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Serological assays for SARS-CoV-2 infection are now widely available for use in diagnostic laboratories. Limited data are available on the performance characteristics in different settings, and at time periods remote from the initial infection. Validation of the Abbott (Architect SARS-CoV-2 IgG), DiaSorin (Liaison SARS-CoV-2 S1/S2 IgG) and Roche (Cobas Elecsys Anti-SARS-CoV-2) assays was undertaken utilising 217 serum samples from 131 participants up to 7 months following COVID-19 infection. The Abbott and DiaSorin assays were implemented into routine laboratory workflow, with outcomes reported for 2764 clinical specimens. Sensitivity and specificity were concordant with the range reported by the manufacturers for all assays. Sensitivity across the convalescent period was highest for the Roche at 95.2-100% (95% CI 81.0-100%), then the DiaSorin at 88.1-100% (95% CI 76.0-100%), followed by the Abbott 68.2-100% (95% CI 53.4-100%). Sensitivity of the Abbott assay fell from approximately 5 months; on this assay paired serum samples for 45 participants showed a significant drop in the signal-to-cut-off ratio and 10 sero-reversion events. When used in clinical practice, all samples testing positive by both DiaSorin and Abbott assays were confirmed as true positive results. In this low prevalence setting, despite high laboratory specificity, the positive predictive value of a single positive assay was low. Comprehensive validation of serological assays is necessary to determine the optimal assay for each diagnostic setting. In this low prevalence setting we found implementation of two assays with different antibody targets maximised sensitivity and specificity, with confirmatory testing necessary for any sample which was positive in only one assay.

Much of our current understanding about novel coronavirus disease 2019 (COVID-19) comes from hospitalised patients. However, the spectrum of mild and subclinical disease has implications for population-level screening and control. Forty-nine participants were recruited from a group of 99 adults repatriated from a cruise ship with a high incidence of COVID-19. Respiratory and rectal swabs were tested by polymerase chain reaction (PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Sera were tested for anti-SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA) and microneutralisation assay. Symptoms, viral shedding and antibody response were examined. Forty-five participants (92%) were considered cases based on either positive PCR or positive ELISA for immunoglobulin G. Forty-two percent of cases were asymptomatic. Only 15% of symptomatic cases reported fever. Serial respiratory and rectal swabs were positive for 10% and 5% of participants respectively about 3 weeks after median symptom onset. Cycle threshold values were high (range 31–45). Attempts to isolate live virus were unsuccessful. The presence of symptoms was not associated with demographics, comorbidities or antibody response. In closed settings, incidence of COVID-19 could be almost double that suggested by symptom-based screening. Serology may be useful in diagnosis of mild disease and in aiding public health investigations.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 106 pfu) and medium (5 × 104 pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 102 pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5–15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease., SARS-CoV-2 induces mild infection in ferret model. Here, Ryan et al. characterise optimal infection dosage inducing upper respiratory tract (UTR) viral shedding, progression time of viral shedding, and pathology in ferrets and finally provide evidence for protection after re-challenge.

In December 2019 an outbreak of coronavirus disease (COVID-19) emerged in Wuhan, China. The causative agent was subsequently identified and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which rapidly spread worldwide causing a pandemic. Currently there are no licensed vaccines or therapeutics available against SARS-CoV-2 but numerous candidate vaccines are in development and repurposed drugs are being tested in the clinic. There is a vital need for authentic COVID-19 animal models to further our understanding of pathogenesis and viral spread in addition to pre-clinical evaluation of candidate interventions.Here we report a dose titration study of SARS-CoV-2 to determine the most suitable infectious dose to use in the ferret model. We show that a high (5×106 pfu) and medium (5×104 pfu) dose of SARS-CoV-2 induces consistent upper respiratory tract (URT) viral RNA shedding in both groups of six challenged animals, whilst a low dose (5×102 pfu) resulted in only one of six displaying signs of URT viral RNA replication. The URT shedding lasted up to 21 days in the high dose animals with intermittent positive signal from day 14. Sequential culls revealed distinct pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung, observed on day 3 in high and medium dosed animals, with presence of mild broncho-interstitial pneumonia on day 7 onwards. No obvious elevated temperature or signs of coughing or dyspnoea were observed although animals did present with a consistent post-viral fatigue lasting from day 9-14 in the medium and high dose groups. After virus shedding ceased, re-challenged ferrets were shown to be fully protected from acute lung pathology. The endpoints of URT viral RNA replication in addition to distinct lung pathology and post viral fatigue were observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease (as displayed by 80% of patients infected with SARS-CoV-2). In addition, intermittent viral shedding on days 14-21 parallel observations reported in a minority of clinical cases.

