Comparison of the Whole Cell Proteome and Secretome of Epidemic Bordetella pertussis Strains From the 2008-2012 Australian Epidemic Under Sulfate-Modulating Conditions.

Sulfate is an important modulator for virulence factor expression in Bordetella pertussis, the causative organism for whooping cough. During infection, sulfate is released when respiratory epithelial cells are damaged which can affect gene expression. The current predominant strains in Australia are found in single nucleotide polymorphism (SNP) cluster I (ptxP3/prn2). It has been reported that ptxP3 strains have higher mRNA expression of virulence genes than ptxP1 strains under intermediate sulfate-modulating conditions (5 mM MgSO4). Our previous proteomic study compared L1423 (cluster I, ptxP3) and L1191 (cluster II, ptxP1) in Thalen-IJssel (THIJS) media without sulfate modulation and identified an upregulation of transport proteins and a downregulation of immunogenic proteins. To determine whether proteomic differences exist between cluster I and cluster II strains in intermediate modulating conditions, this study compared the whole cell proteome and secretome between L1423 and L1191 grown in THIJS media with 5 mM MgSO4 using iTRAQ and high-resolution multiple reaction monitoring (MRM-hr). Two proteins (BP0200 and BP1175) in the whole cell were upregulated in L1423 [fold change (FC) >1.2, false discovery rate (FDR) <0.05]. In the secretome, four proteins from the type III secretion system (T3SS) effectors were downregulated (FC < 0.8, FDR < 0.05) while six proteins, including two adhesins, pertactin (Prn) and tracheal colonization factor A (TcfA), were upregulated which were consistent with our previous proteomic study. The upregulation of Prn and TcfA in SNP cluster I may result in improved adhesion while the downregulation of the T3SS and other immunogenic proteins may reduce immune recognition, which may contribute to the increased fitness of cluster I B. pertussis strains

Translational web robots for pathogen genome analysis

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Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours)

Pertactin-negative and filamentous hemagglutinin-negative Bordetella pertussis, Australia, 2013-2017

During the 2008-2012 pertussis epidemic in Australia, pertactin (Prn)-negative Bordetella pertussis emerged. We analyzed 78 isolates from the 2013-2017 epidemic and documented continued expansion of Prn-negative ptxP3 B. pertussis strains. We also detected a filamentous hemagglutinin- negative and Prn-negative B. pertussis isolate

Enhancing genomics-based outbreak detection of endemic Salmonella enterica serovar Typhimurium using dynamic thresholds.

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens

Community perspectives on the benefits and risks of technologically enhanced communicable disease surveillance systems: a report on four community juries

BACKGROUND:Outbreaks of infectious disease cause serious and costly health and social problems. Two new technologies - pathogen whole genome sequencing (WGS) and Big Data analytics - promise to improve our capacity to detect and control outbreaks earlier, saving lives and resources. However, routinely using these technologies to capture more detailed and specific personal information could be perceived as intrusive and a threat to privacy. METHOD:Four community juries were convened in two demographically different Sydney municipalities and two regional cities in New South Wales, Australia (western Sydney, Wollongong, Tamworth, eastern Sydney) to elicit the views of well-informed community members on the acceptability and legitimacy of: making pathogen WGS and linked administrative data available for public health researchusing this information in concert with data linkage and machine learning to enhance communicable disease surveillance systems Fifty participants of diverse backgrounds, mixed genders and ages were recruited by random-digit-dialling and topic-blinded social-media advertising. Each jury was presented with balanced factual evidence supporting different expert perspectives on the potential benefits and costs of technologically enhanced public health research and communicable disease surveillance and given the opportunity to question experts. RESULTS:Almost all jurors supported data linkage and WGS on routinely collected patient isolates for the purposes of public health research, provided standard de-identification practices were applied. However, allowing this information to be operationalised as a syndromic surveillance system was highly contentious with three juries voting in favour, and one against by narrow margins. For those in favour, support depended on several conditions related to system oversight and security being met. Those against were concerned about loss of privacy and did not trust Australian governments to run secure and effective systems. CONCLUSIONS:Participants across all four events strongly supported the introduction of data linkage and pathogenomics to public health research under current research governance structures. Combining pathogen WGS with event-based data surveillance systems, however, is likely to be controversial because of a lack of public trust, even when the potential public health benefits are clear. Any suggestion of private sector involvement or commercialisation of WGS or surveillance data was unanimously rejected.Chris Degeling, Stacy M. Carter, Antoine M. van Oijen, Jeremy McAnulty, Vitali Sintchenko, Annette Braunack-Mayer ... et al

