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.

In countries with a low prevalence of COVID-19 and a low pre-test probability, confirmation of positive nucleic acid test (NAT) results for SARS-CoV-2 is recommended given the potential for false positive results. As of 24 September 2020, there have been 26,983 confirmed cases and 861 deaths from COVID-19 in Australia. Widespread testing, together with Australia's geographic advantage, border controls, social distancing and public health messaging have all contributed to limit the number of infections. Australia has one of the highest testing rates in the world with 7,441,327 SARS-CoV-2 NATs performed on 6.4% of the population1Australian Government Department of Health Coronavirus (COVID-19) current situation and case numbers.Cited 20 Sep 2020https://www.health.gov.au/news/health-alerts/novel-coronavirus-2019-ncov-health-alert/coronavirus-covid-19-current-situation-and-case-numbers#tests-conducted-and-resultsGoogle Scholar since 22 January 2020, of which 0.4% were positive. The prevalence of laboratory-confirmed COVID-19 has varied between the different jurisdictions in Australia since the pandemic was declared, with the highest overall rate of 0.8% in Victoria in July 2020. During the early phases of the pandemic, SARS-CoV-2 NATs were mainly performed by public health laboratories using 'in-house' developed tests targeting one or more regions of the SARS-CoV-2 genome. Over time, commercial NATs became available, and testing was also undertaken by private laboratories. Ideally and prior to intended use, all SARS-CoV-2 diagnostic assays should be validated to ensure they are fit for purpose. The urgent nature of the pandemic led to expedited assessments of many SARS-CoV-2 tests by regulatory bodies such as the Therapeutic Goods Association in Australia and the Food and Drug Administration in the United States of America, with approvals contingent on the supply of ongoing evidence to support the safety and performance of the assays.2Therapeutic Goods Association COVID-19 testing in Australia - information for health professionals. COVID-19 test performance, Cited 17 Jun 2020https://www.tga.gov.au/covid-19-testing-australia-information-health-professionalsGoogle Scholar, 3Woloshin S. Patel N. Kesselheim A.S. False negative tests for SARS-CoV-2 infection - challenges and implications.N Engl J Med. 2020; 383: e38Crossref PubMed Scopus (566) Google Scholar, 4US Food and Drug Administration Emergency Use Authorization (EUA) information, and list of all current EUAs. Cited 17 Jun 2020.https://www.fda.gov/emergency-preparedness-and-response/mcm-legal-regulatory-and-policy-framework/emergency-use-authorizationGoogle Scholar Initially, the Public Health Laboratory Network (PHLN) Australia recommended that confirmatory testing be performed on samples where SARS-CoV-2 RNA had been detected to ensure that the result was a true positive.5Australian Government Department of Health PHLN guidance on laboratory testing for SARS-CoV-2 (the virus that causes COVID-19).Cited 8 Jun 2020https://www.health.gov.au/resources/publications/phln-guidance-on-laboratory-testing-for-sars-cov-2-the-virus-that-causes-covid-19Google Scholar However, this was not always practical or efficient with substantial testing volumes and in the face of shortages of nucleic acid extraction and testing reagents and consumables. Despite the second wave of infections in Victoria, the prevalence of COVID-19 in Australia remains low (<1%), meaning that in the absence of epidemiological risk factors, the pre-test probability will be low and false positive results will occur even with highly specific NATs. For example, the positive predictive value (PPV) of SARS-CoV-2 NATs with a specificity of 99% is only 50% when the prevalence of infection is 1%.6Australian Government Department of Health PHLN guidance on nucleic acid test result interpretation for SARS-CoV-2.Cited 19 Sep 2020https://www.health.gov.au/resources/publications/phln-guidance-on-nucleic-acid-test-result-interpretation-for-sars-cov-2Google Scholar At the height of the pandemic in Australia in late March, the prevalence of COVID-19 was 2% (range 0.7–3.4% between the different States and Territories),7COVID-19 National Incident Room Surveillance TeamCOVID-19, Australia: Epidemiology Report 9 (Reporting week to 23:59 AEDT 29 March 2020).Commun Dis Intell. 2020; 44https://doi.org/10.33321/cdi.2020.44.29Crossref Scopus (5) Google Scholar indicating that NATs with the same analytical performance will have a PPV of approximately 67% (range 41.2–77.3%). When SARS-CoV-2 was detected by in-house or commercial NATs in NSW Health Pathology (NSWHP) laboratories, samples were sent to NSWHP-Institute of Clinical Pathology and Medical Research, Westmead Hospital, for supplementary testing as part of NSWHP's testing algorithm to minimise false positive results. Supplementary testing was performed using real time reverse-transcriptase polymerase chain reaction (RT-PCR) assays targeting the E, RdRp, M, N, ORF1ab and ORF1b genes8Rahman H. Carter I. Basile K. et al.Interpret with caution: an evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus.J Clin Virol. 