The global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.

Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time reverse transcription-PCR (rRT-PCR) for the rapid detection of measles vaccine strains was developed with high specificity and sensitivity equivalent to that of traditional measles genotyping methods., Rapid differentiation of wild-type measles virus from measles vaccine strains is crucial during a measles outbreak and in a measles elimination setting. A real-time reverse transcription-PCR (rRT-PCR) for the rapid detection of measles vaccine strains was developed with high specificity and sensitivity equivalent to that of traditional measles genotyping methods. The “stressed” minor groove binder-TaqMan probe design approach achieves specificity to vaccine strains only, without compromising sensitivity. This assay, without requiring sequence genotyping, has proved to be extremely useful in outbreak settings for over 4 years at the Regional Measles Reference Laboratory for the Western Pacific Region.

'Tubular aggregates' are morphologically distinct cytoplasmic structures that have been linked to a variety of pathological conditions. This report documents the presence of tubular aggregates in an insect cell line (C6/36 cells derived from Aedes albopictus) following inoculation of the cells with material derived from cell culture passaged homogenized Culex australicus mosquitoes. The tubular aggregates were detected in ∼2% of treated cells and had three morphological forms that were termed primary, secondary and tertiary, with progressively greater levels of structural complexity. The findings indicate that tubular aggregates can be induced in an insect cell culture system by an unidentified agent present in some mosquitoes.

Genotyping by VP1 fragment polymerase chain reaction (PCR) and nucleic acid sequencing to detect enterovirus (EV) genotypes was performed directly on 729 EV PCR positive cerebrospinal fluid (CSF) samples collected between 2007 and 2012 from Victorian hospital inpatients. The overall genotype identification rate from CSF-positive material was 43%. The four most common genotypes identified were Echovirus 6 (24%), Echovirus 30 (17%), Echovirus 25 (10%), and Coxsackievirus A9 (10%), together comprising 61% of all EVs typed. The seasonal distribution of all EVs identified followed the recognized pattern of mainly summer epidemics. Three of the four predominant genotypes were present in each of the 6 years in which the study was conducted, with 20 other EV genotypes also detected, often in only a single year. Genotyping of EVs directly in CSF is faster, simpler and more sensitive than traditional virus neutralization assays performed on EV positive samples.

To the editor: We thank the authors of the letter to the editor for their interest in our report of the detection of a potential new epidemic strain of norovirus [1]. The authors express some doubt as to the validity of our claims of a possible new norovirus GII.4 variant, since the recombinant form (GII.4_NewOrleans_2009/GII.4_Sydney_2012) has been reported previously in a number of instances, including by their own laboratory [2]. Nevertheless we stand by our proposal that a potential new epidemic strain of GII.4 norovirus has arisen, for the reasons outlined below.

A norovirus recombinant GII.P4_NewOrleans_2009/GII.4_Sydney_2012 was first detected in Victoria, Australia, in August 2015 at low frequency, and then re-emerged in June 2016, having undergone genetic changes. Analysis of 14 years’ surveillance data from Victoria suggests a typical delay of two to seven months between first detection of a new variant and occurrence of a subsequent epidemic linked to that variant. We consider that the current recombinant strain has the potential to become a pandemic variant.

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.

