The Genomic Epidemiology of Clinical Burkholderia pseudomallei Isolates in North Queensland, Australia.

Background:Burkholderia pseudomallei, the causative agent of melioidosis, is highly genetically recombinant, resulting in significant genomic diversity. Multiple virulence factors have been associated with specific disease presentations. To date, there are limited data relating to genomic diversity and virulence factors associated with melioidosis cases in North Queensland, Australia. Aim: To describe the genetic diversity of B. pseudomallei and identify virulence factors associated with clinical risk factors and patient outcomes. Methods: Whole genome sequencing of clinical isolates was performed and analysed with clinical data obtained from a retrospective melioidosis cohort study. Results: Fifty-nine distinct sequence types (STs) were identified from the 128 clinical isolates. Six STs comprised 64/128 (50%) isolates. Novel STs accounted for 38/59 (64%) STs, with ST TSV-13 as the most prevalent (n = 7), and were less likely to possess an LPS A genotype or YLF gene cluster (p < 0.001). These isolates were most likely to be found outside the inner city (aOR: 4.0, 95% CI: 1.7-9.0, p = 0.001). ST TSV-13 was associated with increased mortality (aOR: 6.1, 95% CI: 1.2-30.9, p = 0.03). Patients with a history of alcohol excess were less likely to be infected by fhaB3 (aOR 0.2, 95% CI: 0.1-0.7, p = 0.01) or YLF (aOR: 0.4, 95% CI: 0.2-0.9, p = 0.04) positive isolates. Conclusions: There are a significant number of novel sequence types in Townsville, Australia. An emerging novel ST appears to have an association with geographic location and mortality. Ongoing investigation is required to further understand the impact of this ST on the Townsville region.

The effect of climate on melioidosis incidence in Townsville, Australia: a dry tropical region.

BACKGROUND: Townsville is in the dry tropics in Northern Australia and an endemic region for melioidosis. Melioidosis is an infectious disease caused by Burkholderia pseudomallei, a soil dwelling organism. The incidence of melioidosis is associated with high levels of rainfall and has been linked to multiple weather variables in other melioidosis endemic regions such as in Darwin. In contrast to Townsville, Darwin is in the wet-dry tropics in Northern Australia and receives 40% more rainfall. We assessed the relationship between melioidosis incidence and weather conditions in Townsville and compared the patterns to the findings in Darwin and other melioidosis endemic regions. METHOD: Performing a time series analysis from 1996 to 2020, we applied a negative binomial regression model to evaluate the link between the incidence of melioidosis in Townsville and various weather variables. Akaike's information criterion was used to assess the most parsimonious model with best predictive performance. Fourier terms and lagged deviance residuals were included to control long term seasonal trends and temporal autocorrelation. RESULTS: Humidity is the strongest predictor for melioidosis incidence in Townsville. Furthermore, the incidence of melioidosis showed a three-times rise in the Townsville region when >200 mm of rain fell within the fortnight. Prolonged rainfall had more impact than a heavy downpour on the overall melioidosis incident rate. There was no statistically significant increase in incidence with cloud cover in the multivariable model. CONCLUSION: Consistent with other reports, melioidosis incidence can be attributed to humidity and rainfall in Townsville. In contrast to Darwin, there was no strong link between melioidosis cases and cloud cover and nor single large rainfall events.

Design and Development of an Internationally Applicable Educational Video to Increase Community Awareness in Regions with High Prevalence of Melioidosis and Diabetes.

Melioidosis is a neglected tropical disease that causes high morbidity and mortality. Public health awareness is essential for both prevention and early detection of the infection. This project aimed to develop an internationally applicable educational tool to increase community awareness in regions with high prevalence of diabetes and melioidosis. The animation was created with international collaboration. Sixty-four delegates from different cultural backgrounds participated in the survey to evaluate the animation. Feedback was positive, with 85% agreeing that they would use this video for public education and 82% agreeing that the video was culturally appropriate to them in the context of their region. The animation was refined after feedback. To supplement the 3-minute animation, a 13-minute film footage of interviews with clinicians, researchers and patients was also created. These materials have been made available online through the International Melioidosis Network and can be readily downloaded or subtitled in any language using publicly available software, demonstrating the utility of developing low-cost adaptable health education material targeted for widespread use internationally.

