SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low-frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole-genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low-frequency variants. Our analyses reveal that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina Respiratory Viral Oligo panel and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high-quality sequence data from low-viral-load samples for public health and genomic surveillance purposes.

Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus.

INTRODUCTION: There is limited data on the analytical performance of commercial nucleic acid tests (NATs) for laboratory confirmation of COVID-19 infection. METHODS: Nasopharyngeal, combined nose and throat swabs, nasopharyngeal aspirates and sputum was collected from persons with suspected SARS-CoV-2 infection, serial dilutions of SARS-CoV-2 viral cultures and synthetic positive controls (gBlocks, Integrated DNA Technologies) were tested using i) AusDiagnostics assay (AusDiagnostics Pty Ltd); ii) in-house developed assays targeting the E and RdRp genes; iii) multiplex PCR assay targeting endemic respiratory viruses. Discrepant SARS-CoV-2 results were resolved by testing the N, ORF1b, ORF1ab and M genes. RESULTS: Of 52 clinical samples collected from 50 persons tested, respiratory viruses were detected in 22 samples (42 %), including SARS CoV-2 (n = 5), rhinovirus (n = 7), enterovirus (n = 5), influenza B (n = 4), hMPV (n = 5), influenza A (n = 2), PIV-2 (n = 1), RSV (n = 2), CoV-NL63 (n = 1) and CoV-229E (n = 1). SARS-CoV-2 was detected in four additional samples by the AusDiagnostics assay. Using the in-house assays as the "gold standard", the sensitivity, specificity, positive and negative predictive values of the AusDiagnostics assay was 100 %, 92.16 %, 55.56 % and 100 % respectively. The Ct values of the real-time in-house-developed PCR assay targeting the E gene was significantly lower than the corresponding RdRp gene assay when applied to clinical samples, viral culture and positive controls (mean 21.75 vs 28.1, p = 0.0031). CONCLUSIONS: The AusDiagnostics assay is not specific for the detection SARS-CoV-2. Any positive results should be confirmed using another NAT or sequencing. The case definition used to investigate persons with suspected COVID-19 infection is not specific.

Increased prevalence of influenza B/Victoria lineage viruses during early stages of the 2015 influenza season in New South Wales, Australia: implications for vaccination and planning.

During the early weeks of the 2015 Australian influenza season, influenza B accounted for 67% (821/1,234) of all positive influenza tests in New South Wales. Of 81 successive influenza B viruses characterised, 33 (41%) were from children aged < 16 years; 23/81 (28%) belonged to the B/Victoria lineage. This lineage is not contained in the southern hemisphere's 2015 trivalent influenza vaccine. The significant B/Victoria lineage activity in the southern hemisphere suggests that the quadrivalent vaccine should be considered for the northern hemisphere.

Virological diagnosis of Ebolavirus infection.

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.

Hepatitis B immunity in Australia: a comparison of national and prisoner population serosurveys.

In Australia, hepatitis B (HBV) vaccination is recommended for injecting drug users (IDUs), Indigenous adults and prisoners. We compared immunity to HBV in prisoners and the general population obtained from national serosurveys in 2007. Individuals with HBV surface antibody (HBsAb) positive sera were considered immune from past infection [HBV core antibody (HBcAb) positive] or from vaccination (HBcAb negative). Male prisoners aged 18-58 years had a higher HBsAb seroprevalence than the general population (46·4% vs. 39·4%, P = 0·061). Comparison of HBcAb results was possible for males aged 18-29 years. In this group, higher HBsAb seroprevalence was due to past infection (12·9% vs. 3·0%, P < 0·001), rather than vaccine-conferred immunity (35·3% vs. 43·4%, P = 0·097). All prisoner groups, but especially IDUs, those of Indigenous heritage or those with a previous episode of imprisonment had higher levels of immunity from past infection than the general population (19·3%, 33·0%, 17·1%, respectively, vs. 3·0%, P < 0·05). Indigenous prisoners, non-IDUs and first-time entrants had significantly lower levels of vaccine-conferred immunity than the general population (26·4%, 26·2% and 20·7% respectively vs. 43·4%, P < 0·05). Improving prison-based HBV vaccination would prevent transmission in the prison setting and protect vulnerable members of the community who are at high risk of both infection and entering the prison system.

Clinical features of endemic community-acquired psittacosis.

