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.

A One Health investigation of Salmonella enterica serovar Wangata in north-eastern New South Wales, Australia, 2016-2017.

Salmonella enterica serovar Wangata (S. Wangata) is an important cause of endemic salmonellosis in Australia, with human infections occurring from undefined sources. This investigation sought to examine possible environmental and zoonotic sources for human infections with S. Wangata in north-eastern New South Wales (NSW), Australia. The investigation adopted a One Health approach and was comprised of three complimentary components: a case-control study examining human risk factors; environmental and animal sampling; and genomic analysis of human, animal and environmental isolates. Forty-eight human S. Wangata cases were interviewed during a 6-month period from November 2016 to April 2017, together with 55 Salmonella Typhimurium (S. Typhimurium) controls and 130 neighbourhood controls. Indirect contact with bats/flying foxes (S. Typhimurium controls (adjusted odds ratio (aOR) 2.63, 95% confidence interval (CI) 1.06-6.48)) (neighbourhood controls (aOR 8.33, 95% CI 2.58-26.83)), wild frogs (aOR 3.65, 95% CI 1.32-10.07) and wild birds (aOR 6.93, 95% CI 2.29-21.00) were statistically associated with illness in multivariable analyses. S. Wangata was detected in dog faeces, wildlife scats and a compost specimen collected from the outdoor environments of cases' residences. In addition, S. Wangata was detected in the faeces of wild birds and sea turtles in the investigation area. Genomic analysis revealed that S. Wangata isolates were relatively clonal. Our findings suggest that S. Wangata is present in the environment and may have a reservoir in wildlife populations in north-eastern NSW. Further investigation is required to better understand the occurrence of Salmonella in wildlife groups and to identify possible transmission pathways for human infections.

Epidemiology and whole genome sequencing of an ongoing point-source Salmonella Agona outbreak associated with sushi consumption in western Sydney, Australia 2015.

During May 2015, an increase in Salmonella Agona cases was reported from western Sydney, Australia. We examine the public health actions used to investigate and control this increase. A descriptive case-series investigation was conducted. Six outbreak cases were identified; all had consumed cooked tuna sushi rolls purchased within a western Sydney shopping complex. Onset of illness for outbreak cases occurred between 7 April and 24 May 2015. Salmonella was isolated from food samples collected from the implicated premise and a prohibition order issued. No further cases were identified following this action. Whole genome sequence (WGS) analysis was performed on isolates recovered during this investigation, with additional S. Agona isolates from sporadic-clinical cases and routine food sampling in New South Wales, January to July 2015. Clinical isolates of outbreak cases were indistinguishable from food isolates collected from the implicated sushi outlet. Five additional clinical isolates not originally considered to be linked to the outbreak were genomically similar to outbreak isolates, indicating the point-source contamination may have started before routine surveillance identified an increase. This investigation demonstrated the value of genomics-guided public health action, where near real-time WGS enhanced the resolution of the epidemiological investigation.

Pooling sputum samples to improve the feasibility of Xpert® MTB/RIF in systematic screening for tuberculosis.

SETTING: Systematic screening for tuberculosis (TB) using Xpert® MTB/RIF. OBJECTIVE: To determine whether pooling sputum samples for Xpert testing may improve the feasibility and cost-effectiveness of Xpert by reducing the number of Xpert tests required. DESIGN: Mycobacterium tuberculosis-spiked sputum samples at low organism concentrations were used to mimic samples that are more likely to be found in the screening, compared to the diagnostic, setting. Using Xpert, pooled sputum samples were tested from a pooling ratio of 1 in 2 to 1 in 12. RESULTS: A linear relationship between the pooling ratio and the Xpert MTB cycle threshold (Ct) value was found. As the sputum pooling ratio increased, the Ct value also increased. However, the slope of this increase was relatively small. In the majority of the samples pooled (75/96, 78.1%), Xpert was able to detect M. tuberculosis. CONCLUSION: These findings suggest that sputum pooling may be a viable method of improving the feasibility and cost-effectiveness of large-scale sputum testing using Xpert in the TB screening setting.

Identification of genetic markers of resistance to echinocandins, azoles and 5-fluorocytosine in Candida glabrata by next-generation sequencing: a feasibility study.

