Genomic Surveillance of Invasive Meningococcal Disease During a National MenW Outbreak in Australia, 2017-2018.

Background: In Australia, invasive meningococcal disease (IMD) incidence rapidly increased between 2014 and 2017 due to rising serogroup W (MenW) and MenY infections. We aimed to better understand the genetic diversity of IMD during 2017 and 2018 using whole genome sequencing data. Methods: Whole genome sequencing data from 440 Australian IMD isolates collected during 2017 and 2018 and 1737 international MenW:CC11 isolates collected in Europe, Africa, Asia, North America, and South America between 1974 and 2020 were used in phylogenetic analyses; genetic relatedness was determined from single-nucleotide polymorphisms. Results: Australian isolates were as follows: 181 MenW (41%), 144 MenB (33%), 88 MenY (20%), 16 MenC (4%), 1 MenW/Y (0.2%), and 10 nongenogroupable (2%). Eighteen clonal complexes (CCs) were identified, and 3 (CC11, CC23, CC41/44) accounted for 78% of isolates (343/440). These CCs were associated with specific serogroups: CC11 (n = 199) predominated among MenW (n = 181) and MenC (n = 15), CC23 (n = 80) among MenY (n = 78), and CC41/44 (n = 64) among MenB (n = 64). MenB isolates were highly diverse, MenY were intermediately diverse, and MenW and MenC isolates demonstrated the least genetic diversity. Thirty serogroup and CC-specific genomic clusters were identified. International CC11 comparison revealed diversification of MenW in Australia. Conclusions: Whole genome sequencing comprehensively characterized Australian IMD isolates, indexed their genetic variability, provided increased within-CC resolution, and elucidated the evolution of CC11 in Australia.

Defining the phylogenetics and resistome of the major Clostridioides difficile ribotypes circulating in Australia.

Clostridioides difficile infection (CDI) remains a significant public health threat globally. New interventions to treat CDI rely on an understanding of the evolution and epidemiology of circulating strains. Here we provide longitudinal genomic data on strain diversity, transmission dynamics and antimicrobial resistance (AMR) of C. difficile ribotypes (RTs) 014/020 (n=169), 002 (n=77) and 056 (n=36), the three most prominent C. difficile strains causing CDI in Australia. Genome scrutiny showed that AMR was uncommon in these lineages, with resistance-conferring alleles present in only 15/169 RT014/020 strains (8.9 %), 1/36 RT056 strains (2.78 %) and none of 77 RT002 strains. Notably, ~90 % of strains were resistant to MLSB agents in vitro, but only ~5.9 % harboured known resistance alleles, highlighting an incongruence between AMR genotype and phenotype. Core genome analyses revealed all three RTs contained genetically heterogeneous strain populations with limited evidence of clonal transmission between CDI cases. The average number of pairwise core genome SNP (cgSNP) differences within each RT group ranged from 23.3 (RT056, ST34, n=36) to 115.6 (RT002, ST8, n=77) and 315.9 (RT014/020, STs 2, 13, 14, 49, n=169). Just 19 clonal groups (encompassing 40 isolates), defined as isolates differing by ≤2 cgSNPs, were identified across all three RTs (RT014/020, n=14; RT002, n=3; RT056, n=2). Of these clonal groups, 63 % (12/19) comprised isolates from the same Australian State and 37 % (7/19) comprised isolates from different States. The low number of plausible transmission events found for these major RTs (and previously documented populations in animal and environmental sources/reservoirs) points to widespread and persistent community sources of diverse C. difficile strains as opposed to ongoing nationwide healthcare outbreaks dominated by a single clone. Together, these data provide new insights into the evolution of major lineages causing CDI in Australia and highlight the urgent need for enhanced surveillance, and for public health interventions to move beyond the healthcare setting and into a One Health paradigm to effectively combat this complex pathogen.

Obstetric and Neonatal Invasive Meningococcal Disease Caused by Neisseria meningitidis Serogroup W, Western Australia, Australia.

Three mother-baby pairs with invasive meningococcal disease occurred over 7 months in Western Australia, Australia, at a time when serogroup W sequence type 11 clonal complex was the predominant local strain. One mother and 2 neonates died, highlighting the role of this strain as a cause of obstetric and early neonatal death.

Flight-Associated Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 Corroborated by Whole-Genome Sequencing.