Following a large outbreak of community-acquired psittacosis in 2002 in residents of the Blue Mountains, New South Wales, Australia, we reviewed new cases in this area over a 7-year period from 2003 to 2009. Using the 2010 criteria from the Centers for Disease Control National Notifiable Diseases Surveillance System, 85 patients with possible psittacosis were identified, of which 48 were identified as definite or probable infection. Clinical features of these cases are summarized. In addition to Chlamydia-specific serology, specimens, where available, underwent nucleic acid testing for chlamydial DNA using real-time PCR. Chlamydophila psittaci DNA was detected in samples from 23 patients. Four of 18 specimens were culture positive. This is the first description of endemic psittacosis, and is characterized in this location by community-acquired psittacosis resulting from inadvertent exposure to birds. The disease is likely to be under-diagnosed, and may often be mistaken for gastroenteritis or meningitis given the frequency of non-respiratory symptoms, particularly without a history of contact with birds. Clinical characteristics of endemic and outbreak-associated cases were similar. The nature of exposure, risk factors and reasons for the occurrence of outbreaks of psittacosis require further investigation.

EBV & HHV6 reactivation is infrequent and not associated with MS clinical course.

BACKGROUND: Among the environmental factors associated with multiple sclerosis (MS) causation, some of the strongest associations are with Epstein-Barr virus (EBV), and to a lesser extent human herpesvirus 6 (HHV6). Associations with clinical course are less conclusive, however. METHODS: We evaluated serum anti-EBV-EA-R IgG and anti-HHV6 IgM, and EBV and HHV6 viral load (VL) for their associations with relapse, disability, and progression in disability in a prospective cohort of 198 participants with clinically definite MS. RESULTS: Anti-EBV-EA-R IgG was detected in 81.8% of cases at study entry, and titers remained essentially unchanged during the study. Anti-HHV6 IgM was detected in only one participant, and EBV-VL (29%) and HHV6-VL (1.8%) were detected in a minority of samples, and where detected levels were low. Our previously demonstrated association between anti-HHV6 IgG and relapse hazard was not affected by adjustment for parameters of reactivation. We found no evidence that any of the viral markers were associated with disability or progression in disability. In relation to relapse, only EBV-VL was positively associated, although this was strongly influenced by a single individual. CONCLUSION: Using a prospective cohort design, we found no convincing evidence that reactivation parameters of EBV or HHV6 were associated with subsequent MS relapse hazard or progression in disability, confirming previous findings, and indicating that herpesvirus reactivation is not an important driver of relapse or disability in this established MS population.

Test performance characteristics of quantitative nucleic acid testing for polyomaviruses in kidney and kidney-pancreas transplant recipients.

Screening for polyoma BK virus (BK) using nucleic testing (NAT) is recommended for kidney and kidney-pancreas transplant recipients, but the performance characteristics of quantitative BK NAT at different thresholds of plasma BK viral loads are unclear. We aim to evaluate the diagnostic accuracy of quantitative BK NAT as an add-on test to qualitative polyoma NAT for the diagnosis of BK virus-associated nephropathy (BKVAN) in kidney and kidney transplant recipients. We calculated the test sensitivity, specificity, and predictive values at the different thresholds of plasma BK viral load for BKVAN. At the recommended threshold of >1 × 10(3) serum BK copies/mL serum for test positivity, the sensitivity for BKVAN was 92.9% (95% confidence intervals [CI]: 66.1-99.8) and specificity 79.1% (95%: CI 67.4-88.1), with corresponding positive and negative predictive values of 42.0% (95% CI: 24.8-57.7%) and 98.6% (95% CI: 98.3-99.9%), respectively. The overall area under curve for the quantitative BK NAT was 0.92 (95% CI: 0.85-0.97). Quantitative BK NAT displays properties of high sensitivity and specificity that are fit for purpose as an add-on test to qualitative polyomavirus NAT for kidney and kidney-pancreas transplant recipients at risk of BKVAN.

Changes in seroprevalence to hepatitis A in Victoria, Australia: a comparison of three time points.

