Use of Comparative Genomics To Resolve an Unusual Case of Aminoglycoside Susceptibility in the Melioidosis Pathogen Burkholderia pseudomallei in Bangladesh.

Melioidosis is an emerging tropical infectious disease with a rising global burden caused by the environmental bacterium Burkholderia pseudomallei. It is endemic in Southeast and South Asia, including Bangladesh. A rare aminoglycoside-susceptible B. pseudomallei isolate (Y2019) has recently been reported from a melioidosis patient in Dhaka, Bangladesh. To understand the geographical origins of Y2019, we subjected it and 10 other isolates from Bangladesh to whole-genome sequencing. In a phylogenetic tree with a global set of B. pseudomallei genomes, most Bangladeshi genomes clustered tightly within the Asian clade. In contrast, Y2019 was closely related to ST881 isolates from Sarawak, Malaysian Borneo, a gentamicin-sensitive sequence type, suggesting infection in Borneo. Y2019 also contained the same gentamicin sensitivity conferring nonsynonymous mutation in the drug efflux pump encoding the amrB gene. In the absence of a full travel history, whole-genome sequencing and bioinformatics tools have revealed the likely origin of this rare isolate.

Performance of MALDI-TOF MS, real-time PCR, antigen detection, and automated biochemical testing for the identification of Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a disease highly endemic to Southeast Asia and northern Australia, though the area of endemicity is expanding. Cases may occur in returning travelers or, rarely, from imported contaminated products. Identification of B. pseudomallei is challenging for laboratories that do not see this organism frequently, and misidentifications by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and automated biochemical testing have been reported. The in vitro diagnostic database for use with the Vitek MS has recently been updated to include B. pseudomallei and we aimed to validate the performance for identification in comparison to automated biochemical testing with the Vitek 2 GN card, quantitative real-time polymerase chain reaction (qPCR) targeting the type III secretion system, and capsular polysaccharide antigen detection using a lateral flow immunoassay (LFA). We tested a "derivation" cohort including geographically diverse B. pseudomallei and a range of closely related Burkholderia species, and a prospective "validation" cohort of B. pseudomallei and B. cepacia complex clinical isolates. MALDI-TOF MS had a sensitivity of 1.0 and specificity of 1.0 for the identification and differentiation of B. pseudomallei from related Burkholderia species when a certainty cutoff of 99.9% was used. In contrast, automated biochemical testing for B. pseudomallei identification had a sensitivity of 0.83 and specificity of 0.88. Both qPCR and LFA correctly identified all B. pseudomallei isolates with no false positives. Due to the high level of accuracy, we have now incorporated MALDI-TOF MS into our laboratory's B. pseudomallei identification workflow.IMPORTANCEBurkholderia pseudomallei causes melioidosis, a disease associated with high morbidity and mortality that disproportionately affects rural areas in Southeast Asia and northern Australia. The known area of endemicity is expanding and now includes the continental United States. Laboratory identification can be challenging which may result in missed or delayed diagnoses and poor patient outcomes. In this study, we compared mass spectrometry using an updated spectral database with multiple other methods for B. pseudomallei identification and found mass spectrometry highly accurate. We have therefore incorporated this fast and cost-effective method into our laboratory's workflow for B. pseudomallei identification.

Disparities in perioperative mortality outcomes between First Nations and non-First Nations peoples in Australia: protocol for a systematic review and planned meta-analysis.

BACKGROUND: Health inequities persist among First Nations people living in developed countries. Surgical care is pivotal in addressing a significant portion of the global disease burden. Evidence regarding surgical outcomes among First Nations people in Australia is limited. The perioperative mortality rate (POMR) indicates timely access to safe surgery and predicts long-term survival after major surgery. This systematic review will examine POMR among First Nations and non-First Nations peoples in Australia. METHODS: A systematic search strategy using MEDLINE, Embase, Emcare, Global Health, and Scopus will identify studies that include First Nations people and non-First Nations people who underwent a surgical intervention under anaesthesia in Australia. The primary focus will be on documenting perioperative mortality outcomes. Title and abstract screening and full-text review will be conducted by independent reviewers, followed by data extraction and bias assessment using the ROBINS-E tool. Meta-analysis will be considered if there is sufficient homogeneity between studies. The quality of cumulative evidence will be evaluated following the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) criteria. DISCUSSION: This protocol describes the comprehensive methodology for the proposed systematic review. Evaluating disparities in perioperative mortality rates between First Nations and non-First Nations people remains essential in shaping the discourse surrounding health equity, particularly in addressing the surgical burden of disease. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42021258970.

