Genome entropy and network centrality contrast exploration and exploitation in evolution of foodborne pathogens.

Modelling evolution of foodborne pathogens is crucial for mitigation and prevention of outbreaks. We apply network-theoretic and information-theoretic methods to trace evolutionary pathways ofSalmonellaTyphimurium in New South Wales, Australia, by studying whole genome sequencing surveillance data over a five-year period which included several outbreaks. The study derives both undirected and directed genotype networks based on genetic proximity, and relates the network's structural property (centrality) to its functional property (prevalence). The centrality-prevalence space derived for the undirected network reveals a salient exploration-exploitation distinction across the pathogens, further quantified by the normalised Shannon entropy and the Fisher information of the corresponding shell genome. This distinction is also analysed by tracing the probability density along evolutionary paths in the centrality-prevalence space. We quantify the evolutionary pathways, and show that pathogens exploring the evolutionary search-space during the considered period begin to exploit their environment (their prevalence increases resulting in outbreaks), but eventually encounter a bottleneck formed by epidemic containment measures.

SABRes: in silico detection of drug resistance conferring mutations in subpopulations of SARS-CoV-2 genomes.

The emergence of resistance to antiviral drugs increasingly used to treat SARS-CoV-2 infections has been recognised as a significant threat to COVID-19 control. In addition, some SARS-CoV-2 variants of concern appear to be intrinsically resistant to several classes of these antiviral agents. Therefore, there is a critical need for rapid recognition of clinically relevant polymorphisms in SARS-CoV-2 genomes associated with significant reduction of drug activity in virus neutralisation experiments. Here we present SABRes, a bioinformatic tool, which leverages on expanding public datasets of SARS-CoV-2 genomes and allows detection of drug resistance mutations in consensus genomes as well as in viral subpopulations. We have applied SABRes to detect resistance-conferring mutations in 25,197 genomes generated over the course of the SARS-CoV-2 pandemic in Australia and identified 299 genomes containing resistance conferring mutations to the five antiviral therapeutics that retain effectiveness against currently circulating strains of SARS-CoV-2 - Sotrovimab, Bebtelovimab, Remdesivir, Nirmatrelvir and Molnupiravir. These genomes accounted for a 1.18% prevalence of resistant isolates discovered by SABRes, including 80 genomes with resistance conferring mutations found in viral subpopulations. Timely recognition of these mutations within subpopulations is critical as these mutations can provide an advantage under selective pressure and presents an important step forward in our ability to monitor SARS-CoV-2 drug resistance.

Epidemiologic Evidence for Airborne Transmission of SARS-CoV-2 during Church Singing, Australia, 2020.

An outbreak of severe acute respiratory syndrome coronavirus 2 infection occurred among church attendees after an infectious chorister sang at multiple services. We detected 12 secondary case-patients. Video recordings of the services showed that case-patients were seated in the same section, up to 15 m from the primary case-patient, without close physical contact, suggesting airborne transmission.

Genomic Surveillance Enables Suitability Assessment of Salmonella Gene Targets Used for Culture-Independent Diagnostic Testing.

Salmonella is a highly diverse genus consisting of over 2,600 serovars responsible for high-burden food- and waterborne gastroenteritis worldwide. Sensitivity and specificity of PCR-based culture-independent diagnostic testing (CIDT) systems for Salmonella, which depend on a highly conserved gene target, can be affected by single nucleotide polymorphisms (SNPs), indels, and genomic rearrangements within primer and probe sequences. This report demonstrates the value of prospectively collected genomic data for verifying CIDT targets. We utilized the genomes of 3,165 Salmonella isolates prospectively collected and sequenced in Australia. The sequences of Salmonella CIDT PCR gene targets (ttrA, spaO, and invA) were systematically interrogated to measure nucleotide dissimilarity. Analysis of 52 different serovars and 79 multilocus sequencing types (MLST) demonstrated dissimilarity within and between PCR gene targets ranging between 0 and 81.3 SNP/kbp (0 and 141 SNPs). The lowest average dissimilarity was observed in the ttrA target gene used by the Roche LightMix at 2.0 SNP/kbp (range, 0 to 46.7); however, entropy across the gene demonstrates that it may not be the most stable CIDT target. While debate continues over the benefits and pitfalls of replacing bacterial culture with molecular assays, the growing volumes of genomic surveillance data enable periodic regional reassessment and validation of CIDT targets against both prevalent and emerging serovars. If PCR systems are to become the primary screening and diagnostic tool for laboratory diagnosis of salmonellosis, ongoing monitoring of the genomic diversity in PCR target regions is warranted, as is the potential inclusion of two Salmonella PCR targets in frontline diagnostic systems.

Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement.

Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short ß-hairpin, and, although the structural fold is similar to that of PfRh5--the essential invasion ligand in Plasmodium falciparum--its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection.

A Registry for Patients With Asplenia/Hyposplenism Reduces the Risk of Infections With Encapsulated Organisms.

Background: Overwhelming post-splenectomy infection (OPSI) is a serious complication of asplenia. Clinical guidelines recommend numerous measures to reduce the risk of OPSI, but awareness and adherence to preventative measures are generally poor. We aimed to determine whether a registry for asplenic/hyposplenic patients was associated with a reduction in the incidence of infection with encapsulated bacteria. Methods: We performed a retrospective cohort study of asplenic/hyposplenic patients in the state of Victoria, Australia, who registered with Spleen Australia from 2003 through 2014. Spleen Australia provides education, clinical guidance, and annual vaccination reminders to registrants and their healthcare providers. We compared the incidence of infection with Streptococcus pneumoniae, Haemophilus influenzae type B (Hib), and Neisseria meningitidis before and after registration. Registry data were linked with Victorian notifiable disease data on invasive pneumococcal disease (IPD), invasive meningococcal disease (IMD), and Hib between 2000 and 2014. Results: Twenty-seven cases of IPD and 1 of IMD occurred among 3221 registrants. No cases of Hib were reported. The rate of IPD/IMD was 150 per 100000 patient-years prior to registration and 36 per 100000 patient-years after registration; registration was associated with a 69% reduction in the risk of infection (incidence rate ratio, 0.31; 95% confidence interval, 0.12 to 0.83; P = .019). Based on the absolute reduction in incidence, we estimate that Spleen Australia prevents 5-6 invasive infections with encapsulated organisms annually among registrants. Conclusions: Systematic, long-term approaches to post-splenectomy care can significantly reduce the risk of infection with encapsulated organisms among individuals with asplenia/hyposplenism.

Novel Salmonella enterica Serovar Typhimurium Genotype Levels as Herald of Seasonal Salmonellosis Epidemics.

We examined the population dynamics of Salmonella enterica serovar Typhimurium during seasonal salmonellosis epidemics in New South Wales, Australia, during 2009-2016. Of 15,626 isolates, 5%-20% consisted of novel genotypes. Seasons with salmonellosis epidemics were associated with a reduction in novel genotypes in the preceding winter and spring.

Multidrug-Resistant Salmonella enterica 4,[5],12:i:- Sequence Type 34, New South Wales, Australia, 2016-2017.

Multidrug- and colistin-resistant Salmonella enterica serotype 4,[5],12:i:- sequence type 34 is present in Europe and Asia. Using genomic surveillance, we determined that this sequence type is also endemic to Australia. Our findings highlight the public health benefits of genome sequencing-guided surveillance for monitoring the spread of multidrug-resistant mobile genes and isolates.

Distinct patterns of diversity, population structure and evolution in the AMA1 genes of sympatric Plasmodium falciparum and Plasmodium vivax populations of Papua New Guinea from an area of similarly high transmission.

BACKGROUND: As Plasmodium falciparum and Plasmodium vivax co-exist in most malaria-endemic regions outside sub-Saharan Africa, malaria control strategies in these areas must target both species in order to succeed. Population genetic analyses can predict the effectiveness of interventions including vaccines, by providing insight into patterns of diversity and evolution. The aim of this study was to investigate the population genetics of leading malaria vaccine candidate AMA1 in sympatric P. falciparum and P. vivax populations of Papua New Guinea (PNG), an area of similarly high prevalence (Pf = 22.3 to 38.8%, Pv = 15.3 to 31.8%). METHODS: A total of 72 Pfama1 and 102 Pvama1 sequences were collected from two distinct areas, Madang and Wosera, on the highly endemic PNG north coast. RESULTS: Despite a greater number of polymorphic sites in the AMA1 genes of P. falciparum (Madang = 52; Wosera = 56) compared to P. vivax (Madang = 36, Wosera = 34), the number of AMA1 haplotypes, haplotype diversity (Hd) and recombination (R) was far lower for P. falciparum (Madang = 12, Wosera = 20; Hd ≤0.92, R ≤45.8) than for P. vivax (Madang = 50, Wosera = 38; Hd = 0.99, R = ≤70.9). Balancing selection was detected only within domain I of AMA1 for P. vivax, and in both domains I and III for P. falciparum. CONCLUSIONS: Higher diversity in the genes encoding P. vivax AMA1 than in P. falciparum AMA1 in this highly endemic area has important implications for development of AMA1-based vaccines in PNG and beyond. These results also suggest a smaller effective population size of P. falciparum compared to P. vivax, a finding that warrants further investigation. Differing patterns of selection on the AMA1 genes indicate that critical antigenic sites may differ between the species, highlighting the need for independent investigations of these two leading vaccine candidates.

