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.

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.

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.

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.

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.

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.

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.

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.

Surveillance for outbreaks of influenza-like illness in the institutionalized elderly.

Respiratory outbreaks are common in aged-care facilities (ACFs), are both underreported and frequently identified late, and are often associated with considerable burden of illness and death. There is emerging evidence that active surveillance coupled with early and systematic intervention can reduce this burden. Active surveillance for influenza-like illness and rapid diagnosis of influenza were established in 16 ACFs in Sydney, Australia, prior to the winter of 2006. A point-of-care influenza test and laboratory direct immunofluorescence tests for common respiratory viruses were used for diagnosis. We achieved early identification of seven respiratory disease outbreaks, two of which were caused by influenza. For the influenza outbreaks, antiviral treatment and prophylaxis were initiated 4-6 days from symptom onset in the primary case. A simple active surveillance system for influenza was successfully implemented and resulted in early detection of influenza and other respiratory disease outbreaks. This enabled earlier implementation of prevention and control measures and increased the potential effectiveness of anti-influenza chemoprophylaxis.

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.

Influenza vaccination amongst hospital health care workers in Beijing.

BACKGROUND: Annual influenza vaccination is recommended for health care workers (HCWs) in many countries in order to reduce the morbidity associated with influenza in health care settings. However, compliance rates with influenza vaccination are commonly low. AIMS: To evaluate the current vaccination status of HCWs in Beijing, China, and examine their attitudes towards the disease and the vaccine. METHODS: In January 2009, a survey was completed by 1909 HCWs in emergency departments, infection fever clinics, respiratory ward/outpatient's clinics and pediatric medical departments of 24 hospitals in Beijing (99% response rate). RESULTS: Respondents were categorized into three main groups by occupation: nursing (60%, n = 1143), medical (36%, n = 693) and other (4%, n = 73). When examining beliefs about the influenza vaccine, 57% (n = 1081) felt it was safe and 54% (n = 1028) thought it was effective. Less than 18% stated that they had been immunized in 2008; 40% (n = 765) or participants agreed with the statement 'the flu vaccine can cause flu in some people'. CONCLUSIONS: A better understanding of the barriers to vaccination in this population should facilitate the development of programs to make health care facilities a safer environment for both HCWs and patients.

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.

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.

Evidence for late stage compartmentalization of HIV-1 resistance mutations between lymph node and peripheral blood mononuclear cells.

OBJECTIVE: To determine the overall distribution of drug-resistance mutations to nucleoside reverse transcriptase inhibitors of HIV strains recovered from the lymph nodes (LN) and peripheral blood mononuclear cell (PBMC) compartments of four HIV-infected patients receiving zidovudine and didanosine and to compare them with antiretroviral-naive patients. DESIGN: Molecular comparison of major and minor HIV-1 env and pol region variants residing in LN and PBMC compartments. MATERIALS AND METHODS: Proviral DNA sequences were amplified by PCR from both PBMC and LN compartments, cloned into PGEM-T II Easy vector and sequenced. The clones were subjected to molecular and phylogenetic analysis. RESULTS: Comparison of PBMC and LN-derived HIV-1 variants in the env V3 region showed that nucleotide and amino acid variability was a characteristic feature of LN-derived variants. In contrast, a majority of resistance mutations to reverse transcriptase inhibitors were localized in the PBMC compartment rather than in LN, which is thought to be a reservoir of HIV. CONCLUSIONS: Distinct compartmentalization or independent evolution of pol and env gene variants between LN and PBMC could be due to the differential selection pressure imposed by the combination drug regimen, hence the bimodal distribution of resistance variants between LN and PBMC compartments.

Arboviruses associated with human disease in Australia.

Mosquito-borne arboviruses are an important public health issue in Australia. The alphaviruses Ross River and Barmah Forest virus are widespread and active annually, and cause debilitating polyarthritis. The flaviviruses Murray Valley encephalitis, Kunjin and Japanese encephalitis virus are restricted in distribution and activity but may cause life-threatening illness, and dengue viruses are active in some areas.

Viral markers in HIV infection and AIDS.

Viral and immune markers are used for monitoring either progression of human immunodeficiency virus (HIV) disease or response to antiviral therapy. Ideal properties of viral markers are that they are present in all HIV-infected persons at all stages of disease, that they are related to disease pathogenesis, that they can be easily quantitated, that this quantitation correlates rapidly and predictably with both disease stage and response to antivirals, and that they can be developed into rapid, reproducible automated tests. Currently available viral markers include HIV p24 antigenemia (after acid glycine dissociation), anti-p24 antibody titres, quantitative DNA and RNA polymerase chain reaction performed on cells and plasma, and HIV isolate phenotype. In Australia, these markers have been studied in acute HIV seroconversion, in neonatal infection, in body fluids other than blood, and in monitoring of response to antiviral drug therapy.