TOC summary: The greatest challenges were insufficient staff and test reagents., In Australia, the outbreak of pandemic (H1N1) 2009 began in Melbourne, Victoria; in the first 17 days, the Victorian Infectious Diseases Reference Laboratory detected 977 cases. Although the laboratory had a pandemic plan in place, a retrospective evaluation found 3 major variations from plan assumptions: 1) higher peak demand not limited by a case definition, 2) prolonged peak demand because containment attempts continued despite widespread influenza, and 3) unexpected influence of negative test results on public health actions. Although implementation of the plan was generally successful, the greatest challenges were limited availability of skilled staff and test reagents. Despite peak demand of 1,401 tests per day, results were provided within the usual 24 hours of specimen receipt; however, turnaround time seemed slower because of slow transport times (>3 days for 45% of specimens). Hence, effective laboratory capability might be enhanced by speeding transport of specimens and improving transmission of clinical data.

Background. Available data on the etiology of community-acquired pneumonia (CAP) in Australia are very limited. Local treatment guidelines promote the use of combination therapy with agents such as penicillin or amoxycillin combined with either doxycycline or a macrolide. Methods. The Australian CAP Study (ACAPS) was a prospective, multicenter study of 885 episodes of CAP in which all patients underwent detailed assessment for bacterial and viral pathogens (cultures, urinary antigen testing, serological methods, and polymerase chain reaction). Antibiotic agents and relevant clinical outcomes were recorded. Results. The etiology was identified in 404 (45.6%) of 885 episodes, with the most frequent causes being Streptococcus pneumoniae (14%), Mycoplasma pneumoniae (9%), and respiratory viruses (15%; influenza, picornavirus, respiratory syncytial virus, parainfluenza virus, and adenovirus). Antibiotic-resistant pathogens were rare: only 5.4% of patients had an infection for which therapy with penicillin plus doxycycline would potentially fail. Concordance with local antibiotic recommendations was high (82.4%), with the most commonly prescribed regimens being a penicillin plus either doxycycline or a macrolide (55.8%) or ceftriaxone plus either doxycycline or a macrolide (36.8%). The 30-day mortality rate was 5.6% (50 of 885 episodes), and mechanical ventilation or vasopressor support were required in 94 episodes (10.6%). Outcomes were not compromised by receipt of narrowerspectrum β-lactams, and they did not differ on the basis of whether a pathogen was identified. Conclusions. The vast majority of patients with CAP can be treated successfully with narrow-spectrum β-lactam treatment, such as penicillin combined with doxycycline or a macrolide. Greater use of such therapy could potentially reduce the emergence of antibiotic resistance among common bacterial pathogens. Members of the study group are listed at the end of the text.

For pandemic influenza planning, realistic estimates of personal protective equipment (PPE) and antiviral medication required for hospital healthcare workers (HCWs) are vital. In this simulation study, a patient with suspected avian or pandemic influenza (API) sought treatment at 9 Australian hospital emergency departments where patient–staff interactions during the first 6 hours of hospitalization were observed. Based on World Health Organization definitions and guidelines, the mean number of “close contacts” of the API patient was 12.3 (range 6–17; 85% HCWs); mean “exposures” were 19.3 (range 15–26). Overall, 20–25 PPE sets were required per patient, with variable HCW compliance for wearing these items (93% N95 masks, 77% gowns, 83% gloves, and 73% eye protection). Up to 41% of HCW close contacts would have qualified for postexposure antiviral prophylaxis. These data indicate that many current national stockpiles of PPE and antiviral medication are likely inadequate for a pandemic.

The noroviruses are now considered a leading cause of outbreaks of non-bacterial gastroenteritis worldwide. Vaccine strategies against norovirus are currently under consideration but depend on a detailed knowledge of the capsid genotypes. This study examined the incidence of norovirus outbreaks in Victoria over 1 year (2013) and documented the genotypes occurring in the different outbreak settings (healthcare and non-healthcare) and age groups. It was found that 63.1% of gastroenteritis outbreaks were associated with norovirus, thereby showing norovirus to be the major viral cause of illness in gastroenteritis outbreaks. Sixteen capsid genotypes were identified and included GI.2, GI.3, GI.4, GI.6, GI.7, GI.8, GI.9, GII.1, GII.2, GII.3, GII.4, GII.5, GII.6, GII.7, GII.13 and an as yet unclassified GII genotype. All genotypes found in the study, with the exception of GI.9, were detected in the elderly, indicating prior exposure to a norovirus genotype did not appear to confer long term immunity in many cases. The incidence of genotypes GII.1, GII.4 and GII.7 was linked with setting and age. As setting and age were correlated it was not possible to determine which variable was critical with the exception of GII.7, which appeared to be linked to age. The findings indicate that norovirus vaccine strategies should encompass a broad range of genotypes and, as setting or age may be important in determining genotype incidence, this should be taken into account as well.