Comparison of outcomes between intracytoplasmic sperm injection and in vitro fertilization inseminations with preimplantation genetic testing for aneuploidy, analysis of Society for Assisted Reproductive Technology Clinic Outcome Reporting System data.

OBJECTIVE: To evaluate whether insemination via intracytoplasmic sperm injection (ICSI) provides any benefit over in vitro fertilization (IVF) insemination for nonmale factor infertility with respect to preimplantation genetic testing (PGT) results and pregnancy outcome. DESIGN: Retrospective cohort study of the Society for Assisted Reproductive Technology database. SETTINGS: US-based fertility clinics reporting to the Society for Assisted Reprodcutive Technology. PATIENTS: Patients undergoing IVF or ICSI inseminations in nonmale factor PGT for aneuploidy cycles. INTERVENTION: In vitro fertilization vs. ICSI inseminations. MAIN OUTCOME MEASURES: Primary outcomes were the percentage of embryos suitable for transfer and live birth rates (LBRs). Secondary outcomes included subgroup analysis for embryos suitable for transfer on cycles from patients ≥35-year-old vs. <35-year-old, ≤6 oocytes retrieved vs. >6 oocytes retrieved, and unexplained infertility. Additionally, gestational age at delivery and birth weight between IVF and ICSI inseminations were evaluated. RESULTS: A total of 30,446 nonmale factor PGT diagnoses for aneuploidy cycles were evaluated, of which 4,867 were IVF inseminations and 25,579 were ICSI inseminations. Following exclusion criteria and adjustment for any necessary confounding variables, no significant differences existed in embryos suitable for transfer between IVF and ICSI treatment cycles, 41.6% (40.6%, 42.6%) vs. 42.5% (42.0%, 42.9%), respectively, or in LBRs, 50.1% (37.8, 62.4%) vs. 50.8% (38.5%, 62.9%), respectively. CONCLUSION: There were no significant differences in the rates of embryos suitable for transfer and LBRs between IVF and ICSI inseminations in nonmale factor cycles undergoing PGT for aneuploidy.

First documented gymnasium cluster of COVID-19 with whole genome sequencing in Australia.

Background: Transmission of coronavirus disease 2019 (COVID-19) has been demonstrated in fitness settings internationally. We report the first documented case of transmission of COVID-19 in a gymnasium in Australia in 2020. Methods: Case finding and case interviews were conducted among attendees in a Western Sydney gymnasium, Australia. Whole genome sequencing using an amplicon-based approach was performed on all SARS CoV-2 polymerase chain reaction positive samples detected through surveillance. Results: We show that five cases of COVID-19 were linked to the gymnasium, with transmission occurring on 7 July 2020, when the index case transmitted the infection to four other gymnasium attendees through the sharing of an enclosed space. Conclusions: There is an ongoing risk of transmission of COVID-19 within gymnasium environments and they are justifiably classified as a 'high-risk' venue. There may be a need to expand ventilation and space requirements to prevent transmission of COVID-19 in such settings in the context of severe COVID-19 variants or to prevent respiratory disease transmission in general.

Emergent Omicron BR.2.1 sublineage of SARS-CoV-2 in New South Wales, Australia: a subvariant with high fitness but without increased disease severity.

OBJECTIVES: To describe the epidemiology and impact of Omicron BR.2.1, an emergent SARS-CoV-2 Omicron BA.2.75 sublineage displaying high fitness compared to other cocirculating subvariants in New South Wales, Australia. METHODS: From September 01 to November 26, 2022, 4971 SARS-CoV-2 consensus genomes from unique patients were generated, and correlated with international travel and reinfection history, and admission to the intensive care unit. RESULTS: BR.2.1 became the predominant variant by late November, and was responsible for a significantly higher proportion of community-acquired cases during the study period (55.1% vs 38.4%, P < 0.001). Reinfections (defined as occurring between 6 and 24 weeks after a prior diagnosis of COVID-19) were significantly higher among BR.2.1 compared to non-BR.2.1 infected persons (17.0% vs 6.0%, P < 0.001). BR.2.1 cases were also significantly younger compared to non-BR.2.1 (median age 48 years (interquartile range [IQR] 32) vs 53 years (IQR 32), P = 0.004). The proportion of patients admitted to the intensive care unit with BR.2.1 was not significantly higher than other subvariants (2.3% vs 2.0%, P = 0.717). CONCLUSION: Having emerged locally within New South Wales, BR.2.1 caused a significant number of SARS-CoV-2 reinfections, but with disease severity comparable with other currently circulating lineages. Given its rapid rise in prevalence, BR.2.1 has the potential to become established internationally.