Clinical Burkholderia pseudomallei isolates from north Queensland carry diverse bimABm genes that are associated with central nervous system disease and are phylogenomically distinct from other Australian strains.

BACKGROUND: Burkholderia pseudomallei is an environmental gram-negative bacterium that causes the disease melioidosis and is endemic in many countries of the Asia-Pacific region. In Australia, the mortality rate remains high at approximately 10%, despite curative antibiotic treatment being available. The bacterium is almost exclusively found in the endemic region, which spans the tropical Northern Territory and North Queensland, with clusters occasionally present in more temperate climates. Despite being endemic to North Queensland, these infections remain understudied compared to those of the Northern Territory. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to assess the prevalence of central nervous system (CNS) disease associated variant bimABm, identify circulating antimicrobial resistance mutations and genetically distinct strains from Queensland, via comparative genomics. From 76 clinical isolates, we identified the bimABm variant in 20 (26.3%) isolates and in 9 (45%) of the isolates with documented CNS infection (n = 18). Explorative analysis suggests a significant association between isolates carrying the bimABm variant and CNS disease (OR 2.8, 95% CI 1.3-6.0, P = 0.009) compared with isolates carrying the wildtype bimABp. Furthermore, 50% of isolates were identified as novel multi-locus sequence types, while the bimABm variant was more commonly identified in isolates with novel sequence types, compared to those with previously described. Additionally, mutations associated with acquired antimicrobial resistance were only identified in 14.5% of all genomes. CONCLUSIONS/SIGNIFICANCE: The findings of this research have provided clinically relevant genomic data of B. pseudomallei in Queensland and suggest that the bimABm variant may enable risk stratification for the development CNS complications and be a potential therapeutic target.

The epidemiology of melioidosis in Townsville, Australia.

BACKGROUND: Melioidosis in an infection caused by Burkholderia pseudomallei, an organism endemic to tropical and subtropical regions. METHODS: This study describes the epidemiology of melioidosis in Townsville, QLD, Australia, as well as clinical features, risk factors associated with the disease, the burden of infection on the Aboriginal and Torres Strait Islander (ATSI) community and patient outcomes over time. RESULTS: From 1997 to 2020, 128 patients were admitted to Townsville University Hospital. The total annual incidence of infection was 3.2 cases per 100 000 compared with 15.3 per 100 000 in the ATSI population. The majority of cases (n=82 [64%]) were male. Alcohol excess (55%) and diabetes mellitus (48%) were the most common risk factors. Bacteraemia occurred in 87 (70%) patients and pneumonia was the most common focus of infection in 84 (69%). The case fatality rate was 23%, with no difference for the ATSI population (6/32 [19%]). The presence of malignancy was the risk factor most associated with mortality (relative risk 2.7 [95% confidence interval 1.4-5.1], p=0.005). CONCLUSIONS: The ATSI community was overrepresented in this study, however, there was no significant difference in adverse outcomes. The case fatality rate was higher than in other regions in Australia. This discrepancy may relate in part to the different risk groups seen in these settings coupled with potential organism variability.

Melioidosis: Laboratory Investigations and Association with Patient Outcomes.

Melioidosis is an infection caused by the bacterium Burkholderia pseudomallei. The most common presentation is bacteremia occurring in 38-73% of all patients, and the mortality rate ranges from 9% to 42%. Although there is abundant data representing risk factors for infection and patient outcomes, there is limited information regarding laboratory investigations associated with bacteremia and mortality. We assessed a range of baseline and diagnostic investigations and their association with patient outcomes in a retrospective cohort study in Townsville, Australia. 124 patients' medical and laboratory records were reviewed between January 1, 1997 and December 31, 2020. Twenty-seven patients died and 87 patients were bacteremic. The presence of lymphopenia (< 1.5 × 109 cells/L) was the highest risk for bacteremia (relative risk [RR] 2.2; 95% CI: 1.3-3.7, P < 0.001). Factors associated with mortality included lymphopenia, (RR: 1.4; 95% CI: 1.2-1.6, P = 0.004); uremia (RR: 1.7; 95% CI: 1.1-2.5, P = 0.03); and an elevated international normalized ratio (RR: 1.5; 95% CI: 1.2-2.0, P = 0.006). Median incubation to positive blood culture result was 28 hours with 15/82 (18%) positive in ≤ 24 hours. For serological testing during admission only 53/121 (44%) were indirect hemagglutination assay positive, 67/120 (56%) enzyme immunoassay IgG positive, and 23/89 (26%) IgM positive. Simple baseline investigations at time of presentation may be used to stratify patients at high risk for both bacteremia and mortality. This information can be used as a decision aid for early intensive management.