Population sequencing for diversity and transmission analyses.

Genomic diversity in a pathogen population is the foundation for evolution and adaptations in virulence, drug resistance, pathogenesis, and immune evasion. Characterizing, analyzing, and understanding population-level diversity is also essential for epidemiological and forensic tracking of sources and revealing detailed pathways of transmission and spread. For bacteria, culturing, isolating, and sequencing the large number of individual colonies required to adequately sample diversity can be prohibitively time-consuming and expensive. While sequencing directly from a mixed population will show variants among reads, they cannot be linked to reveal allele combinations associated with particular traits or phylogenetic inheritance patterns. Here, we describe the theory and method of how population sequencing directly from a mixed sample can be used in conjunction with sequencing a very small number of colonies to describe the phylogenetic diversity of a population without haplotype reconstruction. To demonstrate the utility of population sequencing in capturing phylogenetic diversity, we compared isogenic clones to population sequences of Burkholderia pseudomallei from the sputum of a single patient. We also analyzed population sequences of Staphylococcus aureus derived from different people and different body sites. Sequencing results confirm our ability to capture and characterize phylogenetic diversity in our samples. Our analyses of B. pseudomallei populations led to the surprising discovery that the pathogen population is highly structured in sputum, suggesting that for some pathogens, sputum sampling may preserve structuring in the lungs and thus present a non-invasive alternative to understanding colonization, movement, and pathogen/host interactions. Our analyses of S. aureus samples show how comparing phylogenetic diversity across populations can reveal directionality of transmission between hosts and across body sites, demonstrating the power and utility for characterizing the spread of disease and identification of reservoirs at the finest levels. We anticipate that population sequencing and analysis can be broadly applied to accelerate research in a broad range of fields reliant on a foundational understanding of population diversity.

Engagement and partnership with consumers and communities in the co-design and conduct of Research: Lessons from the INtravenous iron polymaltose for First Nations Australian patients with high FERRitin levels on haemodialysis (INFERR) clinical trial.

BACKGROUND: Engagement and partnership with consumers and communities throughout research processes produces high quality research meeting community needs and promoting translation of research into improved policy and practice. Partnership is critical in research involving Aboriginal and/or Torres Strait Islander people (First Nations Peoples) to ensure cultural safety. We present lessons from the design, implementation and progress of the National Health and Medical Research Council funded INtravenous iron polymaltose for First Nations Australian patients with high FERRitin levels on hemodialysis (INFERR) clinical trial. MAIN BODY: The trial was designed to understand the benefits and harms of iron therapy in First Nations Australians on haemodialysis with anaemia and hyperferritinaemia. The lack of evidence for treatment was discussed with patients who were potential participants. A key element ensuring safe conduct of the INFERR trial was the establishment of the Indigenous Reference Groups (IRGs) comprising of dialysis patients based in the Top End of Australia and Central Australia. Two IRGs were needed based on advice from First Nations communities and researchers/academics on the project regarding local cultural differences and approaches to trial conduct. The IRGs underpin culturally safe trial conduct by providing input into study materials and translating study findings into effective messages and policies for First Nations dialysis patients. Throughout the trial conduct, the IRGs' role has developed to provide key mechanisms for advice and guidance regarding research conduct both in this study and more broadly. Support provided to the IRGs by trial First Nations Research Officers and independent First Nations researchers/academics who simplify research concepts is critical. The IRGs have developed feedback documents and processes to participants, stakeholders, and the renal units. They guarantee culturally safe advice for embedding findings from the trial into clinical practice guidelines ensuring evidence-based approaches in managing anaemia in haemodialysis patients with hyperferritinaemia. CONCLUSION: Active consumer and community partnership is critical in research conduct to ensure research impact. Strong partnership with consumers in the INFERR clinical trial has demonstrated that First Nations Consumers will engage in research they understand, that addresses health priorities for them and where they feel respected, listened to, and empowered to achieve change.