OBJECTIVES: Multi-antifungal drug resistance in Candida glabrata is increasing. We examined the feasibility of next-generation sequencing (NGS) to investigate the presence of antifungal drug resistance markers in C. glabrata. METHODS: The antifungal susceptibility of 12 clinical isolates and one ATCC strain of C. glabrata was determined using the Sensititre YeastOne® YO10 assay. These included three isolate pairs where the second isolate of each pair had developed a rise in drug MICs. Single nucleotide polymorphisms (SNPs) in genes known to be linked to echinocandin, azole and 5-fluorocytosine resistance were analysed in all isolates through NGS. RESULTS: High-quality non-synonymous SNPs in antifungal resistance genes such as FKS1, FKS2, CgCDR1, CgPDR1 and FCY2 were identified. For two of three isolate pairs, there was a >60-fold rise in MICs to all echinocandins in the second isolate from each pair; one echinocandin-resistant isolate harboured a mutation in FKS1 (S629P) and the other in FKS2 (S663P). Of the third pair, both the 5-fluorocytosine-susceptible, and resistant isolates had a mutation in FCY2 (A237T). SNPs in CgPDR1 were found in pan-azole-resistant isolates. SNPs in other genes linked to azole resistance (CgCDR1, ERG9 and CgFLR1) were present in both azole-susceptible and azole-resistant isolates. SNPs were also identified in Candida adhesin genes EPA1, EPA6, PWP2 and PWP5 but their presence was not associated with higher drug MICs. CONCLUSIONS: Genome-wide analysis of antifungal resistance markers was feasible and simultaneously revealed mutation patterns of genes implicated in resistance to different antifungal drug classes.

Tuberculosis and HIV co-infection in Vietnam.

UNLABELLED: Tuberculosis (TB) and human immunodeficiency virus (HIV) infection are leading causes of disease and death in Vietnam, but TB/HIV disease trends and the profile of co-infected patients are poorly described. METHODS: We examined national TB and HIV notification data to provide a geographic overview and describe relevant disease trends within Vietnam. We also compared the demographic and clinical profiles of TB patients with and without HIV infection. RESULTS: During the past 10 years (2005-2014) cumulative HIV case numbers and deaths increased to 298,151 and 71,332 respectively, but access to antiretroviral therapy (ART) improved and new infections and deaths declined. From 2011-2014 routine HIV testing of TB patients increased from 58.9% to 72.5% and of all TB patients diagnosed with HIV in 2014, 2,803 (72.4%) received ART. The number of multidrug resistant (MDR)-TB cases enrolled for treatment increased almost 3-fold (578 to 1,532) from 2011-2014. The rate of HIV co-infection in MDR and non-MDR TB cases (51/1,532; 3.3% vs 3,774/100,555; 3.8%; OR 0.77, 95% CI 0.7-1.2) was similar in 2014. CONCLUSIONS: The care of TB/HIV co-infected patients have shown sustained improvement in Vietnam. Rising numbers of MDR-TB cases is a concern, but this is not "driven" by HIV co-infection.

Spatiotemporal evidence for cross-border spread of MDR-TB along the Trans-Siberian Railway line.

BACKGROUND: Mongolia has the fifth highest incidence of tuberculosis (TB) in the Western Pacific Region, with high rates of multidrug-resistant TB (MDR-TB). OBJECTIVE: To examine the recent spatiotemporal dynamics of MDR-TB in Mongolia. METHODS: All MDR-TB cases diagnosed from 2004 to 2012, identified from the National Tuberculosis Control Programme database, were included in the study. Cases diagnosed from 2006 to 2012 were further examined using spatial scan statistics. RESULTS: Few MDR-TB cases (n = 29) were diagnosed before the programmatic management of MDR-TB was introduced in 2006. During 2006-2012, 1106 MDR-TB cases were detected, at an annualised rate of 5.9 cases per 100 000 population. Most (>80%) cases were identified in the 15-44 year age group; 45% were among those aged 15-29 years. Case notification rates were highest in the capital city, Ulaanbaatar, with an increasing trend over time in all locations. Three MDR-TB hotspots were identified, all in close proximity to the Trans-Siberian Railway line. The majority of the MDR-TB isolates were resistant to all first-line drugs tested. CONCLUSION: Spatiotemporal analysis indicates likely cross-border spread of MDR-TB along the Trans-Siberian Railway line, with subsequent spatial expansion across Mongolia. The frequency of MDR-TB among young patients with pan-resistance to all first-line drugs suggests ongoing MDR-TB transmission within the community.