In Search of the Holy Grail: A Specific Diagnostic Test for Rheumatic Fever.

Current diagnosis of Acute Rheumatic Fever and Rheumatic Heart Disease (ARF/RHD) relies on a battery of clinical observations aided by technologically advanced diagnostic tools and non-specific laboratory tests. The laboratory-based assays fall into two categories: those that (1) detect "evidence of preceding streptococcal infections" (ASOT, anti-DNAse B, isolation of the Group A Streptococcus from a throat swab) and (2) those that detect an ongoing inflammatory process (ESR and CRP). These laboratory tests are positive during any streptococcal infection and are non-specific for the diagnosis of ARF/RHD. Over the last few decades, we have accumulated considerable knowledge about streptococcal biology and the immunopathological mechanisms that contribute to the development, progression and exacerbation of ARF/RHD. Although our knowledge is incomplete and many more years will be devoted to understanding the exact molecular and cellular mechanisms involved in the spectrum of clinical manifestations of ARF/RHD, in this commentary we contend that there is sufficient understanding of the disease process that using currently available technologies it is possible to identify pathogen associated peptides and develop a specific test for ARF/RHD. It is our view that with collaboration and sharing of well-characterised serial blood samples from patients with ARF/RHD from different regions, antibody array technology and/or T-cell tetramers could be used to identify streptococcal peptides specific to ARF/RHD. The availability of an appropriate animal model for this uniquely human disease can further facilitate the determination as to whether these peptides are pathognomonic. Identification of such peptides will also facilitate testing of potential anti-streptococcal vaccines for safety and avoid potential candidates that may pre-dispose potential vaccine recipients to adverse outcomes. Such peptides can also be readily incorporated into a universally affordable point of care device for both primary and tertiary care.

The Burkholderia pseudomallei intracellular 'TRANSITome'.

Prokaryotic cell transcriptomics has been limited to mixed or sub-population dynamics and individual cells within heterogeneous populations, which has hampered further understanding of spatiotemporal and stage-specific processes of prokaryotic cells within complex environments. Here we develop a 'TRANSITomic' approach to profile transcriptomes of single Burkholderia pseudomallei cells as they transit through host cell infection at defined stages, yielding pathophysiological insights. We find that B. pseudomallei transits through host cells during infection in three observable stages: vacuole entry; cytoplasmic escape and replication; and membrane protrusion, promoting cell-to-cell spread. The B. pseudomallei 'TRANSITome' reveals dynamic gene-expression flux during transit in host cells and identifies genes that are required for pathogenesis. We find several hypothetical proteins and assign them to virulence mechanisms, including attachment, cytoskeletal modulation, and autophagy evasion. The B. pseudomallei 'TRANSITome' provides prokaryotic single-cell transcriptomics information enabling high-resolution understanding of host-pathogen interactions.

Diagnosis of melioidosis: the role of molecular techniques.

Melioidosis is an emerging infectious disease with an estimated global burden of 4.64 million disability-adjusted life years per year. A major determinant related to poor disease outcomes is delay to diagnosis due to the fact that identification of the causative agent Burkholderia pseudomallei may be challenging. Over the last 25 years, advances in molecular diagnostic techniques have resulted in the potential for rapid and accurate organism detection and identification direct from clinical samples. While these methods are not yet routine in clinical practice, laboratory diagnosis of infectious diseases is transitioning to culture-independent techniques. This review article aims to evaluate molecular methods for melioidosis diagnosis direct from clinical samples and discuss current and future utility and limitations.

Correlation between serum cryptococcal antigen titre and meningitis in immunocompetent patients.