In this paper, we present the importance of actively involving consumers in the planning, implementation and conduct of research using the example of a clinical trial among Aboriginal and/or Torres Strait Australians (First Nations Australians) who have kidney disease and are currently receiving haemodialysis. The study assesses how safe and effective it is for people on dialysis to receive iron given through the vein during dialysis when they have anaemia and high levels of a blood test called ferritin, a test used routinely to measure iron levels. Two consumer reference groups of First Nations patients on dialysis, one based in the Top End of Australia and the other based in Central Australia, are supported by First Nations Research Officers and Research Academics to make sure that the research is performed in a way that involves, respects and values First Nations participation, culture, and knowledge. Active consumer and community partnership in this study has supported robust research governance processes which we believe are crucial for knowledge translation to have a positive impact for patients.

Epidemiology and genetic diversity of Burkholderia pseudomallei from Riau Province, Indonesia.

Melioidosis is a bacterial infection caused by Burkholderia pseudomallei, that is common in tropical and subtropical countries including Southeast Asia and Northern Australia. The magnitude of undiagnosed and untreated melioidosis across the country remains unclear. Given its proximity to regions with high infection rates, Riau Province on Sumatera Island is anticipated to have endemic melioidosis. This study reports retrospectively collected data on 68 culture-confirmed melioidosis cases from two hospitals in Riau Province between January 1, 2009, and December 31, 2021, with full clinical data available on 41 cases. We also describe whole genome sequencing and genotypic analysis of six isolates of B. pseudomallei. The mean age of the melioidosis patients was 49.1 (SD 11.5) years, 85% were male and the most common risk factor was diabetes mellitus (78%). Pulmonary infection was the most common presentation (39%), and overall mortality was 41%. Lung as a focal infection (aOR: 6.43; 95% CI: 1.13-36.59, p = 0.036) and bacteremia (aOR: 15.21; 95% CI: 2.59-89.31, p = 0.003) were significantly associated with death. Multilocus sequence typing analysis conducted on six B.pseudomallei genomes identified three sequence types (STs), namely novel ST1794 (n = 3), ST46 (n = 2), and ST289 (n = 1). A phylogenetic tree of Riau B. pseudomallei whole genome sequences with a global dataset of genomes clearly distinguished the genomes of B. pseudomallei in Indonesia from the ancestral Australian clade and classified them within the Asian clade. This study expands the known presence of B. pseudomallei within Indonesia and confirms that Indonesian B. pseudomallei are genetically linked to those in the rest of Southeast Asia. It is anticipated that melioidosis will be found in other locations across Indonesia as laboratory capacities improve and standardized protocols for detecting and confirming suspected cases of melioidosis are more widely implemented.

Melioidosis Knowledge Awareness in Three Distinct Groups in the Tropical Northern Territory of Australia.

Melioidosis is a potentially life-threatening infection. This study aimed to assess the melioidosis knowledge among distinct participant groups in the tropical Top End of the Northern Territory (NT) of Australia. Participants were categorised into three groups: NT medical students and health research staff (Group 1: Hi-Ed), Aboriginal Rangers and Aboriginal Healthcare Workers (Group 2: Rangers/AHWs), and patients with a history of melioidosis infection (Group 3: Patients). A questionnaire was developed to collect data on demographics, risk and protective factor awareness, and knowledge acquisition sources. We used responses to calculate indices for risk knowledge (RKI), protective knowledge (PKI), overall melioidosis knowledge (MKI), and information sources (ISI). We found that 93.6% of participants in Group 1 (Hi-Ed) said that they had heard of melioidosis, followed by 81.5% in Group 3 (Patients), and 72.0% in Group 2 (Rangers/AHWs). Group 1 (Hi-Ed) participants demonstrated greater knowledge of risk-increasing behaviours but had gaps in knowledge of clinical risks like diabetes. Multiple regression revealed that the number of resources used was the only significant predictor of MKI. There are varying melioidosis knowledge levels across different NT participant groups. Targeted educational interventions are needed to enhance melioidosis awareness. A weblink with an interactive summary of our analysis can be found under Results part.