Multidrug-resistant tuberculosis in New South Wales, Australia, 1999-2010: a case series report.

SETTING: The emergence of multidrug-resistant tuberculosis (MDR-TB) threatens the ongoing control of tuberculosis (TB). The Australian state of New South Wales (NSW) has low TB and MDR-TB incidence. OBJECTIVE: To examine the epidemiology and the clinical and public health management of MDR-TB in NSW. DESIGN: A retrospective case-series analysis of MDR-TB diagnosed in NSW between 1999 and 2010 was undertaken. A standardised questionnaire was used to collect information from the public health surveillance system, medical records and the State Mycobacterium Reference Laboratory about clinical features, drug susceptibility, treatment regimens, hospitalisation, risk factors for tuberculous infection, contact tracing and patient outcomes. RESULTS: Fifty-five cases of culture-confirmed MDR-TB, including two cases of extensively drug-resistant TB, were diagnosed. All cases were reviewed by an expert management panel. Fifty cases (91%) were foreign-born, and 50 cases (91%) had fully supervised treatment. Of the 55 cases, 46 (84%) successfully completed treatment, 3 (5%) died of TB and 3 (5%) required surgery. No MDR-TB cases were reported among contacts. CONCLUSION: Using a multidisciplinary, expert guided, case-management approach, the NSW TB Control Program achieved excellent MDR-TB outcomes. The impact of global increases in MDR-TB requires sustained commitment to TB in all settings.

Tuberculosis and HIV co-infection-focus on the Asia-Pacific region.

Tuberculosis (TB) is the leading opportunistic disease and cause of death in patients with HIV infection. In 2013 there were 1.1 million new TB/HIV co-infected cases globally, accounting for 12% of incident TB cases and 360,000 deaths. The Asia-Pacific region, which contributes more than a half of all TB cases worldwide, traditionally reports low TB/HIV co-infection rates. However, routine testing of TB patients for HIV infection is not universally implemented and the estimated prevalence of HIV in new TB cases increased to 6.3% in 2013. Although HIV infection rates have not seen the rapid rise observed in Sub-Saharan Africa, indications are that rates are increasing among specific high-risk groups. This paper reviews the risks of TB exposure and progression to disease, including the risk of TB recurrence, in this vulnerable population. There is urgency to scale up interventions such as intensified TB case-finding, isoniazid preventive therapy, and TB infection control, as well as HIV testing and improved access to antiretroviral treatment. Increased awareness and concerted action is required to reduce TB/HIV co-infection rates in the Asia-Pacific region and to improve the outcomes of people living with HIV.

Whole genome sequencing in clinical and public health microbiology.

Genomics and whole genome sequencing (WGS) have the capacity to greatly enhance knowledge and understanding of infectious diseases and clinical microbiology.The growth and availability of bench-top WGS analysers has facilitated the feasibility of genomics in clinical and public health microbiology.Given current resource and infrastructure limitations, WGS is most applicable to use in public health laboratories, reference laboratories, and hospital infection control-affiliated laboratories.As WGS represents the pinnacle for strain characterisation and epidemiological analyses, it is likely to replace traditional typing methods, resistance gene detection and other sequence-based investigations (e.g., 16S rDNA PCR) in the near future.Although genomic technologies are rapidly evolving, widespread implementation in clinical and public health microbiology laboratories is limited by the need for effective semi-automated pipelines, standardised quality control and data interpretation, bioinformatics expertise, and infrastructure.

Transmission of Mycobacterium tuberculosis from an Asian elephant (Elephas maximus) to a chimpanzee (Pan troglodytes) and humans in an Australian zoo.