Serological data provide an important measure of past exposure and immunity to hepatitis A virus (HAV) infection in a population. National serosurveys from developed countries have typically indicated a decline in HAV seroprevalence over time as sanitation levels improve. We examined trends in the seroepidemiology of HAV antibodies in Victoria, Australia, drawing on cross-sectional samples taken at three time points over a 20-year period. Stored sera from 1988 (n=753), 1998 (n=1091), and 2008 (n=791) from persons aged 1-69 years were obtained from the state of Victoria, Australia. The within-year population adjusted results show a significant trend of increasing population HAV seroprevalence over time from 34.3% (95% CI 31.7-36.9) in 1988, to 40.0% (95% CI 37.1-42.8) in 1998 and 55.1% (95% CI 52.1-58.1) in 2008, P<0.0001. A particularly noticeable rise in population seroprevalence was observed between 1998 and 2008 for those aged 5-39 years. The increase in HAV seropositivity over time is in contrast to the declining rates of disease notification in Australia. Based on comparisons with other Australian data, it appears the increase in population seroprevalence over the last two decades is unlikely to be due to endemic transmission of infection. Instead, other factors, including increases in travel to HAV endemic regions, migration to Australia from HAV endemic regions and vaccine uptake are more likely causes. Ongoing monitoring of serological HAV profiles in the population is required to determine future policy direction to prevent increased burden.

Frequency of oseltamivir resistance in Sydney, during the Newcastle outbreak of community transmitted oseltamivir-resistant influenza A(H1N1)pdm09 virus, Australia, June to August 2011.

Although oseltamivir-resistant pandemic influenza A(H1N1)pdm09 is uncommon in immunocompetent individuals, a recent report from Newcastle, Australia, showed the first sustained community spread, from June to August 2011, of oseltamivir-resistant influenza A(H1N1)pdm09 virus carrying the H275Y neuraminidase (NA) mutation. To determine the frequency and the extent of this viral variant spread in the nearest major city to Newcastle, we performed a sequencebased genotypic assessment on samples from 143 oseltamivir untreated and 23 oseltamivir post-treatment individuals with influenza collected contemporaneously in Sydney, 120 km southwest of Newcastle. The detection of two of 143 (1.4%) community-derived samples containing H275Y suggests a low prevalence of oseltamivir-resistant influenza A(H1N1)pdm09 virus in the general community and no convincing evidence of spread of the NA H275Y-bearing influenza A(H1N1)pdm09 virus. In oseltamivir treated patients, oseltamivir-resistant influenza A(H1N1)pdm09 virus continue to emerge with three of 23 (13%) post-treatment samples containing the H275Y mutation. The observation of signature mutations and distinct phylogenetic relationship in full-length sequences of haemagglutinin and neuraminidase genes derived from 2011 strains against 2009 strains indicates continued genetic evolution and antigenic drift of the influenza A(H1N1)pdm09 viruses circulating in Australia.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Epstein-Barr virus and multiple sclerosis.

This review of the considerable evidence linking Epstein-Barr virus (EBV) infection to risk and disease progression in multiple sclerosis (MS) builds on the background to the virus and its interactions with the human host available in the online supplement (see supplement, available online only). The evidence for a similarity in the geographic patterns of occurrence of MS and EBV infection (with infectious mononucleosis or EBV specific serology used as surrogate markers), when reviewed critically, is very limited. There is strong evidence however that people with MS are more likely to report a past history of infectious mononucleosis (thought to represent initial EBV infection at an older age), and higher titres of EBV specific antibodies are associated with an increased risk of developing MS. Elevated levels of the latter are apparent many years before MS onset (compared with non-MS controls) and there is a dose-response relationship between MS risk and antibody titre, with antibodies to the EBV nuclear antigen-1 particularly important. The evidence in relation to EBV DNA load in blood or CSF is conflicting, as is that in relation to T cell responses to EBV. Several hypotheses that have been proposed to explain the links between EBV and MS risk are reviewed and gaps requiring further research are identified.

Current and past Epstein-Barr virus infection in risk of initial CNS demyelination.

OBJECTIVES: To assess risk of a first clinical diagnosis of CNS demyelination (FCD) in relation to measures of Epstein-Barr virus (EBV) infection within the context of other known risk factors. METHODS: This was a multicenter incident case-control study. FCD cases (n = 282) aged 18-59 years and controls (n = 558, matched on age, sex, and region) were recruited from 4 Australian centers between November 1, 2003, and December 31, 2006. A nested study (n = 215 cases, n = 216 controls) included measurement of whole blood quantitative EBV DNA load and serum EBV-specific antibodies. Conditional logistic regression was used to analyze case-control differences. RESULTS: There were no significant case-control differences in the proportion with detectable EBV DNA (55.8% vs 50.5%, respectively, p = 0.28), or in quantitative EBV DNA load (p = 0.33). Consistent with previous work, higher anti-EBV-specific immunoglobulin G (IgG) titers and a history of infectious mononucleosis were associated with increased FCD risk and there was an additive interaction with HLA-DRB1*1501 status. We found additional interactions between high anti-EBNA IgG titer and SNPs in HLA-A (adjusted odds ratios [AOR] = 19.84 [95% confidence interval (CI) 5.95 to 66.21] for both factors compared to neither) and CTLA-4 genes (AOR = 0.31 [95% CI 0.13 to 0.76] for neither factor compared to both). EBV DNA load was lower at higher serum 25-hydroxyvitamin D concentrations in controls (r = -0.17, p = 0.01). An adverse effect of higher EBV DNA load on FCD risk was increased with higher 25-hydroxyvitamin D concentration (p[interaction] = 0.02). CONCLUSION: Past infection with EBV, but not current EBV DNA load in whole blood, is significantly associated with increased FCD risk. These associations appear to be modified by immune-related gene variants.