Severe flea-borne typhus complicated by hemophagocytic lymphohistiocytosis: A case report and review of literature.

Flea-borne typhus (FBT), also known as murine typhus, is a zoonotic infection caused by R. typhi with world-wide distribution. In the United States, the infection is uncommon but remains endemic in some areas, including Los Angeles County. It typically manifests as a benign acute febrile illness but can be complicated in a minority of cases. Associated hemophagocytic lymphohistiocytosis (HLH) has been described in a limited number of cases. Here, we present a case of a patient with FBT complicated by HLH treated empirically with doxycycline with subsequent resolution of HLH. Also included is a review of the literature of other published cases.

Broad spectrum SARS-CoV-2-specific immunity in hospitalized First Nations peoples recovering from COVID-19.

Indigenous peoples globally are at increased risk of COVID-19-associated morbidity and mortality. However, data that describe immune responses to SARS-CoV-2 infection in Indigenous populations are lacking. We evaluated immune responses in Australian First Nations peoples hospitalized with COVID-19. Our work comprehensively mapped out inflammatory, humoral and adaptive immune responses following SARS-CoV-2 infection. Patients were recruited early following the lifting of strict public health measures in the Northern Territory, Australia, between November 2021 and May 2022. Australian First Nations peoples recovering from COVID-19 showed increased levels of MCP-1 and IL-8 cytokines, IgG-antibodies against Delta-RBD and memory SARS-CoV-2-specific T cell responses prior to hospital discharge in comparison with hospital admission, with resolution of hyperactivated HLA-DR+ CD38+ T cells. SARS-CoV-2 infection elicited coordinated ASC, Tfh and CD8+ T cell responses in concert with CD4+ T cell responses. Delta and Omicron RBD-IgG, as well as Ancestral N-IgG antibodies, strongly correlated with Ancestral RBD-IgG antibodies and Spike-specific memory B cells. We provide evidence of broad and robust immune responses following SARS-CoV-2 infection in Indigenous peoples, resembling those of non-Indigenous COVID-19 hospitalized patients.

Melioidosis in Timor-Leste: First Case Description and Phylogenetic Analysis.

Burkholderia pseudomallei, the causative agent of melioidosis, has not yet been reported in Timor-Leste, a sovereign state northwest of Australia. In the context of improved access to diagnostic resources and expanding clinical networks in the Australasian region, we report the first 3 cases of culture-confirmed melioidosis in Timor-Leste. These cases describe a broad range of typical presentations, including sepsis, pneumonia, multifocal abscesses, and cutaneous infection. Phylogenetic analysis revealed that the Timor-Leste isolates belong to the Australasian clade of B. pseudomallei, rather than the Asian clade, consistent with the phylogeographic separation across the Wallace Line. This study underscores an urgent need to increase awareness of this pathogen in Timor-Leste and establish diagnostic laboratories with improved culture capacity in regional hospitals. Clinical suspicion should prompt appropriate sampling and communication with laboratory staff to target diagnostic testing. Local antimicrobial guidelines have recently been revised to include recommendations for empiric treatment of severe sepsis.

Robust and prototypical immune responses toward COVID-19 vaccine in First Nations peoples are impacted by comorbidities.

High-risk groups, including Indigenous people, are at risk of severe COVID-19. Here we found that Australian First Nations peoples elicit effective immune responses to COVID-19 BNT162b2 vaccination, including neutralizing antibodies, receptor-binding domain (RBD) antibodies, SARS-CoV-2 spike-specific B cells, and CD4+ and CD8+ T cells. In First Nations participants, RBD IgG antibody titers were correlated with body mass index and negatively correlated with age. Reduced RBD antibodies, spike-specific B cells and follicular helper T cells were found in vaccinated participants with chronic conditions (diabetes, renal disease) and were strongly associated with altered glycosylation of IgG and increased interleukin-18 levels in the plasma. These immune perturbations were also found in non-Indigenous people with comorbidities, indicating that they were related to comorbidities rather than ethnicity. However, our study is of a great importance to First Nations peoples who have disproportionate rates of chronic comorbidities and provides evidence of robust immune responses after COVID-19 vaccination in Indigenous people.