Mycobacterium tuberculosis is primarily a pathogen of humans. Infections have been reported in animal species and it is emerging as a significant disease of elephants in the care of humans. With the close association between humans and animals, transmission can occur. In November 2010, a clinically healthy Asian elephant in an Australian zoo was found to be shedding M. tuberculosis; in September 2011, a sick chimpanzee at the same zoo was diagnosed with tuberculosis caused by an indistinguishable strain of M. tuberculosis. Investigations included staff and animal screening. Four staff had tuberculin skin test conversions associated with spending at least 10 hours within the elephant enclosure; none had disease. Six chimpanzees had suspected infection. A pathway of transmission between the animals could not be confirmed. Tuberculosis in an elephant can be transmissible to people in close contact and to other animals more remotely. The mechanism for transmission from elephants requires further investigation.

Ureaplasma urealyticum is significantly associated with non-gonococcal urethritis in heterosexual Sydney men.

We investigated the prevalence of various genital organisms in 268 men with (cases) and 237 men without (controls) urethral symptoms/signs (urethral discharge, dysuria and/or urethral irritation) from two sexual health clinics in Sydney between April 2006 and November 2007. The presence of urethral symptoms/signs was defined as non-gonococcal urethritis (NGU) for this study. Specific aims were to investigate the role of Ureaplasma urealyticum in NGU and the prevalence of Mycoplasma genitalium in our population. Multiplex polymerase chain reaction-based reverse line blot (mPCR/RLB) assay was performed to detect 14 recognized or putative genital pathogens, including Chlamydia trachomatis, M. genitalium, U. urealyticum and U. parvum. U. urealyticum was associated with NGU in men without another urethral pathogen (odds ratio [OR] 2.0, 95% confidence interval [CI] 1.1-3.8; P = 0.04); this association remained after controlling for potential confounding by age and history of unprotected vaginal sex in the last four weeks (OR 2.0, 95% CI: 1.1-3.9; P = 0.03). C. trachomatis (OR 7.5, P < 0.001) and M. genitalium (OR 5.5, P = 0.027) were significantly associated with NGU. The prevalence of M. genitalium was low (4.5% cases, 0.8% controls). U. urealyticum is independently associated with NGU in men without other recognized urethral pathogens. Further research should investigate the role of U. urealyticum subtypes among heterosexual men with NGU.

The changing nature of clinical decision support systems: a focus on consumers, genomics, public health and decision safety.

OBJECTIVES: To review the recent research literature in clinical decision support systems (CDSS). METHODS: A review of recent literature was undertaken, focussing on CDSS evaluation, consumers and public health, the impact of translational bioinformatics on CDSS design, and CDSS safety. RESULTS: In recent years, researchers have concentrated much less on the development of decision technologies, and have focussed more on the impact of CDSS in the clinical world. Recent work highlights that traditional process measures of CDSS effectiveness, such as document relevance are poor proxy measures for decision outcomes. Measuring the dynamics of decision making, for example via decision velocity, may produce a more accurate picture of effectiveness. Another trend is the broadening of user base for CDSS beyond front line clinicians. Consumers are now a major focus for biomedical informatics, as are public health officials, tasked with detecting and managing disease outbreaks at a health system, rather than individual patient level. Bioinformatics is also changing the nature of CDSS. Apart from personalisation of therapy recommendations, translational bioinformatics is creating new challenges in the interpretation of the meaning of genetic data. Finally, there is much recent interest in the safety and effectiveness of computerised physician order entry (CPOE) systems, given that prescribing and administration errors are a significant cause of morbidity and mortality. Of note, there is still much controversy surrounding the contention that poorly designed, implemented or used CDSS may actually lead to harm. CONCLUSIONS: CDSS research remains an active and evolving area of research, as CDSS penetrate more widely beyond their traditional domain into consumer decision support, and as decisions become more complex, for example by involving sequence level genetic data.

The Centre for Health Informatics at the University of New South Wales--a clinical informatics research centre.

OBJECTIVES: Building a sustainable health system in the 21st Century will require the reinvention of much of the present day system, and the intelligent use of information and communication technologies (ICT) to deliver high quality, safe, efficient and affordable health care. The Centre for Health Informatics (CHI) is Australia's largest academic research group in this emerging discipline. METHODS: Our research is underpinned by a planning process, based on different future scenarios for the health system, which helps us identify longer-term problems needing a sustained research effort. A research competency matrix is used to ensure that the Centre has the requisite core capabilities in the research methods and tools needed to pursue our research program. RESULTS: The Centre's work is internationally recognized for its contributions in the development of intelligent search systems to support evidence-based healthcare, developing evaluation methodologies for ICT, and in understanding how communication shapes the safety and quality of health care delivery. Centre researchers also are working on safety models and standards for ICT in healthcare, mining complex gene micro array, medical literature and medical record data, building health system simulation methods to model the impact of health policy changes, and developing novel computational methods to automate the diagnosis of 3-D medical images. CONCLUSIONS: Any individual research group like CHI must necessarily focus on a few areas to allow it to develop sufficient research capacity to make novel and internationally significant contributions. As CHI approaches the end of its first decade, it is becoming clear that developing capacity becomes increasingly challenging as the research territory changes under our feet, and that the Centre will continue to evolve and shift its focus in the years to come.