To investigate potential transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during a domestic flight within Australia, we performed epidemiologic analyses with whole-genome sequencing. Eleven passengers with PCR-confirmed SARS-CoV-2 infection and symptom onset within 48 hours of the flight were considered infectious during travel; 9 had recently disembarked from a cruise ship with a retrospectively identified SARS-CoV-2 outbreak. The virus strain of those on the cruise and the flight was linked (A2-RP) and had not been previously identified in Australia. For 11 passengers, none of whom had traveled on the cruise ship, PCR-confirmed SARS-CoV-2 illness developed between 48 hours and 14 days after the flight. Eight cases were considered flight associated with the distinct SARS-CoV-2 A2-RP strain; the remaining 3 cases (1 with A2-RP) were possibly flight associated. All 11 passengers had been in the same cabin with symptomatic persons who had culture-positive A2-RP virus strain. This investigation provides evidence of flight-associated SARS-CoV-2 transmission.

Meningococcal Disease-Associated Prophage-Like Elements Are Present in Neisseria gonorrhoeae and Some Commensal Neisseria Species.

Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.

Respiratory viral infections in Western Australians with cystic fibrosis.

BACKGROUND: Viral respiratory infections (VRI) in people living with Cystic fibrosis (CF) is less well understood than respiratory bacterial infections, particularly adults with CF and few studies have compared children with adults. This study evaluated the frequency of respiratory viruses in patients with cystic fibrosis (CF) in Western Australia (WA). We determined the VRI in CF and compared them with non-CF patients. Further, we compared CF patients that were hospitalised with those that were not. PATIENTS/METHODS: Nucleic acid from sputum of 157 CF and 348 non-CF patients was analysed for influenzavirus A (Flu A) and B, (Flu B), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), human rhinovirus (RV), and parainfluenza viruses (PIV 1-3) by RT-PCR, during the 2016 winter respiratory season. RESULTS: No significant difference in the frequency of respiratory virus detection between CF and non-CF patients was found. RV was the most frequently detected virus in CF patients, and in hospitalised CF. RSV and hMPV were found less frequently in CF patients and RSV was not found in any hospitalised CF patient. A trend for fewer influenzavirus detections in adult CF patients was observed, however the trend was opposite for paediatric patients. RV and Flu A were the most common viruses detected in hospitalised CF patients. CONCLUSION: There was no significant difference in VRI between CF and non-CF patients. RV and influenza A were most commonly found in hospitalised CF patients, suggesting that infection with these viruses may contribute to hospitalisation for CF respiratory exacerbations.

Genomic epidemiology and population structure of Neisseria gonorrhoeae from remote highly endemic Western Australian populations.

BACKGROUND: Neisseria gonorrhoeae causes gonorrhoea, the second most commonly notified sexually transmitted infection in Australia. One of the highest notification rates of gonorrhoea is found in the remote regions of Western Australia (WA). Unlike isolates from the major Australian population centres, the remote community isolates have low rates of antimicrobial resistance (AMR). Population structure and whole-genome comparison of 59 isolates from the Western Australian N. gonorrhoeae collection were used to investigate relatedness of isolates cultured in the metropolitan and remote areas. Core genome phylogeny, multilocus sequencing typing (MLST), N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) in addition to hierarchical clustering of sequences were used to characterize the isolates. RESULTS: Population structure analysis of the 59 isolates together with 72 isolates from an international collection, revealed six population groups suggesting that N. gonorrhoeae is a weakly clonal species. Two distinct population groups, Aus1 and Aus2, represented 63% of WA isolates and were mostly composed of the remote community isolates that carried no chromosomal AMR genotypes. In contrast, the Western Australian metropolitan isolates were frequently multi-drug resistant and belonged to population groups found in the international database, suggesting international transmission of the isolates. CONCLUSIONS: Our study suggests that the population structure of N. gonorrhoeae is distinct between the communities in remote and metropolitan WA. Given the high rate of AMR in metropolitan regions, ongoing surveillance is essential to ensure the enduring efficacy of the empiric gonorrhoea treatment in remote WA.

Differences in the population structure of Neisseria meningitidis in two Australian states: Victoria and Western Australia.

