of the 16 confirmed measles cases, 11 were young adults aged between 20–35 years and 15 were not age-appropriately vaccinated. The most common genotype detected was D9 (11/16), followed by D4 (1/16) and D8 (1/16). Two imported cases were from the Philippines (D4) and Italy (D9). Of six disease markers, respiratory swab PCR and serum IgM gave the highest percentage (100%) of positive samples for confirmed cases followed by urine PCR (90.9%), serum PCR (66.6%), urine IFA (54.5%) and respiratory IFA (46.2%). Conclusions Measles should be considered in the differential diagnosis of a presentation with fever and rash, even in countries in the elimination phase of measles control. Genotyping is a powerful molecular-epidemiological tool to assist low incidence countries towards eradication goals. Improving vaccination coverage remains essential, particularly in young adults and travellers.

Summary Understanding respiratory pathogen transmission is essential for public health measures aimed at reducing pathogen spread. Particle generation and size are key determinant for pathogen carriage, aerosolisation, and transmission. Production of infectious respiratory particles is dependent on the type and frequency of respiratory activity, type and site of infection and pathogen load. Further, relative humidity, particle aggregation and mucus properties influence expelled particle size and subsequent transmission. Review of 26 studies reporting particle sizes generated from breathing, coughing, sneezing and talking showed healthy individuals generate particles between 0.01 and 500 μm, and individuals with infections produce particles between 0.05 and 500 μm. This indicates that expelled particles carrying pathogens do not exclusively disperse by airborne or droplet transmission but avail of both methods simultaneously and current dichotomous infection control precautions should be updated to include measures to contain both modes of aerosolised transmission.

A sudden increase in the rate of asthma exacerbations has been observed among young children in many countries 2-3 weeks after their return-to-school following the summer holidays. These exacerbations are frequently associated with human rhinovirus (hRV) infections, with possible interactions with allergen sensitisation, allergen exposure and medication use. It was originally proposed that the sudden increase resulted from new strains of respiratory viruses acquired during the holidays spreading rapidly on return to school. While there is compelling evidence implicating hRV in these exacerbations, recent observations on virus transmission, infection patterns and immune responses to both viruses and allergens have led us to propose an additional hypothesis for this increase in exacerbations. We propose that classrooms typically provide persistent exposure to a mixture of airborne viruses, viral proteins, endotoxin, community allergens and other human-derived aerosols - a modern miasma. During the preceding school term, this exposure establishes and maintains a level of immune tolerance and herd immunity, which then declines during the two-month holidays due to lack of such exposure, creating a transitory window of susceptibility to viral infections and asthma. The return to school re-establishes exposure to these aerosols resulting in an acceleration of exacerbations, until the tolerance and herd immunity are re-established. Thus, the peak in return-to-school asthma is more a function of a transitory increase in susceptibility due to a temporary lack of this complex exposure, than it is to novel, locally endemic strains of hRV.

The impact of fibrosis stage on chronic hepatitis C virus (HCV) treatment response was explored in CHARIOT, a study of high dose peginterferon alfa-2a (PEG-IFNalpha-2a) induction therapy in treatment naïve genotype 1 infection.Eight hundred and ninety-six patients were randomised 1:1 to 360 microg (n=448) or 180 microg (n=448) PEG-IFNalpha-2a weekly with RBV 1000-1200 mg/day for 12 weeks followed by 36 weeks of 180 microg PEG-IFNalpha-2a weekly plus RBV 1000-1200 mg/day. Virological responses were assessed at week 4, 8, 12, 24, 48 (end of therapy), and 24 weeks following therapy (sustained virological response, SVR). As previously reported, there was no significant difference in SVR in the induction (53%) and standard (50%) arms, therefore the pooled study population was used for analysis of SVR and relapse.A marked step-wise decline in SVR was evident by fibrosis stage: F0 (70%); F1 (60%); F2 (51%); F3 (31%); F4 (10%) (p0.0001). Early virological responses were lower among F3/4 patients, including rapid virological response (RVR) (21% vs. 34% for F3/4 and F0-2, respectively) (p=0.0072), and the RVR positive predictive value was also lower (63% vs. 80%). Virological relapse rates were similar in early disease stages (F0, 16%; F1, 23%; F2, 26%), but increased markedly in advanced fibrosis (F3, 50%; F4, 80%) (p0.0001). Cumulative PEG-IFNalpha-2a and ribavirin doses were similar among patients with F3/4 and F0-2 within treatment arms through week 4, 8, 12, and week 24.Low virological response in hepatitis C genotype 1 patients with advanced fibrosis is not explained by inadequate cumulative PEG-IFN and ribavirin doses.