Pangenome Analysis of a Salmonella Enteritidis Population Links a Major Outbreak to a Gifsy-1-Like Prophage Containing Anti-Inflammatory Gene gogB.

A major outbreak of the globally significant Salmonella Enteritidis foodborne pathogen was identified within a large clinical data set by a program of routine WGS of clinical presentations of salmonellosis in New South Wales, Australia. Pangenome analysis helped to quantify and isolate prophage content within the accessory partition of the pangenome. A prophage similar to Gifsy-1 (henceforth GF-1L) was found to occur in all isolates of the outbreak core SNP cluster, and in three other isolates. Further analysis revealed that the GF-1L prophage carried the gogB virulence factor. These observations suggest that GF-1L may be an important marker of virulence for S. Enteritidis population screening and, that anti-inflammatory, gogB-mediated virulence currently associated with Salmonella Typhimurium may also be displayed by S. Enteritidis. IMPORTANCE We examined 5 years of genomic and epidemiological data for the significant global foodborne pathogen, Salmonella enterica. Although Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) is the leading cause of salmonellosis in the USA and Europe, prior to 2018 it was not endemic in the southern states of Australia. However, in 2018 a large outbreak led to the endemicity of S. Enteritidis in New South Wales, Australia, and a unique opportunity to study this phenomenon. Using pangenome analysis we uncovered that this clone contained a Gifsy-1-like prophage harboring the known virulence factor gogB. The prophage reported has not previously been described in S. Enteritidis isolates.

Understanding the population genetics of Plasmodium vivax is essential for malaria control and elimination.

Traditionally, infection with Plasmodium vivax was thought to be benign and self-limiting, however, recent evidence has demonstrated that infection with P. vivax can also result in severe illness and death. Research into P. vivax has been relatively neglected and much remains unknown regarding the biology, pathogenesis and epidemiology of this parasite. One of the fundamental factors governing transmission and immunity is parasite diversity. An understanding of parasite population genetic structure is necessary to understand the epidemiology, diversity, distribution and dynamics of natural P. vivax populations. In addition, studying the population structure of genes under immune selection also enables investigation of the dynamic interplay between transmission and immunity, which is crucial for vaccine development. A lack of knowledge regarding the transmission and spread of P. vivax has been particularly highlighted in areas where malaria control and elimination programmes have made progress in reducing the burden of Plasmodium falciparum, yet P. vivax remains as a substantial obstacle. With malaria elimination back on the global agenda, mapping of global and local P. vivax population structure is essential prior to establishing goals for elimination and the roll-out of interventions. A detailed knowledge of the spatial distribution, transmission and clinical burden of P. vivax is required to act as a benchmark against which control targets can be set and measured. This paper presents an overview of what is known and what is yet to be fully understood regarding P. vivax population genetics, as well as the importance and application of P. vivax population genetics studies.

COVID-19 Infection With the Omicron SARS-CoV-2 Variant in a Cohort of Kidney and Kidney Pancreas Transplant Recipients: Clinical Features, Risk Factors, and Outcomes.

BACKGROUND: Since November 2021, a new variant of concern (VOC), the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage B.1.1.529 (Omicron) has emerged as the dominant coronavirus disease 2019 (COVID-19) infection worldwide. We describe the clinical presentation, risk factors, and outcomes in a cohort of kidney and kidney pancreas transplant recipients with COVID-19 caused by Omicron infection. METHODS: We included all kidney and kidney pancreas transplant recipients diagnosed with SARS-CoV-2 Omicron infections between December 26, 2021, and January 14, 2022, in a single transplant center in Australia. Identification of the VOC Omicron was confirmed using phylogenetic analysis of SARS-CoV-2 sequences. RESULTS: Forty-one patients with kidney (6 living and 33 deceased) and kidney pancreas transplants were diagnosed with the VOC Omicron (lineage B.1.1.529/BA.1) infection during the study period. The mean age (SD) at the time of diagnosis was 52 (11.1) y; 40 (out of 41) (98%) had received at least 2 doses of COVID-19 vaccine. Cough was the most frequent symptom (80.5%), followed by myalgia (70.7%), sore throat (63.4%), and fever (58.5%). After a follow-up time of 30 d, 1 (2.4%) patient died, 2 (4.9%) experienced multiorgan failure, and 5 (12.2%) had respiratory failure; 11 (26.8%) patients developed other superimposed infections. Compared with recipients who did not receive sotrovimab antibody therapy, the odds ratio (95% confidence interval) for hospitalization among patients who received sotrovimab was 0.05 (0.005-0.4). CONCLUSIONS: Despite double or triple dose vaccination, VOC Omicron infections in kidney and kidney pancreas transplant recipients are not necessarily mild. Hospitalization rates remained high (around 56%), and sotrovimab use may prevent hospitalization.