Laboratory diagnosis is important to distinguish influenza from other respiratory virus infections. It will be especially important in detecting the first cases of pandemic influenza. Good quality respiratory tract sampling is needed to maximise diagnostic yield in influenza infection. In the appropriate clinical setting, pandemic strain-specific nucleic acid testing is the initial test of choice for suspected pandemic influenza. It is more sensitive than virus isolation, and more sensitive and specific than serology, immunofluorescence and other antigen detection methods. Virus isolation is needed to monitor new influenza strains and for vaccine development. Analysis of influenza isolates is undertaken by the World Health Organization Global Influenza Surveillance Network. Monitoring for antiviral resistance will be needed with widespread use of neuraminidase inhibitors for treatment and prophylaxis during a pandemic.

In Victoria (Australia) surveillance for mumps and rubella has historically been passive, with most notified cases clinically diagnosed. In July 2001, the Victorian Department of Human Services implemented an enhanced surveillance system focusing on improved laboratory testing. We tested 85% of notifications and only 9% of all mumps and 27% of rubella notifications were laboratory confirmed. While most notified cases were children who had been clinically diagnosed, we found most laboratory-confirmed cases were in adults. The positive predictive value of the clinical case definition was low: mumps (10%); rubella (22%). These results highlight the value of laboratory confirmation of the diagnosis when mumps and rubella are rare, failure to do so is likely to overestimate disease incidence.

Nineteen distinct measles virus (MV) strains associated with nine different genotypes were identified in five Australian states (Victoria, New South Wales, Queensland, Northern Territory and Western Australia) between 1999 and 2001. One of the strains identified is likely to represent a new genotype within the clade D viruses (proposed to be d9). No evidence for an indigenous MV strain was found. When epidemiologic information associated with the index case was available for the outbreaks, it usually supported introduction of the virus from overseas, with the main source being South East Asia. Changes in the circulation of MV in Australia since the early 1970s were also observed. Prior to the introduction of measles vaccine, the majority of the population acquired immunity through infection with wild-type virus in early childhood. Nowadays in Australia, young adults are at most risk of infection. The age range of cases in the study period was from 1 month to 48 years, with the majority (59%) of cases from individuals aged 18–30 years.

Results: Of 2144 surveillance samples tested, 1235 (57.6%) were positive for at least one virus. The most common were influenza A strains (17.9%), with pandemic influenza A(H1N1) 2009 and seasonal H3N2 strain accounting for 52% and 43% of these, respectively. Other virus detections included: rhinovirus (12.4%), enterovirus (8.9%), influenza B (8.3%), adenovirus (5.3%), parainfluenza (4.7%), respiratory syncytial virus (RSV) (3.9%), human coronavirus (3.0%) and human metapneumovirus (0.3%). The detection rate was greatest in the 0–5 year age group. Viral co-infections were identified in 148 (6.9%) cases. Discussion: The outbreak in 2009 of the influenza A(H1N1) pandemic strain provided a practical test of the laboratory’s pandemic plan. This study shows that the availability of appropriate equipment and molecular-based testing can contribute to important individual and public health outcomes in geographical locations susceptible to emerging infections.

This chapter describes cases of lymphocytic choriomeningitis virus (LCMV) and LCMV-like infections in three clusters of solid organ transplant recipients. It discusses some of the implications for organ transplant units. Infections in humans can occur through exposure to rodent urine, although nosocomial or laboratory spread has been documented. LCMV is known to occur primarily in Europe and the Americas but has been isolated from mice in other areas such as Japan and China. In Wisconsin in 2003 and in Rhode Island in 2005, there were two clusters of transplant-associated infections caused by LCMV. Arenavirus-like virus particles were seen using electron microscopy, and indirect fluorescent antibody testing (IFAT) confirmed LCMV as the cause. The clinical and diagnostic features of the three clusters of cases have implications for donor selection and organ transplantation services. Reverse transcription polymerase chain reaction (RT-PCR) testing is unlikely to be useful, as the three donors all tested negative using this technique. Although transplant-related infections with arenaviruses are rare, the consequences seen in the clusters are devastating. Exclusion of donors who have spent any time in areas where LCMV or LCMV-like viruses are endemic is likely to prove problematic. This is likely to exclude donors who are not infected in times where there is a shortage of human donors.