Neisseria meningitidis is the causative agent of invasive meningococcal disease (IMD). A recombinant vaccine called Bexsero® incorporates four subcapsular antigens (fHbp, NHBA, NadA and PorA) which are used to assign a Bexsero® antigen sequence type (BAST) to each meningococcal strain. The vaccine elicits an immune response against combinations of variants of these antigens which have been grouped into specific BAST profiles that have been shown to have different distributions within geographical locations thus potentially affecting the efficacy of the vaccine. In this study, invasive meningococcal disease isolates from the western seaboard of Australia (Western Australia; WA) were compared to those from the south-eastern seaboard (Victoria; VIC) from 2008 to 2012. Whole-genome sequencing (WGS) of 131 meningococci from VIC and 70 meningococci from WA were analysed for MLST, FetA and BAST profiling. Serogroup B predominated in both jurisdictions and a total of 10 MLST clonal complexes (cc) were shared by both states. Isolates belonging to cc22, cc103 and cc1157 were unique to VIC whilst isolates from cc60 and cc212 were unique to WA. Clonal complex 41/44 represented one-third of the meningococcal population in each state but the predominant ST was locally different: ST-6058 in VIC and ST-146 in WA. Of the 108 BAST profiles identified in this collection, only 9 BASTs were simultaneously observed in both states. A significantly larger proportion of isolates in VIC harboured alleles for the NHBA-2 peptide and fHbp-1, antigenic variants predicted to be covered by the Bexsero® vaccine. The estimate for vaccine coverage in WA (47.1% [95% CI: 41.1-53.1%]) was significantly lower than that in VIC (66.4% [95% CI: 62.3-70.5%]). In conclusion, the antigenic structure of meningococci causing invasive disease in two geographically distinct states of Australia differed significantly during the study period which may affect vaccine effectiveness and highlights the need for representative surveillance when predicting potential impact of meningococcal B vaccines.

M protein gene (emm type) analysis of group A Streptococcus isolates recovered during an acute glomerulonephritis outbreak in northern Western Australia.

Certain M protein types of group A streptococcus (GAS) are known to cause acute post-streptococcal glomerulonephritis (APSGN). Outbreaks of APSGN can occur regularly in tropical regions but the emm types responsible are geographically and temporally diverse. GAS isolates from Western Australia (WA) were analysed for emm type and emm cluster during the period of increased APSGN activity in the tropical northern Kimberley region of WA. Although emm types 49, 75 and 108 and corresponding emm clusters E3, E6 and D4 were more common in WA during the outbreak there was no predominant circulating emm type or cluster found to correspond to the APSGN activity. This is consistent with the high diversity of GAS strains found during APSGN outbreaks in other countries. Potential vaccine coverage of the new 30-valent M-protein GAS vaccine was 70%.

Clonal Expansion of New Penicillin-Resistant Clade of Neisseria meningitidis Serogroup W Clonal Complex 11, Australia.

In Western Australia, Neisseria meningitidis serogroup W clonal complex 11 became the predominant cause of invasive meningococcal disease in 2016. We used core-genome analysis to show emergence of a penicillin-resistant clade that had the penA_253 allele. This new penicillin-resistant clade might affect treatment regimens for this disease.

The implications of endemic IMP-4 carbapenemase for clinical laboratory susceptibility testing.

A local predominance of carbapenemase producing Enterobacteriaceae with low minimum inhibitory concentrations (MIC) to meropenem prompted a review of methods available for carbapenemase detection. We report on results using two selective media, temocillin discs, CarbaNP test, GeneXpert Carba-R assay and an in-house PCR assay.

Enterovirus D68 disease and molecular epidemiology in Australia.

BACKGROUND: Enterovirus D68 (EV-D68) has received considerable recent attention as a cause of widespread respiratory illness. Neurological syndromes such as acute flaccid paralysis following EV-D68 infection have also been reported in a small number of cases. OBJECTIVES: To summarize the clinical and epidemiological characteristics of laboratory confirmed EV-D68 cases in Australia. STUDY DESIGN: We combined EV-D68 data acquired through laboratory surveillance in Western Australia with cases from national enterovirus surveillance and regional acute flaccid paralysis (AFP) surveillance. Clinical data was obtained for EV-D68 cases and capsid protein sequences were used for phylogenetic analysis. RESULTS: Sporadic cases of EV-D68 were recorded in Australia since 2008, with peaks in activity during 2011 and 2013. EV-D68 was primarily associated with respiratory disease, but was also detected in cerebrospinal fluid of one patient and faeces of two patients presenting with AFP. CONCLUSIONS: EV-D68 has been circulating in Western Australia and is likely to have also been present in the wider region for a number of years, causing primarily respiratory disease. Detection of EV-D68 in cerebrospinal fluid of one patient and in faeces of two AFP cases reinforces the association between EV-D68 and neurological disease.