The aim of this paper was to determine the correlation between serum cryptococcal antigen and a diagnosis of cryptococcal meningitis in the immunocompetent cohort. A retrospective multicentre analysis of immunocompetent patients diagnosed and treated for cryptococcal meningitis between January 2000 and December 2017 was performed. Sixty-seven of the 143 cases of cryptococcosis occurred in immunocompetent patients. The serum cryptococcal antigen titre was significantly higher in the meningitis group [1 : 256 (IQR: 64-1024)] compared with that for non-meningitis patients [1 : 64 (IQR: 8-256)], P=0.012. The relative risk of meningitis with a serum cryptococcal antigen (CRAG) >1 : 64 was 1.8 (95 % CI: 1.15-2.82). This study demonstrates a clear correlation between serum cryptococcal antigen titre and meningitis. While the serum titre is not definitive for meningitis, in resource-limited settings or cases where lumbar puncture may be contraindicated, this evidence may aid diagnosis and subsequent therapeutic decisions.

Paediatric thoracic empyema in the tropical North Queensland region of Australia: Epidemiological trends over a decade.

AIM: The Townsville Hospital and Health Service is the regional referral centre for children in the north of Queensland. Aboriginal and Torres Strait Islander (ATSI) people make up 7-10% of the population. Increasing numbers of children with paediatric thoracic empyema (pTE) are being referred to Townsville Hospital and Health Service for management. This study aims to describe the incidence rates, epidemiology, microbiology and trends of this disease in North Queensland over a 10-year period. METHODS: A retrospective chart review of all children (1 month to 16 years), admitted in the years 2007-2016, with community-acquired pTE was conducted. International Classification of Diseases codes were used to identify the patients. Epidemiological and microbiological data were extracted from records. RESULTS: Of the 123 cases identified, incidence rates per 100 000 were 8.5 (95% confidence interval (CI) 8.4-8.6) in all children and much higher at 19.8 (95% CI: 19.5-21.9) in ATSI children. The under 5 years age group had the highest rate (24.5; 95% CI: 24.4-24.6). There was a progressive rise in incidence during the 10-year period, with the highest incidence of 15.2 (95% CI: 15.1-15.2) occurring in 2016. A pathogen was isolated in 76% of cases. Non-multi-resistant methicillin-resistant Staphylococcus aureus was the most common pathogen isolated in 22 of 64 ATSI children (34%), while Streptococcus pneumoniae was the most common pathogen isolated in 27 of 59 non-ATSI children (46%). CONCLUSIONS: A high and increasing incidence of pTE in North Queensland is being observed. ATSI children have higher incidence rates and are more likely to have non-multi-resistant methicillin-resistant Staphylococcus aureus as a causative agent.

Group G Streptococcus Induces an Autoimmune Carditis Mediated by Interleukin 17A and Interferon γ in the Lewis Rat Model of Rheumatic Heart Disease.

Acute rheumatic fever and rheumatic heart disease (ARF/RHD) have long been described as autoimmune sequelae of Streptococcus pyogenes or group A streptococcal (GAS) infection. Both antibody and T-cell responses against immunodominant GAS virulence factors, including M protein, cross-react with host tissue proteins, triggering an inflammatory response leading to permanent heart damage. However, in some ARF/RHD-endemic regions, throat carriage of GAS is low. Because Streptococcus dysgalactiae subspecies equisimilis organisms, also known as ß-hemolytic group C streptococci and group G streptococci (GGS), also express M protein, we postulated that streptococci other than GAS may have the potential to initiate or exacerbate ARF/RHD. Using a model initially developed to investigate the uniquely human disease of ARF/RHD, we have discovered that GGS causes interleukin 17A/interferon γ-induced myocarditis and valvulitis, hallmarks of ARF/RHD. Remarkably the histological, immunological, and functional changes in the hearts of rats exposed to GGS are identical to those exposed to GAS. Furthermore, antibody cross-reactivity to cardiac myosin was comparable in both GGS- and GAS-exposed animals, providing additional evidence that GGS can induce and/or exacerbate ARF/RHD.

A survey of the antibiotic prescribing practices of doctors in an Australian Emergency Department.