The amniotic fluid cytokine profile has been shown to be indicative of various disease states, and changes may be associated with preterm labor or infection. Anti-inflammatory cytokine profiles may be essential for successful normal pregnancy. However, there are currently few normative data on the concentration of cytokines in amniotic fluids during pregnancy. The aim of this study was to provide new amniotic fluid cytokine data for future comparative studies in disease states, notably in utero viral infections, and to compare these with maternal serum levels. Amniotic fluid was obtained from 100 pregnant women undergoing elective amniocentesis at the Royal Hospital for Women, Randwick. Concentrations of 27 cytokines were simultaneously measured in amniotic fluid and a subset of matching maternal sera (n=33) using a multiplex bead-based immunoassay system (Bio-Plex, Bio-Rad). To exclude infection, nested multiplex PCR targeting 17 known congenital infectious agents were performed on all amniotic fluid and maternal serum samples, and serological testing was also performed against some of these agents. Maternal serum concentration was positively correlated with amniotic fluid levels for MIP-1beta (r=0.39, P=0.027). IL-1ra was positively correlated to maternal age (r=0.210, P=0.036), and mean IL-5 levels were significantly higher in amniotic fluids from pregnancies with male fetuses than those with female fetuses (P=0.036). Normal amniotic fluid concentrations for five cytokines (IL-6, IL-8, IP-10, MCP-1, IL-1ra) were found to be significantly elevated over maternal serum concentrations in matched pairs (P0.05). Concentrations of 12 cytokines (eotaxin, IFN-gamma, IL-9, IL-12, IL-15, IL-17, MIP-1alpha, MIP-1beta, RANTES, TNF-alpha, VEGF, PDGF bb) were significantly elevated in maternal serum compared to paired amniotic fluid at midtrimester (P0.05). Amniotic fluid may be more representative of the fetal cytokine profile than cytokine analysis on antenatal sera as it represents predominantly fetal urinary and respiratory secretions. This study provides new normative data for multiple cytokine levels in amniotic fluid and maternal sera at 14-16 weeks gestation, and is a valuable tool for future diagnostic and comparative studies.

Background It is well established that human papillomavirus (HPV) infection is highly related to the development of precursor lesions of cervical cancer and uterine cancers. However, for a pre-cancerous lesion to develop, a persistent infection with a high-risk type HPV is necessary. The Digene Hybrid Capture II (hcII) assay is the only FDA approved method used in conjunction with cytology for HPV screening of women older than 30. The hcII has moderate sensitivity (64.7%) and is dependent on the cellular content of samples, rendering occasionally false positive and false negative results. Objective This study aims to evaluate the performance of a new HPV diagnostic kit (High-Risk HPV detection kit, manufactured by Human Genetic Signatures (HGS), Sydney, Australia). Methods The method under evaluation was assessed by comparing the results obtained from testing 834 cervical specimens with the HGS method and the Digene hcII method, using genotyping as the reference standard. Results Results of the study showed that the specificity and positive predictive value of the HGS High-Risk HPV detection test are significantly greater than those of the Digene hcII test. Overall the HGS HPV assay provides a more accurate system for the detection of high-risk HPV strains, with simpler technical use compared with PCR-sequencing methods.

Background The emergence of human cytomegalovirus (CMV) antiviral resistance plays a significant role in disease progression in immunocompromised patients who have received antiviral therapy. Objectives To determine the pattern of antiviral-resistant CMV strains in a highly immunocompromised child. Study design Retrospective specimens of blood and urine were analysed using PCR-sequencing to identify antiviral-resistant CMV strains containing UL97 or UL54 mutations. Results CMV strains resistant to antiviral agents contributed to disease in a bone marrow transplant recipient with X-linked severe combined immunodeficiency (SCID) treated with ganciclovir (GCV) and foscarnet (FOS). Retrospective analyses detected GCV-resistant CMV (L595S) in a specimen taken after disease progression. This GCV-resistant CMV strain persisted for 1 year, after which time it was no longer detected even though the patient continued to receive GCV. A FOS-resistant strain (T700A) then emerged even though no FOS had been administered in the preceding year. Conclusion The detection of antiviral-resistant CMV did not follow the patterns found in other patients tested for antiviral resistance, including emergence of a FOS-resistant strain in the absence of antiviral-selective pressure. These findings indicate the patient's underlying immunosuppressive condition should be considered for diagnosis and management of resistant CMV.