Exploring Cefiderocol Resistance Mechanisms in Burkholderia pseudomallei.

Cefiderocol is a siderophore cephalosporin designed mainly for treatment of infections caused by ß-lactam and multidrug-resistant Gram-negative bacteria. Burkholderia pseudomallei clinical isolates are usually highly cefiderocol susceptible, with in vitro resistance found in a few isolates. Resistance in clinical B. pseudomallei isolates from Australia is caused by a hitherto uncharacterized mechanism. We show that, like in other Gram-negatives, the PiuA outer membrane receptor plays a major role in cefiderocol nonsusceptibility in isolates from Malaysia.

Development and evaluation of a multiplex serodiagnostic bead assay (BurkPx) for accurate melioidosis diagnosis.

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative soil bacterium well recognized in Southeast Asia and northern Australia. However, wider and expanding global distribution of B. pseudomallei has been elucidated. Early diagnosis is critical for commencing the specific therapy required to optimize outcome. Serological testing using the indirect hemagglutination (IHA) antibody assay has long been used to augment diagnosis of melioidosis and to monitor progress. However, cross reactivity and prior exposure may complicate the diagnosis of current clinical disease (melioidosis). The goal of our study was to develop and initially evaluate a serology assay (BurkPx) that capitalized upon host response to multiple antigens. Antigens were selected from previous studies for expression/purification and conjugation to microspheres for multiantigen analysis. Selected serum samples from non-melioidosis controls and serial samples from culture-confirmed melioidosis patients were used to characterize the diagnostic power of individual and combined antigens at two times post admission. Multiple variable models were developed to evaluate multivariate antigen reactivity, identify important antigens, and determine sensitivity and specificity for the diagnosis of melioidosis. The final multiplex assay had a diagnostic sensitivity of 90% and specificity of 93%, which was superior to any single antigen in side-by-side comparisons. The sensitivity of the assay started at >85% for the initial serum sample after admission and increased to 94% 21 days later. Weighting antigen contribution to each model indicated that certain antigen contributed to diagnosis more than others, which suggests that the number of antigens in the assay can be decreased. In summation, the BurkPx assay can facilitate the diagnosis of melioidosis and potentially improve on currently available serology assays. Further evaluation is now required in both melioidosis-endemic and non-endemic settings.

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.

Disguised as Ovarian Tumor: A Rare Case of Uterine Abscess Due to Hypervirulent Klebsiella pneumoniae Infection.

Hypervirulent Klebsiella pneumoniae (hvKp) causes invasive infections in the community setting. We report a rare case of uterine abscess due to hvKp, which appeared as a large-sized ovarian tumor-like pelvic mass. A timely laboratory warning of possible hvKp prompted correct diagnosis and helped guide perioperative decision making, contributing to successful treatment.

Melioidosis in the remote Katherine region of northern Australia.

Melioidosis is endemic in the remote Katherine region of northern Australia. In a population with high rates of chronic disease, social inequities, and extreme remoteness, the impact of melioidosis is exacerbated by severe weather events and disproportionately affects First Nations Australians. All culture-confirmed melioidosis cases in the Katherine region of the Australian Top End between 1989-2021 were included in the study, and the clinical features and epidemiology were described. The diversity of Burkholderia pseudomallei strains in the region was investigated using genomic sequencing. From 1989-2021 there were 128 patients with melioidosis in the Katherine region. 96/128 (75%) patients were First Nations Australians, 72/128 (56%) were from a very remote region, 68/128 (53%) had diabetes, 57/128 (44%) had a history of hazardous alcohol consumption, and 11/128 (9%) died from melioidosis. There were 9 melioidosis cases attributable to the flooding of the Katherine River in January 1998; 7/9 flood-associated cases had cutaneous melioidosis, five of whom recalled an inoculating event injury sustained wading through flood waters or cleaning up after the flood. The 126 first-episode clinical B. pseudomallei isolates that underwent genomic sequencing belonged to 107 different sequence types and were highly diverse, reflecting the vast geographic area of the study region. In conclusion, melioidosis in the Katherine region disproportionately affects First Nations Australians with risk factors and is exacerbated by severe weather events. Diabetes management, public health intervention for hazardous alcohol consumption, provision of housing to address homelessness, and patient education on melioidosis prevention in First Nations languages should be prioritised.