Treat or test first? Decision analysis of empirical antiviral treatment of influenza virus infection versus treatment based on rapid test results.

BACKGROUND: neuraminidase (NA) inhibitors have recently become available for treatment of influenza. Rapid antigen detection assays at 'point-of-care' may improve the accuracy of clinical diagnosis, but the value of these techniques in assisting with the appropriate use of antivirals remains controversial. OBJECTIVE: to compare the diagnostic utilities of two management strategies for influenza, empirical antiviral therapy versus therapy based on a positive rapid test result in pre-epidemic and epidemic periods. STUDY DESIGN: a threshold decision analytic model was designed to compare these competing strategies and sensitivity analysis performed to examine the impact of diagnostic variables on the expected utility of the decision with a range of prior probabilities of infection between 1 and 50%. RESULTS: on the basis of the calculated sensitivity (77%) and specificity (95%) of a point-of-care test for influenza, pre-treatment testing was preferred and cost-effective in non-epidemic stage of the influenza cycle. The alternative strategy of empirical treatment produces a higher utility value during epidemics, but may result in overuse of antivirals for low-risk populations. The two strategies had equivalent efficacy when the probability of influenza was 42%. CONCLUSIONS: Patients with flu-like illness, who present outside the influenza outbreak and are considered to be at low risk for influenza-related complications, should be tested to confirm the diagnosis before starting antiviral treatment with a NA inhibitor. The most important variables in the model were the accuracy of the clinical diagnosis and the pre-test probability of influenza. A threshold probability of influenza of 42% would dictate changing from the rapid testing strategy to a 'treat regardless' strategy.

What do physicians think about evidence-based antibiotic use in critical care? A survey of Australian intensivists and infectious disease practitioners.

BACKGROUND: The analysis of factors that influence prescribing decisions is increasingly important. Antibiotic use is often based on limited evidence and lack of information about clinical decision-making processes is an important obstacle to improving antibiotic utilization. AIMS: To compare the attitudes of intensive care unit practitioners (ICUP) and infectious disease practitioners (IDP) to antibiotic use and to the evidence-based information support. METHOD: A postal survey conducted between March and July 2000 of ICUP and IDP representing all States and Territories in Australia. RESULTS: One hundred and fifty-three of 224 clinicians returned the questionnaire (68.3% response rate). In choosing an antibiotic, IDP placed significantly more weight than ICUP on the in vitro susceptibility of the pathogen (P = 0.001), antibiotic cost (P = 0.05) and possible development of antibiotic resistance (P = 0.007). More than 95% of both groups believed that unit-specific antibiotic susceptibility of endemic pathogens was an essential factor in rational prescribing, but only 68.5% of IDP and 38.7% of ICUP use microbiology laboratory databases. When in doubt about appropriate antibiotic use, 63.8% of ICUP seek and 76.3% usually follow the advice of IDP. Both groups agree that published antibiotic guidelines are useful, but IDP were more likely to consult them. ICUP were more likely to believe that guidelines are used to control clinicians rather than to improve quality of care (P = 0.001). A greater proportion of IDP (71.2%) than ICUP (52.5%) believed that antibiotic prescribing in their intensive care unit (ICU) was evidence based but most (91.8% and 86.9%, respectively) agreed that it should be. CONCLUSIONS: Australian clinicians have positive views about evidence-based prescribing and antibiotic guidelines. However, there are clinically significant differences in prescribing behaviour between ICUP and IDP. These may be explained by different disease spectra managed by each group or different cultures, training and/or cognitive styles. Improvements in the understanding of physicians' information and decision support needs are required to strengthen evidence-based prescribing.