Previously, we identified the glycoprotein gO gene, UL74, as a hypervariable locus in the human cytomegalovirus (HCMV) genome [Virology 293 (2002) 281]. Here, we analyze gO from 50 isolates from congenitally infected newborns, transplant recipients, and HIV/AIDS patients from Italy, Australia, and UK. These are compared to four gO groups described from USA transplantation patients [J. Virol. 76 (2002) 10841]. Phylogenetic analyses identified seven genotypes. Divergence between genotypes was up to 55% and within 3%. Discrete linkage was shown between seven hypervariable gO and gN genotypes, but not with gB. This suggests interactions, while gN and gO are known to form complexes with distinct conserved glycoproteins gM, gH/gL, respectively, both are involved in fusogenic entry and exit. Codon-based maximum likelihood models showed evidence for sites of positive selection. Further analyses of disease relationships should take into account these newly defined gO/gN groups.

The role of cytotoxic T lymphocytes (CTL) in limiting viral replication and producing hepatocellular injury in patients with chronic hepatitis C virus (HCV) infection is controversial.Intrahepatic and peripheral blood HCV-specific CTL activity against the entire HCV polyprotein was assessed in 26 patients. CTL responses were assessed after effector lymphocytes were re-stimulated for 6 days in vitro using HCV-vaccinia virus-infected autologous cells expressing HCV antigens. Serum and hepatic viral loads were measured and immunohistochemistry for CD3 and CD8 was performed to localise and enumerate effector cells in liver.A positive CTL response was detected in 39/52 (75%) of assays conducted with intrahepatic mononuclear cells and 21/52 (40%) of peripheral blood assays (P0.001). The presence of an intrahepatic CTL response was associated with low hepatic viral load (P=0.004). Hepatic lobular infiltration by CD8(+)T cells correlated weakly with serum alanine aminotransferase levels (r=0.42, P=0.04) and no relationship was demonstrated between CTL activity and histological evidence of liver damage.HCV-specific CTL activity is found more commonly in liver than in blood. An inverse relationship between CTL responses and viral load supports the hypothesis that HCV-specific CTL limit viral replication in patients with chronic HCV infection.

The aim of this study was to determine the isolation trends of common and emerging pathogens in children over a 12-month period. The study group included 412 children under 6 years with diarrhoea who were either hospitalised, or seen in the outpatients department of The Sydney Children's Hospital. Pathogens were detected in 137 (33%) samples, with rotavirus most common (40%), followed by adenovirus (26%), astrovirus (12%), Campylobacter jejuni (12%), Salmonella spp. (10%) and Giardia lamblia (< 1 %). Giardia-specific antigen (GSA) was detected in 11 of 382 (3%) using an enzyme immunoassay (EIA), and this included four samples in which cysts of G. lamblia were detected by microscopy. Using electron microscopy (EM), viruses were detected in 29 of 120 (24%) samples from hospitalised children and 53 of 171 (31%) outpatients (P = 0.23). Amongst this subset, Norwalk-like viruses (NLVs) were detected by RT-PCR in 10 samples including six of 14 with small round viruses, one of seven with small viral-like particles (SVLPs), and three of 126 EM-negative samples. Lactoferrin, detected by EIA, was 59% more likely to be positive in samples infected with salmonella/campylobacter than in samples in which bacterial pathogens were not isolated. As an indicator for infection with these bacterial agents, the assay showed a sensitivity and specificity of 95 and 40.3%, respectively. A routine microbiological analysis of stools from children of this age group should include a screen for foodborne bacterial agents and rotavirus. Tests for adenovirus, astrovirus and NLVs should be secondary. The cost-effectiveness of including the EIAs for lactoferrin and G. lamblia in the routine testing protocol needs to be evaluated.