SARS-CoV-2 Within-Host and in vitro Genomic Variability and Sub-Genomic RNA Levels Indicate Differences in Viral Expression Between Clinical Cohorts and in vitro Culture.

Background: Low frequency intrahost single nucleotide variants (iSNVs) of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) have been increasingly recognised as predictive indicators of positive selection. Particularly as growing numbers of SARS-CoV-2 variants of interest (VOI) and concern (VOC) emerge. However, the dynamics of subgenomic RNA (sgRNA) expression and its impact on genomic diversity and infection outcome remain poorly understood. This study aims to investigate and quantify iSNVs and sgRNA expression in single and longitudinally sampled cohorts over the course of mild and severe SARS-CoV-2 infection, benchmarked against an in vitro infection model. Methods: Two clinical cohorts of SARS-CoV-2 positive cases in New South Wales, Australia collected between March 2020 and August 2021 were sequenced. Longitudinal samples from cases hospitalised due to SARS-CoV-2 infection (severe) (n = 16) were analysed and compared with cases that presented with SARS-CoV-2 symptoms but were not hospitalised (mild) (n = 23). SARS-CoV-2 genomic diversity profiles were also examined from daily sampling of culture experiments for three SARS-CoV-2 variants (Lineage A, B.1.351, and B.1.617.2) cultured in VeroE6 C1008 cells (n = 33). Results: Intrahost single nucleotide variants were detected in 83% (19/23) of the mild cohort cases and 100% (16/16) of the severe cohort cases. SNP profiles remained relatively fixed over time, with an average of 1.66 SNPs gained or lost, and an average of 4.2 and 5.9 low frequency variants per patient were detected in severe and mild infection, respectively. sgRNA was detected in 100% (25/25) of the mild genomes and 92% (24/26) of the severe genomes. Total sgRNA expressed across all genes in the mild cohort was significantly higher than that of the severe cohort. Significantly higher expression levels were detected in the spike and the nucleocapsid genes. There was significantly less sgRNA detected in the culture dilutions than the clinical cohorts. Discussion and Conclusion: The positions and frequencies of iSNVs in the severe and mild infection cohorts were dynamic overtime, highlighting the importance of continual monitoring, particularly during community outbreaks where multiple SARS-CoV-2 variants may co-circulate. sgRNA levels can vary across patients and the overall level of sgRNA reads compared to genomic RNA can be less than 1%. The relative contribution of sgRNA to the severity of illness warrants further investigation given the level of variation between genomes. Further monitoring of sgRNAs will improve the understanding of SARS-CoV-2 evolution and the effectiveness of therapeutic and public health containment measures during the pandemic.

Genome-wide networks reveal emergence of epidemic strains of Salmonella Enteritidis.

OBJECTIVES: To enhance monitoring of high-burden foodborne pathogens, there is opportunity to combine pangenome data with network analysis. METHODS: Salmonella enterica subspecies Enterica serovar Enteritidis isolates were referred to the New South Wales (NSW) Enteric Reference Laboratory between August 2015 and December 2019 (1033 isolates in total), inclusive of a confirmed outbreak. All isolates underwent whole genome sequencing. Distances between genomes were quantified by in silico multiple-locus variable-number tandem repeat analysis (MLVA) as well as core single nucleotide polymorphisms (SNPs), which informed the construction of undirected networks. Centrality-prevalence spaces were generated from the undirected networks. Components on the undirected SNP network were considered alongside a phylogenetic tree representation. RESULTS: Outbreak isolates were identified as distinct components on the MLVA and SNP networks. The MLVA network-based centrality-prevalence space did not delineate the outbreak, whereas the outbreak was delineated in the SNP network-based centrality-prevalence space. Components on the undirected SNP network showed a high concordance to the SNP clusters based on phylogenetic analysis. CONCLUSIONS: Bacterial whole-genome data in network-based analysis can improve the resolution of population analysis. High concordance of network components and SNP clusters is promising for rapid population analyses of foodborne Salmonella spp. owing to the low overhead of network analysis.