2020; 127: 104374Crossref PubMed Scopus (34) Google Scholar using either referred nucleic acid extract and/or nucleic acid re-extracted from the original sample using the MagNA Pure 96 instrument (Roche Diagnostics, Germany). The final result was determined as the consensus of the results from testing the six targets above. Of 122 samples referred from both internal and external laboratories for SARS-CoV-2 confirmatory testing from 14 July to 24 September 2020, we identified a false positive rate of 11% (13/122). Only two of the 13 cases defined as false positive had SARS-CoV-2 serology and/or respiratory tract PCR results available, both testing negative for SARS-CoV-2-specific serology and one patient testing positive for rhinovirus. False positive results may not always be easily identified, and laboratory staff (alone, or in conjunction with clinicians and/or public health physicians) should remain vigilant for their presence. Suspicion should arise if there are discrepant clinico-epidemiological findings (particularly problematic when there are many asymptomatic infections); unexpected laboratory results (such as discordant results where only one SARS-CoV-2 target is detected in assays with multiple targets, and/or RT-PCR results with high cycle threshold values) or contamination (for example, when a batch of samples test positive); incorrect results from external quality assurance programs; warnings from diagnostic companies about potential contaminated assays or reagents9Bustin S.A. Nolan T. RT-qPCR testing of SARS-CoV-2: a primer.Int J Mol Sci. 2020; 21: E3004Crossref PubMed Scopus (120) Google Scholar; or when supplemental NATs on other platforms, the use of different SARS-CoV-2 targets, SARS-CoV-2-specific serology, or genomic sequencing do not concur with the initial NAT result. In the context of Australia's low prevalence of COVID-19 and thus low pre-test probability for infection, we recommend that all positive SARS-CoV-2 NAT results be confirmed by supplementary testing on the original nucleic acid extract and/or re-extraction of nucleic acid from the original sample (if available) and tested using another assay(s) with different gene targets and/or lower limits of detection10US Food and Drug Administration SARS-CoV-2 reference panel comparative data.Cited 21 Sep 2020https://www.fda.gov/medical-devices/coronavirus-covid-19-and-medical-devices/sars-cov-2-reference-panel-comparative-dataGoogle Scholar (Fig. 1). Repeat respiratory tract sampling (including sputum if available), especially in asymptomatic individuals with no identified epidemiological links, is an approach that has been implemented by laboratories following the release of the PHLN guidance document on NAT result interpretation for SARS-CoV-2.6Australian Government Department of Health PHLN guidance on nucleic acid test result interpretation for SARS-CoV-2.Cited 19 Sep 2020https://www.health.gov.au/resources/publications/phln-guidance-on-nucleic-acid-test-result-interpretation-for-sars-cov-2Google Scholar Sera should also be collected for the detection of SARS-CoV-2-specific antibodies (Fig. 1). Serology testing may not always confirm acute SARS-CoV-2 infection (particularly if collected early in the illness course, as SARS-CoV-2-specific antibodies appear around 10 days after disease onset) but can be useful if positive. However, confirmation of infection requires convalescent sera to be collected to demonstrate seroconversion or a four-fold or greater rise in antibody titres between the acute and convalescent samples. Serology may also be used for retrospective diagnosis of SARS-CoV-2 where NAT testing was not performed or was inconclusive. A combination of these approaches will assist with the recognition of reinfection, with SARS-CoV-2-specific antibody assays still to be validated.11European Centre for Disease Prevention and ControlThreat Assessment Brief. Reinfection with SARS-CoV: considerations for public health response.21 Sep 2020https://www.ecdc.europa.eu/sites/default/files/documents/Re-infection-and-viral-shedding-threat-assessment-brief.pdfGoogle Scholar Timely identification of true false positive SARS-CoV-2 NAT results is important as unrecognised false positive results can lead to unnecessary quarantining and contact tracing, delays in the recognition and treatment of the true illness, significant patient anxiety and concern, potential exposure to nosocomial infection from other patients with confirmed COVID-19, wastage of personal protective equipment, and inaccurate statistics regarding local prevalence of infection. The authors state that there are no conflicts of interest to disclose.