Molecular epidemiology of Neisseria gonorrhoeae using multi-antigen sequence typing and pulse-field gel electrophoresis in highly endemic Western Australian populations.

BACKGROUND: The remote and indigenous populations of Western Australia (WA) have one of the highest notification rates of gonorrhoea in the world. Despite this, the low rate of antimicrobial resistance in Neisseria gonorrhoeae from these regions permits the use of amoxycillin as empirical therapy. We describe the first molecular epidemiological study of gonococci isolated from this population using two different typing platforms. METHODS: Pulse-field gel electrophoresis (PFGE), Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) and antimicrobial susceptibility tests were performed on 128 consecutive N. gonorrhoeae isolates cultured between January 2011 and December 2013. To highlight clusters isolates were evaluated based on their tbpB sequence types. RESULTS: No predominant NG-MAST or PFGE types were found. A total of 67 distinct PFGE pulsotypes were identified amongst the 128 isolates in this study with 20 PFGE pulsotypes representing 78 isolates. A total of 59 NG-MAST sequence types were found, represented by 45 porB alleles and 28 tbpB alleles with 13 tbpB genomogroups from 45 NG-MAST sequence types. TbpB genomogroup 29, represented by 45 isolates, was by far the most common genomogroup overall. CONCLUSIONS: Results from this study suggest that gonococcal epidemiology in WA is quite different between remote regions and major population centres and, in some cases, geographically restricted. It is likely that isolates originating from endemic regions of WA mostly represent independent, small sexual networks with an infrequent interchange between other communities and regions. Given the high rate of antimicrobial resistance elsewhere in Australia, ongoing surveillance is essential to ensure the enduring efficacy of amoxycillin empiric use in the remote regions of WA.

Acute meningoencephalitis associated with echovirus 9 infection in Sri Lanka, 2009.

The aetiology of acute meningoencephalitis in Sri Lankan children and adults is poorly understood. This study was carried out to determine pathogens responsible for meningoencephalitis in Sri Lanka. A hospital-based cross-sectional study was performed using cerebrospinal fluid samples (22 adult and 17 pediatric) collected from August to December 2009 from patients clinically diagnosed with acute meningoencephalitis at two tertiary care hospitals in Sri Lanka. Routine microbiology for bacterial pathogens together with in-house RT-PCR and PCR assays for the detection of dengue viruses, Japanese encephalitis virus, West Nile virus, chikungunya virus, enteroviruses, mumps virus, measles virus, herpes simplex viruses types 1 and 2, and varicella zoster virus were performed. Bacterial pathogens were not isolated from any patient specimens. However, from nine of the paediatric patients aged 1 month to 10 years (mean age 5.2 years) echovirus 9 (E-9; family Picornaviridae, genus Enterovirus,species Enterovirus B ) was detected by RT-PCR. All nine patients presented with fever, six had headache, and seven had vomiting. Neck stiffness indicating meningitis was present in six of the patients. Phylogenetic analysis of partial VP1 and VP4-VP2 genes showed these E-9 strains to be most closely related to E-9 strains detected in CSF from Korea and France in 2005 and 2006. The remaining patients were negative for all other viruses tested. E-9 was the most common cause of acute meningoencephalitis in the tested paediatric population from Sri Lanka in 2009, which likely reflects circulation of this E-9 strain between Europe and Asia over several years.

Neisseria meningitidis porA, fetA and fHbp gene distribution in Western Australia 2000 to 2011.

BACKGROUND: PorA, fetA and fHbp are three antigen encoding genes useful for meningococcal typing and FHbp is an important component of meningococcal B vaccines. METHODS: We performed sequence analysis of meningococcal porA, fetA and fHbp genes on 128 isolates from Western Australia, relating results to age, gender, race and geographic region. RESULTS: We found predominantly PorA subtypes P1.22,14-16 (n = 23) and P1.7-2,4 (n = 19); FetA subtypes F1-5 (n = 41), F3-6 (n = 11), F5-1 (n = 10), F5-2 (n = 9), F5-5 (n = 8), F3-3 (n = 8); and FHbp variant groups 1 (n = 65) and 2 (n = 44). PorA P1.22,14-16 and FHbp variant group 2 were associated with younger age and aboriginality. CONCLUSIONS: FHbp modular groups of the bivalent recombinant FHbp vaccine and the multicomponent 4CMenB vaccine make up 8.3% and 47.7% respectively of the examined serogroup B isolates from 2000-2011, however to estimate vaccine efficacy requires an account of all vaccine antigens and their levels of expression.