Summary Aim Serological testing for human cytomegalovirus (CMV) immunoglobulin G (IgG) continues to play a major role in the evaluation of tissue and organ transplant donors and recipients, screening blood donors and in estimating the risk of infection in pregnant women. In this study we evaluated the performance and technical characteristics of two semiautomated commercial CMV IgG immunoassays. Methods One hundred consecutive sera submitted to a referral serology laboratory for CMV IgG antibody testing were assayed using two automated CMV IgG immunoassays: the Architect (Abbott Diagnostics) and Elecsys (Roche Diagnostics) immunoassays. Because this patient population has a high rate (∼87%) of seropostivity, we took a further 50 sera that were CMV IgG negative on Architect and tested on the Elecsys assay, in order to assess the specificity of the assays. Results The assays showed an excellent qualitative agreement with two (2%) discrepant results in the 100 patients tested initially, with all of the 50 additional sera negative on Architect also negative on the Elecsys. However, there was no correlation between the optical density of results generated by the instruments for the set of samples. Conclusion Both Elecsys and Architect immunoassays are convenient for detection of CMV IgG antibody, with near identical qualitative results.

Cytomegaloviruses likely encode numerous gene products involved in regulating virus–host cell interactions and pathogenesis. We previously identified a region of murine cytomegalovirus (MCMV) within HindIII-J and -I that regulates pathogenesis of the virus [open reading frames (ORFs) M139–M141] or is likely required for MCMV replication (ORFs m142 and m143). As a prerequisite for further studies on the structure and function of this gene region, we mapped the transcripts encoded within MCMV HindIII-I. Probes for ORFs M140 and M141 hybridized to 5.4- and 7.0-kb RNA, respectively, which were transcribed with early kinetics and were 3′ coterminal with HindIII-J ORF M139. Probes representing ORFs m142, m143, or m144 hybridized to 3′ coterminal transcripts of 1.8, 3.8, and 5.1 kb, respectively. ORFs m142 and m143 were transcribed with immediate-early kinetics but were most abundantly expressed at early times. Probes for the rightmost end of HindIII-I hybridized to a 5.1-kb early/late RNA corresponding to m144 and to a 1.8-kb early RNA transcribed from m145. All of the major transcripts were polyadenylated and therefore are likely coding. Additional minor transcripts of intermediate sizes were also detected. ORFs M139–m143 showed homology to the betaherpesvirus-specific HCMV US22 gene family. Because deletion of these viral genes results in attenuated or helper-dependent phenotypes, this conserved region of US22 family genes may have a role in virus replication as well as in the pathogenesis of betaherpesviruses in their natural hosts.

Summary Human cytomegalovirus (CMV) remains an important cause of illness in immunocompromised individuals and the most common viral cause of congenital malformation. The tests available for diagnosis of CMV include serology, antigen detection, virus culture, tissue histopathology and nucleic acid detection. The diagnosis of CMV remains difficult because of the issues of virus latency, virus infection versus clinical disease and virus reactivation. The tests available and the use of these tests are undergoing significant changes. This Broadsheet presents a review of these tests, particularly in the diagnosis of congenital infection and infection in pregnant women and immuncompromised individuals.

Background and objectives Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. Study design A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995–2007. Results and conclusions The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

CSF-PCR is currently the most sensitive test to diagnose encephalitis due to cytomegalovirus or herpes simplex virus. However, false negative results sometimes arise due to inhibitors in CSF. We have shown that the inhibitory effects may be due to increased levels of proteins and increased cell numbers, but are not due to cellular DNA. Simple techniques were used to remove the inhibitors and successfully apply these methods to CSF specimens that gave equivocal results when tested untreated.

Background Viruses are frequently associated with acute exacerbations of asthma, but the extent to which they contribute to the level of day-to-day symptom control is less clear. Objective We sought to explore the relationship between viral infections, host and environmental factors, and respiratory symptoms in children. Methods Sixty-seven asthmatic children collected samples twice weekly for an average of 10 weeks. These included nasal wash fluid and exhaled breath for PCR-based detection of viral RNA, lung function measurements, and records of medication use and asthma and respiratory symptoms in the previous 3 days. Atopy, mite allergen exposure, and vitamin D levels were also measured. Mixed-model regression analyses were performed. Results Human rhinoviruses (hRVs) were detected in 25.5% of 1232 nasal samples and 11.5% of breath samples. Non-hRV viruses were detected in less than 3% of samples. hRV in nasal samples was associated with asthma symptoms (cough and phlegm: odds ratio = 2.0; 95% CI = 1.4-2.86, P = .0001; wheeze and chest tightness: odds ratio = 2.34, 95% CI = 1.55-3.52, P