BACKGROUND: Appropriate antibiotic prescribing is important for good patient care and reducing the development of resistance. There has been limited research into doctors' prescribing practices. The aim of this study is to evaluate antibiotic prescribing practices in an Australian emergency department compared with the Therapeutic Guidelines. METHODS: A case vignette survey was sent to emergency department doctors at The Townsville Hospital between February and May 2016. Antibiotic choices were assessed for appropriateness using the National Antimicrobial Prescribing Survey guidelines. Factors associated with antibiotic choice were assessed according to case, clinician experience and rationale. Data was analysed using a non-parametric Kruskal-Wallis test. Post-hoc analysis of variance was performed using Dunn test with Bonferroni correction for multiple simultaneous comparisons, with p < 0.05 considered significant. RESULTS: 197 of 274 antibiotic choices (72%) were appropriate with 149 (54%) optimal. Antibiotic choice was more likely to be appropriate for a urinary tract infection (UTI) compared with severe pyelonephritis (p < 0.01), severe cellulitis (p < 0.01), moderate community-acquired pneumonia (CAP) (p < 0.01) and sepsis (p < 0.01), and was more likely to be appropriate for cellulitis than CAP (p = 0.03) and sepsis (p = 0.02). Antibiotic choices were more likely to be appropriate when doctors reported basing antibiotic choice on the Therapeutic Guidelines compared with current hospital practice (p = 0.02). No significant difference was found in antibiotic appropriateness in relation to grade of doctor (p = 0.34). CONCLUSION: This study demonstrates generally poor antibiotic prescribing compliance with the Therapeutic Guidelines across all grades of clinician. Antibiotic prescribing was more likely to be appropriate if based on the Therapeutic Guidelines and in less severe infection.

The double burden: a new-age pandemic meets an ancient infection.

Tuberculosis is responsible for significant morbidity and mortality in the tropics. Active TB develops when host defences are impaired. Epidemiological evidence and studies addressing the double burden of communicable and non-communicable diseases demonstrate a clear association between diabetes and susceptibility to TB, treatment failure and complications. The immune mechanisms involved in host-pathogen interactions in co-morbid TB-diabetes are not well defined and require further investigation. This combined with the increase in diabetes predominately in low- and middle-income countries where TB is prevalent has major health implications.

Improved diagnosis of melioidosis using a 2-dimensional immunoarray.

Melioidosis is caused by the Gram negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days obtaining a result, hindering successful treatment of acute disease. The existing indirect haemagglutination assay (IHA) has several disadvantages, in that approximately half of patients later confirmed culture positive are not diagnosed at presentation and a subset of patients are persistently seronegative. We have developed 2 serological assays, an enzyme-linked immunosorbent assay (ELISA), and a 2-dimensional immunoarray (2DIA), capable of detecting antibodies in patient sera from a greater proportion of IHA-negative patient subsets. The 2DIA format can distinguish between different LPS serotypes. Currently, the 2DIA has a sensitivity and specificity of 100% and 87.1%, respectively, with 100% of culture-positive, IHA-negative samples detected. The ELISA has a sensitivity and specificity of 86.2% and 93.5%, respectively, detecting 67% of culture-positive, IHA-negative samples. The ELISA and 2DIA tests described here are more rapid and reliable for serological testing compared to the existing IHA.

ELISA and immuno-polymerase chain reaction assays for the sensitive detection of melioidosis.

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei. The gold standard for diagnosis is culture, which requires at least 3-4 days to obtain a result, hindering successful treatment of acute disease. An indirect haemagglutination assay (IHA) is often used but lacks sensitivity. Approximately half of patients later confirmed culture positive are not detected by IHA at presentation and a subset of patients persistently continue to be IHA negative. More rapid and reliable serologic testing for melioidosis is essential and will improve diagnosis and patient outcome. We have developed an ELISA and a quantitative immuno-polymerase chain reaction assay capable of detecting melioidosis-specific antibodies and demonstrate their validity with IHA-negative sera from patients with melioidosis. These new sensitive assays are based upon a secreted antigenic fraction from B. pseudomallei and will be ideal for the diagnosis of melioidosis in patients in nonendemic regions returning from endemic tropical areas and for seroepidemiologic surveys.