The role of diverse infectious agents, particularly Norwalk-like viruses (NLV), in three successive gastro-enteritis outbreaks in one setting (a restaurant) was evaluated. Methods included standard bacteriological tests, specific tests for Escherichia coli, tests for verocytotoxins, electron microscopy (EM) for viruses and reverse transcription-PCR (RT-PCR) methodology for NLV. No pathogenic bacteria were detected. Verocytotoxin genes, although detected by PCR in the first outbreak, could not be confirmed in the E. coli isolated, so they did not appear to be of significance. NLV was the main agent detected in each of the three outbreaks. DNA sequencing and phylogenetic analysis of the amplified products obtained from the RT-PCR positive specimens indicated that only one NLV strain was involved in each outbreak, but the NLV strains responsible for the three outbreaks were different from each other. PCR technology for detection of NLV proved highly sensitive, but failed to detect one specimen which was positive by EM. The restaurant associated with the outbreaks is a Mediterranean-style restaurant where food from a common platter is typically eaten with fingers. The findings indicate that NLV was introduced by guests or staff and was not due to a long-term reservoir within the setting.

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.

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.

The aim of the study was to compare the sensitivity of a norovirus RT-PCR method using two manual RNA extraction methods (Qiagen and Roche) and two automated RNA extraction methods (Qiagen and Corbett). All four RNA extraction methods gave similar sensitivities although the automated methods, especially the Corbett, required significantly less labour than the manual methods. The automated methods also enabled RNA extraction of approximately two to three times the number of specimens in a given time period compared to manual methods.

An avian influenza quality assurance program was used to provide information for laboratories on the sensitivity and specificity of their avian influenza nucleic acid testing. Most laboratories were able to correctly detect clinically relevant amounts of influenza virus (H5N1), and results improved as each subsequent panel was tested.

Background. No conventional immunosuppressive agent preferentially inhibits antibody production. Studies in experimental animals and in human cells in vitro suggested mycophenolate mofetil (MMF) might have such an effect. If this was the case in vivo it could have significant implications in terms of both MMF toxicity and the rational design of immunotherapeutic regimens. Methods. Subjects were renal transplant recipients (25 patients treated with prednisolone, cyclosporine and azathioprine, and 13 treated with prednisolone, cyclo­ sporine and MMF) and 20 normal controls. The three groups received influenza vaccination, and the anti­ body response to it was measured 4-6 weeks later using a standard haemagglutination assay. Results. MMF profoundly suppressed the humoral immune response to influenza vaccination when added to prednisolone and cyclosporine. This effect could be seen when comparing the rise in the mean titre of antibody after vaccination. It was also reflected in the number of patients mounting responses deemed to be clinically protective by either demonstrating a 4-fold rise in titre or an increase in titre to ~ 40. Conclusions. Suppression of the humoral immune response by MMF has implications for the design of immunization protocols to protect the immuno­ suppressed, and raises the possibility that MMF use may be accompanied by more or different infections than complicate more conventional immunosuppres­ sion. More importantly, consideration should be given to harnessing the relatively specific effect of MMF on antibody production to treat antibody-mediated diseases.

Objectives: To describe the epidemiology of HIV in Victoria between 1997 and 2002 using HIV surveillance data. Methods: All HIV diagnoses notified to the Victorian HIV Registry from 1997 to 2002 were described. Results: The average annual number of HIV notifications rose from 160 during 1997–99 to 216 during 2000–02, with the number of infections from men who have sex with men (MSM) increasing by 41%. Notifications from MSM acquired from casual or anonymous partners increased from 65 in 1997–99 to 92 in 2000–02. Infections attributable to heterosexual contact increased from an average number of 30 during 1997–99 to 46 during 2000–02, a 53% increase. Conclusions: This rise in HIV notifications in Victoria threatens this State's progress in controlling the HIV epidemic. Implications: The surveillance data demonstrate a need to implement effective, innovative and evidence-based programs for HIV prevention.