•Ct values of E gene were significantly lower than RdRp gene target.•COVID-19 case definition not specific, other respiratory viruses in 42 % of samples.•AusDiagnostics assay sensitive but not specific for the detection of SARS-CoV-2.

There are minimal UK data on the prevalence of genital tract infections in HIV‐infected pregnant women. British HIV Association guidelines suggest sexually transmitted infection (STI) screening as early as possible in pregnancy with consideration given to repeat at 28 weeks’ gestation. A retrospective case notes review of HIV-infected pregnant women at four South London HIV Centres (1 January 2004–1 January 2014) was carried out. Five hundred and ninety-eight pregnancies in 384 patients were identified. Median age 32 years (interquartile range [IQR] 27–36) and 96% (n = 346) were heterosexually infected. HIV was diagnosed antenatally in 21% of pregnancies (n = 107). Seventy-seven per cent of women (n = 384) were of Black African ethnicity and 75% were born in sub-Saharan Africa with 14% UK-born. The majority of pregnancies (279/507) were reported to be unplanned with 42 women proceeding to termination of pregnancy. A regular male partner was reported in 95% of pregnancies (n = 539) with median relationship duration (n = 347) of four years (IQR 1.5–7.0); 11/324 (3.4%) women reported additional sexual partners during the pregnancy. 76.6% (n = 427) of women had an initial STI screen which was done in the first trimester in 52.1%; 32.1% of women had a repeat STI screen in pregnancy, 96% of which was done in the third trimester. Overall, 61 (14.3%) women were diagnosed with at least one STI during their pregnancy. Vaginal candidiasis and bacterial vaginosis were diagnosed in 27.6% (n = 100) and 21.7% (n = 73) of pregnancies, respectively. STI prevalence was low and obstetric outcomes favourable in this cohort of women. Further information about STI prevalence in this population may impact future screening guidelines.

SUMMARY 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.

Introduction Rates of new HIV diagnoses are increasing in Australia, with evidence of an increasing proportion of non-B subtypes reflecting a growing impact of sexual networks, migration and travel. This present study aims to further define HIV-1 subtype diversity and investigate HIV-1 transmission networks within Australia. Methods The Australian Molecular Epidemiology Network (AMEN) HIV collaborating sites in Western Australia, South Australia, Victoria, Queensland and Western Sydney, provided baseline HIV-1 partial pol sequence, age and gender information for a total of 4929 patients during 2005–2012. HIV-1 phylogenetic analyses utilised MEGA V6, with a stringent classification of transmission clusters (bootstrap ≥98%, genetic distance ≤1.5%). Results HIV-1 B subtype represented 74.9% of 4929 sequences (WA 59.3%, SA 68.6%, W Syd 75.2%, Vic 75.7%, Qld 82.3%), with a greater proportion of clusters compared to non-B subtypes (27.6% vs 22.4% of sequences, p = 0.003), larger cluster size (36.0% with >2 sequences vs 24.8% of non-B clusters, p = 0.03) and more male-only groups (90%). The largest cluster comprised 29 B subtype sequences from Vic + WA (age range 23–70 years). HIV-1 subtype C networks (38 groups) included more female/male groups (73.6%) and a smaller proportion of groups >2 (16%), while CRF01_AE networks (44 groups) included 59.1% male-only groups, with groups >2 accounting for 22.7%. Conclusion This nationwide study of HIV-1 sequences involving 4929 patients’ highlights the increasing diversity of HIV-1 subtypes within the Australian epidemic, as well as differences in transmission networks within Australia that are associated with these HIV-1 subtypes. These findings provide epidemiological insights not readily available using standard surveillance methods and can inform the development of effective strategies for prevention of new HIV-1 diagnoses across Australian state boundaries. Disclosure of interest statement None declared.

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.

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.

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.

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 .

A. C. Hurt, K. Hardie, N. J. Wilson, Y. M. Deng, M. Osbourn, S. K. Leang, R. T. C. Lee, P. Iannello, N. Gehrig, R. Shaw, P. Wark, N. Caldwell, R. C. Givney, L. Xue, S. Maurer-Stroh, D. E. Dwyer, B. Wang, D. W. Smith, A. Levy, R. Booy, R. Dixit, T. Merritt, A. Kelso, C. Dalton, D. Durrheim, and I. G. Barr WHO Collaborating Centre for Reference and Research on Influenza, North Melbourne, Victoria; Hunter New England Population Health, Newcastle, New South Wales; Hunter Area Pathology Service, A Division of Pathology North, Newcastle, New South Wales, Australia; Bioinformatics Institute (BII), Agency for Science, Technology and Research (A*STAR), Singapore; Centre for Asthma and Respiratory Disease, University of Newcastle, New South Wales, Australia; School of Biological Sciences, Nanyang Technological University (NTU); National Public Health Laboratory (NPHL), Ministry of Health, Singapore; Centre for Infectious Diseases and Microbiology Laboratory Services, ICPMR, Westmead Hospital and University of Sydney, New South Wales; PathWest Laboratory Medicine, Nedlands, Western Australia; School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, Western Australia; National Centre for Immunisation Research and Surveillance of Vaccine Preventable Disease, Kids Research Institute, The Children’s Hospital at Westmead, New South Wales; Sydney Institute for Emerging infections and Biosecurity (SEIB), University of Sydney, New South Wales, Australia; and Academic Unit of Child Health, Queen Mary University of London, London, United Kingdom

Herpes zoster (also called shingles) is becoming more common as the population ages.It should be part of the differential diagnosis of a localised unilateral vesicular rash, or a pruritic or painful area before the rash appears.Early management with antivirals and analgesia is important and may reduce the incidence of postherpetic neuralgia.Preventing herpes zoster with vaccination is the best way to avoid postherpetic neuralgia and other complications.