What is the Role of Lateral Flow Immunoassay for the Diagnosis of Melioidosis?

Background: Culture of Burkholderia pseudomallei remains the gold standard for diagnosis of melioidosis but is not possible in many resource-limited settings where melioidosis is endemic. Direct identification of B. pseudomallei antigen in clinical samples has been developed using a lateral flow immunoassay (LFA) targeting B. pseudomallei capsular polysaccharide. Methods: We summarized the findings from the 8 studies to date of the Active Melioidosis Detect (AMD) LFA and compared these with our results from 232 patients with culture-confirmed melioidosis. We have also optimized the methodology for testing different clinical samples. Results: Sensitivity and specificity for different samples were broadly similar in our study to those published from Thailand, India, Laos, and Malaysia. One hundred thirty of 232 (56%) of our melioidosis patients were positive on 1 or more AMD tests: 27% for serum (rising to 39% in those with bacteremic melioidosis and 68% in those with septic shock), 63% for urine (72% in bacteremic melioidosis and 90% in septic shock), 85% in sputum that was culture positive, and 83% in pus that was culture positive. Heating sputum and pus samples increased sensitivity. Faint false-positive urine bands seen on earlier AMD versions were not seen when retested using the most recent version, AMD-Plus. Conclusions: While the sensitivity of melioidosis LFA is low overall for blood samples, there is potential for use as a rapid diagnostic: testing serum and urine from those with severe sepsis who may have melioidosis and testing sputum and pus samples from clinically relevant scenarios. Prospective studies of patients with sepsis and other clinical presentations resembling melioidosis are required to ascertain if the specificity of AMD-PLUS is adequate to enable diagnosis of melioidosis with a high positive predictive value.

Melioidosis of the central nervous system; impact of the bimABm allele on patient presentation and outcome.

BACKGROUND: The autotransporter protein Burkholderia intracellular motility A (BimA) facilitates the entry of Burkholderia pseudomallei into the central nervous system (CNS) in mouse models of melioidosis. Its role in the pathogenesis of human cases of CNS melioidosis is incompletely defined. METHODS: Consecutive culture-confirmed cases of melioidosis at two sites in tropical Australia after 1989 were reviewed. Demographic, clinical and radiological data of the patients with CNS melioidosis were recorded. The bimA allele (bimABm or bimABp) of the B. pseudomallei isolated from each patient was determined. RESULTS: Of the 1587 cases diagnosed at the two sites during the study period, 52 (3.3%) had confirmed CNS melioidosis; 20 (38.5%) had a brain abscess, 18 (34.6%) had encephalomyelitis, 4 (7.7%) had isolated meningitis and 10 (19.2%) had extra-meningeal disease. Among the 52 patients, there were 8 (15.4%) deaths; 17/44 (38.6%) survivors had residual disability. The bimA allele was characterized in 47/52; 17/47 (36.2%) had the bimABm allele and 30 (63.8%) had the bimABp allele. Patients with a bimABm variant were more likely to have a predominantly neurological presentation (odds ratio (OR) (95% confidence interval (CI)): 5.60 (1.52-20.61), p=0.01), to have brainstem involvement (OR (95%CI): 7.33 (1.92-27.95), p=0.004) and to have encephalomyelitis (OR (95%CI): 4.69 (1.30-16.95), p=0.02. Patients with a bimABm variant were more likely to die or have residual disability (odds ratio (95%CI): 4.88 (1.28-18.57), p=0.01). CONCLUSIONS: The bimA allele of B. pseudomallei has a significant impact on the clinical presentation and outcome of patients with CNS melioidosis.