We report the case of an elderly woman excreting high levels (about 5 x 10(5) virions per gram of faeces) of Norwalk-like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342-nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

A range of laboratory methods is now available for the detection of norovirus, a major cause of gastroenteritis. Recently, a commercial immunochromatographic assay for norovirus detection, the RIDA(®)QUICK assay, has become available, but there is still only limited information on its efficacy. This study examined the sensitivity and specificity of the RIDA(®)QUICK assay, using faecal material received for testing in a major diagnostic/reference laboratory in Australia. The sensitivity of the assay was found to be 83% and the specificity was 100%. No false positive norovirus results were found and the assay did not cross-react with common faecal viruses such as rotavirus, astrovirus, sapovirus and adenovirus. The assay was less reliable for genogroup I (GI) noroviruses than for genogroup II (GII) noroviruses. Genotypes detected by the assay included GII.1, GII.2, GII.3, GII.4, GII.6 and GII.7. The assay failed to detect any GI specimens in the test group. Genotypes not detected included GI.4 and GI.6. The assay was simple and quick to perform. It is valuable in a point-of-care situation or as a backup in a laboratory where a rapid initial norovirus result is required.

Data collected over winter 2009 by five World Health Organisation National Influenza Centres in the southern hemisphere were used to examine the circulation of pandemic and seasonal influenza A strains during the first pandemic wave in the southern hemisphere. There is compelling evidence that the pandemic influenza A(H1N1) 2009 virus significantly displaced seasonal influenza A(H1N1) and, to a lesser extent, A(H3N2) viruses circulating in the southern hemisphere. Complete replacement of seasonal influenza A strains, however, was not observed during the first pandemic wave.

Victoria was the first Australian state to report widespread transmission of pandemic H1N1 2009 influenza. Notifiable laboratory-confirmed influenza and a general practitioner sentinel surveillance system measuring influenza-like illness (ILI), including laboratory confirmation of influenza as the cause of ILI, were used to assess the pandemic. The pandemic influenza A(H1N1)v virus quickly became the dominant circulating strain and notification rates were highest in children and young adults. Despite a high number of notified cases, comparison of ILI rates suggested the season peaked in late June, was similar in magnitude to 2003 and 2007 and less severe than 1997. The majority of clinical presentations were mild, but one quarter of hospitalised cases required admission to intensive care. Given the low proportion of imported cases in the Victorian pandemic, the rapid increase in cases with no travel history and the low median age of cases notified during the phases of intense surveillance, we suggest there may have been silent importations of pandemic virus into Victoria before the first case was recognised. The usefulness of a general practitioner sentinel surveillance system to provide a comparable assessment of influenza and ILI activity over time was clearly demonstrated, and the need for similar hospital and mortality surveillance systems for influenza in Victoria was highlighted.

In a pandemic, many current national stockpiles of PPE and antiviral medications are likely inadequate., For pandemic influenza planning, realistic estimates of personal protective equipment (PPE) and antiviral medication required for hospital healthcare workers (HCWs) are vital. In this simulation study, a patient with suspected avian or pandemic influenza (API) sought treatment at 9 Australian hospital emergency departments where patient–staff interactions during the first 6 hours of hospitalization were observed. Based on World Health Organization definitions and guidelines, the mean number of “close contacts” of the API patient was 12.3 (range 6–17; 85% HCWs); mean “exposures” were 19.3 (range 15–26). Overall, 20–25 PPE sets were required per patient, with variable HCW compliance for wearing these items (93% N95 masks, 77% gowns, 83% gloves, and 73% eye protection). Up to 41% of HCW close contacts would have qualified for postexposure antiviral prophylaxis. These data indicate that many current national stockpiles of PPE and antiviral medication are likely inadequate for a pandemic.