SARS-CoV-2 neutralizing antibodies: Longevity, breadth, and evasion by emerging viral variants.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) antibody neutralization response and its evasion by emerging viral variants and variant of concern (VOC) are unknown, but critical to understand reinfection risk and breakthrough infection following vaccination. Antibody immunoreactivity against SARS-CoV-2 antigens and Spike variants, inhibition of Spike-driven virus-cell fusion, and infectious SARS-CoV-2 neutralization were characterized in 807 serial samples from 233 reverse transcription polymerase chain reaction (RT-PCR)-confirmed Coronavirus Disease 2019 (COVID-19) individuals with detailed demographics and followed up to 7 months. A broad and sustained polyantigenic immunoreactivity against SARS-CoV-2 Spike, Membrane, and Nucleocapsid proteins, along with high viral neutralization, was associated with COVID-19 severity. A subgroup of "high responders" maintained high neutralizing responses over time, representing ideal convalescent plasma donors. Antibodies generated against SARS-CoV-2 during the first COVID-19 wave had reduced immunoreactivity and neutralization potency to emerging Spike variants and VOC. Accurate monitoring of SARS-CoV-2 antibody responses would be essential for selection of optimal responders and vaccine monitoring and design

Detection of viral respiratory pathogens in mild and severe acute respiratory infections in Singapore.

To investigate the performance of laboratory methods and clinical case definitions in detecting the viral pathogens for acute respiratory infections (ARIs) from a prospective community cohort and hospital inpatients, nasopharyngeal swabs from cohort members reporting ARIs (community-ARI) and inpatients admitted with ARIs (inpatient-ARI) were tested by Singleplex Real Time-Polymerase Chain Reaction (SRT-PCR), multiplex RT-PCR (MRT-PCR) and pathogen-chip system (PathChip) between April 2012 and December 2013. Community-ARI and inpatient-ARI was also combined with mild and severe cases of influenza from a historical prospective study as mild-ARI and severe-ARI respectively to evaluate the performance of clinical case definitions. We analysed 130 community-ARI and 140 inpatient-ARI episodes (5 inpatient-ARI excluded because multiple pathogens were detected), involving 138 and 207 samples respectively. Detection by PCR declined with days post-onset for influenza virus; decrease was faster for community-ARI than for inpatient-ARI. No such patterns were observed for non-influenza respiratory virus infections. PathChip added substantially to viruses detected for community-ARI only. Clinical case definitions discriminated influenza from other mild-ARI but performed poorly for severe-ARI and for older participants. Rational strategies for diagnosis and surveillance of influenza and other respiratory virus must acknowledge the differences between ARIs presenting in community and hospital settings

Infectious Disease Ontology

Technological developments have resulted in tremendous increases in the volume and diversity of the data and information that must be processed in the course of biomedical and clinical research and practice. Researchers are at the same time under ever greater pressure to share data and to take steps to ensure that data resources are interoperable. The use of ontologies to annotate data has proven successful in supporting these goals and in providing new possibilities for the automated processing of data and information. In this chapter, we describe different types of vocabulary resources and emphasize those features of formal ontologies that make them most useful for computational applications. We describe current uses of ontologies and discuss future goals for ontology-based computing, focusing on its use in the field of infectious diseases. We review the largest and most widely used vocabulary resources relevant to the study of infectious diseases and conclude with a description of the Infectious Disease Ontology (IDO) suite of interoperable ontology modules that together cover the entire infectious disease domain