Genomic Epidemiology Links Burkholderia pseudomallei from Individual Human Cases to B. pseudomallei from Targeted Environmental Sampling in Northern Australia.

Each case of melioidosis results from a single event when a human is infected by the environmental bacterium Burkholderia pseudomallei. Darwin, in tropical northern Australia, has the highest incidences of melioidosis globally, and the Darwin Prospective Melioidosis Study (DPMS) commenced in 1989, documenting all culture-confirmed melioidosis cases. From 2000 to 2019, we sampled DPMS patients' environments for B. pseudomallei when a specific location was considered to be where infection occurred, with the aim of using genomic epidemiology to understand B. pseudomallei transmission and infecting scenarios. Environmental sampling was performed at 98 DPMS patient sites, where we collected 975 environmental samples (742 soil and 233 water). Genotyping matched the clinical and epidemiologically linked environmental B. pseudomallei for 19 patients (19%), with the environmental isolates cultured from soil (n = 11) and water (n = 8) sources. B. pseudomallei isolates from patients and their local environments that matched on genotyping were subjected to whole-genome sequencing (WGS). Of the 19 patients with a clinical-environmental genotype match, 17 pairs clustered on a Darwin core genome single-nucleotide polymorphism (SNP) phylogeny, later confirmed by single sequence typing (ST) phylogenies and pairwise comparative genomics. When related back to patient clinical scenarios, the matched clinical and environmental B. pseudomallei pairs informed likely modes of infection: percutaneous inoculation, inhalation, and ingestion. Targeted environmental sampling for B. pseudomallei can inform infecting scenarios for melioidosis and dangerous occupational and recreational activities and identify hot spots of B. pseudomallei presence. However, WGS and careful genomics are required to avoid overcalling the relatedness between clinical and environmental isolates of B. pseudomallei.

Addressing personal protective equipment (PPE) decontamination: Methylene blue and light inactivates severe acute respiratory coronavirus virus 2 (SARS-CoV-2) on N95 respirators and medical masks with maintenance of integrity and fit.

OBJECTIVE: The coronavirus disease 2019 (COVID-19) pandemic has resulted in shortages of personal protective equipment (PPE), underscoring the urgent need for simple, efficient, and inexpensive methods to decontaminate masks and respirators exposed to severe acute respiratory coronavirus virus 2 (SARS-CoV-2). We hypothesized that methylene blue (MB) photochemical treatment, which has various clinical applications, could decontaminate PPE contaminated with coronavirus. DESIGN: The 2 arms of the study included (1) PPE inoculation with coronaviruses followed by MB with light (MBL) decontamination treatment and (2) PPE treatment with MBL for 5 cycles of decontamination to determine maintenance of PPE performance. METHODS: MBL treatment was used to inactivate coronaviruses on 3 N95 filtering facepiece respirator (FFR) and 2 medical mask models. We inoculated FFR and medical mask materials with 3 coronaviruses, including SARS-CoV-2, and we treated them with 10 µM MB and exposed them to 50,000 lux of white light or 12,500 lux of red light for 30 minutes. In parallel, integrity was assessed after 5 cycles of decontamination using multiple US and international test methods, and the process was compared with the FDA-authorized vaporized hydrogen peroxide plus ozone (VHP+O3) decontamination method. RESULTS: Overall, MBL robustly and consistently inactivated all 3 coronaviruses with 99.8% to >99.9% virus inactivation across all FFRs and medical masks tested. FFR and medical mask integrity was maintained after 5 cycles of MBL treatment, whereas 1 FFR model failed after 5 cycles of VHP+O3. CONCLUSIONS: MBL treatment decontaminated respirators and masks by inactivating 3 tested coronaviruses without compromising integrity through 5 cycles of decontamination. MBL decontamination is effective, is low cost, and does not require specialized equipment, making it applicable in low- to high-resource settings.