Background: Although rotavirus is a major cause of gastroenteritis in children, its role in adult gastroenteritis and the sensitivity of different methods for its detection in specimens collected from adults are less well understood. Objectives: (1) To examine the frequency and seasonality of rotavirus-associated gastroenteritis outbreaks in aged-care facilities in Victoria, Australia. (2) To determine rotavirus type in these outbreaks. (3) To determine whether other enteropathogenic agents are present in specimens from these outbreaks. (4) To examine the sensitivity of different methods (electron microscopy (EM), reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA) and latex agglutination (LA)) for the detection of rotavirus in specimens from adults. Study design: Specimens from gastroenteritis outbreaks in aged-care facilities forwarded to this laboratory for the years 1997–2000 were tested for enteropathogenic agents by various methods. Epidemiological, clinical and seasonal data from the rotavirus-positive outbreaks were analysed. Results: Rotavirus was detected by EM in 18 out of 29 individuals associated with seven out of 53 (13%) gastroenteritis outbreaks in aged-care facilities; norovirus was detected in 22 outbreaks (42%) and astrovirus in one outbreak (2%). No mixed viral infection was found in any outbreak. All rotaviruses were typed as Group A by RT-PCR. The rotaviruses in the seven outbreaks were G-typed as follows: G2 (three outbreaks), G4 (two outbreaks), G1 (one outbreak) and G9 (one outbreak). The rotavirus-associated outbreaks were concentrated in mid-winter to mid-spring. The relative sensitivities of the Group A rotavirus detection methods (for the 29 specimens tested) were EM (18), first-round RT-PCR (11), second-round PCR (19), EIA-visual (19), EIA-photometric (19) and LA (13). Conclusions: In Victoria, Australia, outbreaks of gastroenteritis associated with rotavirus are quite common in aged-care facilities. They involve Group A rotavirus and have a winter/spring seasonality. G-types G1, G2, G4 and G9 were all detected. EIA, second-round PCR and EM proved sensitive methods for rotavirus detection whereas first-round RT-PCR and LA did not.

Two separate cases of acute hepatitis C virus (HCV) infection following medical procedures, arthroscopy and colonoscopy, are reported. In both episodes, patient risk factors were reviewed, and staff and other patients' sera were tested for HCV antibodies and RNA. HCV RNA positive samples were genotyped, sequenced, and subjected to phylogenetic analysis. No risk factors for HCV infection were identified for either case except for medical procedures. HCV RNA positive patients were identified preceding both cases on the respective theatre lists. HCV infection in a second low risk patient was also identified. Nucleic acid sequencing and phylogenetic analysis of HCV from the two putative source patients and the three recipient patients demonstrated a high degree of relatedness respectively. The results suggest that patient-to-patient transmission occurred in both episodes via contamination of intravenous anaesthetic ampoules with HCV used on multiple patients. Injectable medication ampoules should not be used for more than one patient.

Endemic gastroenteritis associated with the Norwalk-like viruses (NLVs) is little understood. This study tested for NLV in gastroenteritis cases in 257 households in Melbourne, Australia, for the period September 1997 to February 1999 by a reverse transcription hemi-nested polymerase chain reaction. Positive samples were studied by nucleotide sequencing and phylogenetic analysis. NLV was detected in 73 (11.4%) of 638 faecal specimens tested. Twelve (1.9%) were NLV genogroup 1 (G1) and 61 (9.6%) NLV genogroup 2 (G2). Gastroenteritis symptoms associated with NLV G2/no other pathogens were significantly more severe than where no NLV was detected. NLV G1 and NLV G2 were detected in adults and children, males and females. NLV G2 incidence showed a marked seasonal periodicity with significant peaks in the Australian late spring/early summer periods. NLV G1 seasonality was significantly different from that of NLV G2. Seven major NLV clusters were identified by phylogenetic analysis.