Evaluation of a clinical scoring system and directed laboratory testing for respiratory virus infection in hematopoietic stem cell transplant recipients.

A simple clinical screening (CS) tool for respiratory virus (RV) infection was introduced and evaluated in a single hematology ward, as part of a strategy to reduce nosocomial RV infection. Up to 6 clinical symptoms or signs were scored and a predefined threshold score of ≥ 2 prompted paired nose/throat swab (NTS) collection for RV testing. The criterion standard for RV infection was positive immunofluorescence (IF) or polymerase chain reaction (PCR) for 7 and 15 viruses, respectively. The tool was shown to be most beneficial at excluding infection at a threshold score of 1 (negative predictive value [NPV] 89%, [95% confidence interval 78-96%], sensitivity 85% [70-94%], specificity 35% [27-43%]), compared with a score of 2 (NPV 85% [76-91%], sensitivity 63% [46-77%], specificity 57% [48-65%]) at a prevalence of 22%. The tool's ability to diagnose infection was limited (positive predictive value 27% and 29% at thresholds 1 and 2). The sensitivity of IF compared with PCR was 45% for the 7 viruses common to both, and 23% for the extended virus panel detected by PCR. An algorithm incorporating CS, paired NTS collection at a threshold of 1 symptom or sign, and sensitive testing including PCR can guide infection control measures in hospitalized hematopoietic stem cell transplant recipients.

Oseltamivir-resistant influenza viruses circulating during the first year of the influenza A(H1N1) 2009 pandemic in the Asia-Pacific region, March 2009 to March 2010.

During the first year of the influenza A(H1N1) 2009 pandemic, unprecedented amounts of the neuraminidase inhibitors, predominantly oseltamivir, were used in economically developed countries for the treatment and prophylaxis of patients prior to the availability of a pandemic vaccine. Due to concerns about the development of resistance, over 1,400 influenza A(H1N1) 2009 viruses isolated from the Asia-Pacific region during the first year of the pandemic (March 2009 to March 2010) were analysed by phenotypic and genotypic assays to determine their susceptibility to the neuraminidase inhibitors. Amongst viruses submitted to the World Health Organization Collaborating Centre for Reference and Research in Melbourne, Australia,oseltamivir resistance was detected in 1.3% of influenza A(H1N1) 2009 strains from Australia and 3.1% of strains from Singapore, but none was detected in specimens received from other countries in Oceania or south-east Asia, or in east Asia. The overall frequency of oseltamivir resistance in the Asia-Pacific region was 16 of 1,488 (1.1%). No zanamivir-resistant viruses were detected. Of the 16 oseltamivir-resistant isolates detected, nine were from immunocompromised individuals undergoing oseltamivir treatment and three were from immunocompetent individuals undergoing oseltamivir treatment. Importantly, four oseltamivir-resistant strains were from immunocompetent individuals who had not been treated with oseltamivir, demonstrating limited low-level community transmission of oseltamivir-resistant strains. Even with increased use of oseltamivir during the pandemic, the frequency of resistance has been low, with little evidence of community-wide spread of the resistant strains. Nevertheless, prudent use of the neuraminidase inhibitors remains necessary, as does continued monitoring for drug-resistant influenza viruses.

The impact of the pandemic influenza A(H1N1) 2009 virus on seasonal influenza A viruses in the southern hemisphere, 2009.

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.

The burden of infectious gastroenteritis in elderly residents and staff of long-term care facilities, Australia.

We estimated the incidence of gastroenteritis in 16 Australian long-term care facilities. During 12 months' surveillance, 245 (96%) of 254 episodes of gastroenteritis among long-term care residents were associated with 17 outbreaks in 11 facilities. Incidence in long-term care residents was 0.64 episodes per 1,000 bed-days (95% confidence interval, 0.29-1.42).