Noroviruses are a major cause of both sporadic and epidemic gastroenteritis in humans, but the mechanisms by which norovirus circulates within the community are poorly understood. In this study, we examined the hypothesis that asymptomatic people act as a reservoir for norovirus. Faecal specimens from 399 asymptomatic individuals were tested for norovirus by reverse transcription polymerase chain reaction (RT-PCR) methodology, and no norovirus was detected. The failure to detect norovirus was not apparently due to the test sample being resistant to norovirus infection, nor to the presence of PCR inhibitors in the test sample. The findings suggest that, if norovirus is carried by asymptomatic people, the carriage rate is very low; the upper bound (95% confidence interval, binomial distribution) of the carriage rate was only 0.8%. Thus, it is unlikely that asymptomatic people are an important reservoir for norovirus.

A multiplex reverse transcription-PCR-enzyme hybridization assay (RT-PCR-EHA; Hexaplex; Prodesse Inc., Waukesha, Wis.) was used for the simultaneous detection of human parainfluenza virus types 1, 2, and 3, influenza virus types A and B, and respiratory syncytial virus types A and B. One hundred forty-three respiratory specimens from 126 patients were analyzed by RT-PCR-EHA, and the results were compared to those obtained by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of 143 (5.6%) samples were positive by viral culture and/or IF. Eight samples were positive by both RT-PCR-EHA and conventional methods, while nine samples were RT-PCR-EHA positive and viral culture and IF negative. Eight of the nine samples with discordant results were then independently tested by a different multiplex RT-PCR assay for influenza virus types A and B, and all eight proved to be positive. In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was able to detect more positive samples, which would otherwise have been missed by routine methods, we suggest that this multiplex RT-PCR-EHA provides a highly sensitive and specific means of diagnostic detection of major respiratory viruses.

The present study describes a heminested multiplex reverse transcription (RT)-PCR assay which enables simultaneous detection and differentiation of Norwalk-like virus (NLV) genogroups from clinical fecal samples without the need to perform sequencing or hybridization. The assay developed was able to detect concentrations of fewer than 100 viral particles per 5 μl of clarified fecal extract and could differentiate the two genogroups with a specificity of 100%. Although the multiplex RT-PCR assay failed to detect NLV in about 3% of the fecal samples which were NLV positive by electron microscopy (EM), the assay was approximately six times more sensitive than EM for NLV detection.

The re-use of solutions used in surgical procedures provides an opportunity for transmission of infectious agents should a breakdown in good work practices occur. Agents that are blood-borne are particularly important in this respect. We examined whether a hepatitis C virus surrogate (bovine viral diarrhoeal virus) could survive exposure to Propafol and Fentanyl, drugs commonly used for induction of anaesthesia and analgesia, respectively. Testing involved the spiking of ampoules of these solutions with a high-titred preparation of virus. Following incubation of this mixture at ambient temperature for various periods of time, attempts were made to isolate the virus in cell cultures. Our results showed that the surrogate virus survived for up to 2 hours without loss of titre in these solutions, suggesting that the hepatitis C virus would also survive and could also be transmitted in a surgical setting under some circumstances.

At various times postonset of rash, 74 patients positive for measles virus-specific immunoglobulin M provided samples for detection of measles virus RNA by a reverse transcriptase PCR. Of lymphocytes, urine, throat swab, and serum specimens, throat swab specimens were optimal for detection of measles virus RNA during the first 2 weeks after the rash.

Summary Ebolaviruses, and the other viral causes of haemorrhagic fevers (VHF) have always posed special problems for diagnostic laboratories. These arise from the rarity of human infections, minimal documented experience with test delivery and interpretation, the paucity of established commercial or in-house assays, the lack of clinical material for test development and validation, the high level containment required for handling live virus, the ongoing evolution of the viruses, and the high personal and public health requirements for accurate diagnosis. This article addresses the current situation and the ongoing challenges associated with delivering timely, high quality and safe testing within Australia for people exposed as part of the current major outbreak of Ebolavirus disease (EVD) in Western Africa. The members of the Public Health Laboratory Network have developed deliverable and reliable nucleic acid detection tests, and also have the laboratory capacity to handle the live viruses if necessary. However delivering and maintaining these services necessitates high levels of experience in developing and applying tests for exotic and emerging infections, strong national and international links and collaborations, ongoing monitoring and reassessment of test design and performance, innovative approaches to generation of positive control material, and a regular quality assurance program.