Molecular types of Cryptococcus neoformans and Cryptococcus gattii in Western Australia and correlation with antifungal susceptibility.

Cryptococcus neoformans and Cryptococcus gattii species complexes have a worldwide distribution; however, there is geographical variation in the prevalence of different molecular types. Additionally, antifungal susceptibility differences between molecular types have been demonstrated. This study investigates the distribution of cryptococcal molecular types among human clinical isolates over a 10-year period from a Western Australian population. Molecular type was determined based on polymorphisms in the phospholipase gene locus identified through amplification and sequencing. Minimum inhibitory concentrations (MICs) were identified for fluconazole, 5-fluorocytosine, posaconazole, itraconazole, voriconazole, and amphotericin B. Most isolates were C. neoformans complex (42) of which over half were molecular type VNI (22) followed by VNII (20). Among the remaining C. gattii complex (13) the majority were VGI (11) with VGII (2) uncommonly found. All isolates demonstrated low MICs to antifungal agents including fluconazole. Geometric mean MIC values against 5-fluorocytosine for VNI (1.741 mg/l) were significantly higher than those for VGI (0.47 mg/l, P = .002). Similarly fluconazole geometric mean MICs against fluconazole for VNI (2.3 mg/l) were significantly higher than VNII (0.87 mg/l, P = .036). These data reveal the presence of four molecular types (VNI, VNII, VGI and VGII) within clinical Western Australian cryptococcal isolates and, while elevated antifungal MICs were not encountered, significant molecular type dependent differences in susceptibility were found.

Identification of multiple species and subpopulations among Australian clinical Sporothrix isolates using whole genome sequencing.

Whole genome sequencing (WGS) was used to demonstrate the wide genetic variability within Sporothrix schenckii sensu lato and establish that there are two main species of Sporothrix within Australian clinical isolates-S. schenckii sensu stricto and Sporothrix globosa. We also demonstrated southwest Western Australia contained genetically similar S. schenckii ss strains that are distinct from strains isolated in the eastern and northern states of Australia. Some genetic clustering by region was also noted for northern NSW, Queensland, and Northern Territory. Phylogenetic analysis of WGS data provided greater phylogenetic resolution compared to analysis of the calmodulin gene alone.

Legionnaires' Disease Outbreak on a Merchant Vessel, Indian Ocean, Australia, 2015.

Two cases of Legionnaires' disease and 1 of Pontiac fever occurred among the crew of a merchant ship operating off the shores of Australia. PCR assays identified potential sources in the ship's cabins. Modification of maritime regulations for Legionnaires' disease prevention in commercial vessels is needed for nonpassenger merchant ships.

Clinical features and outcome of patients with cutaneous melioidosis during a nosocomial outbreak in a temperate region of Australia.

Six cases of cutaneous melioidosis from southwestern Australia, a non-endemic region occurred as a result of Burkholderia pseudomallei contamination of normal saline that was used for irrigating superficial wounds. Treatment with parenteral meropenem, given by continuous infusion for 2 weeks, followed by oral antibiotics was successful in all cases.

Volatile-sulfur-compound profile distinguishes Burkholderia pseudomallei from Burkholderia thailandensis.

Solid-phase microextraction gas chromatography-mass spectrometry (SPME-GCMS) was used to show that dimethyl sulfide produced by Burkholderia pseudomallei is responsible for its unusual truffle-like smell and distinguishes the species from Burkholderia thailandensis. SPME-GCMS can be safely used to detect dimethyl sulfide produced by agar-grown B. pseudomallei.

Plasma cfDNA predictors of established bacteraemic infection.

Introduction. Increased plasma cell-free DNA (cfDNA) has been reported for various diseases in which cell death and tissue/organ damage contribute to pathogenesis, including sepsis. Gap Statement. While several studies report a rise in plasma cfDNA in bacteraemia and sepsis, the main source of cfDNA has not been identified. Aim. In this study, we wanted to determine which of nuclear, mitochondrial or bacterial cfDNA is the major contributor to raised plasma cfDNA in hospital subjects with bloodstream infections and could therefore serve as a predictor of bacteraemic disease severity. Methodology. The total plasma concentration of double-stranded cfDNA was determined using a fluorometric assay. The presence of bacterial DNA was identified by PCR and DNA sequencing. The copy numbers of human genes, nuclear ß globin and mitochondrial MTATP8, were determined by droplet digital PCR. The presence, size and concentration of apoptotic DNA from human cells were established using lab-on-a-chip technology. Results. We observed a significant difference in total plasma cfDNA from a median of 75 ng ml-1 in hospitalised subjects without bacteraemia to a median of 370 ng ml-1 (P=0.0003) in bacteraemic subjects. The copy numbers of nuclear DNA in bacteraemic also differed between a median of 1.6 copies µl-1 and 7.3 copies µl-1 (P=0.0004), respectively. In contrast, increased mitochondrial cfDNA was not specific for bacteraemic subjects, as shown by median values of 58 copies µl-1 in bacteraemic subjects, 55 copies µl-1 in other hospitalised subjects and 5.4 copies µl-1 in healthy controls. Apoptotic nucleosomal cfDNA was detected only in a subpopulation of bacteraemic subjects with documented comorbidities, consistent with elevated plasma C-reactive protein (CRP) levels in these subjects. No bacterial cfDNA was reliably detected by PCR in plasma of bacteraemic subjects over the course of infection with several bacterial pathogens. Conclusions. Our data revealed distinctive plasma cfDNA signatures in different groups of hospital subjects. The total cfDNA was significantly increased in hospital subjects with laboratory-confirmed bloodstream infections comprising nuclear and apoptotic, but not mitochondrial or bacterial cfDNAs. The apoptotic cfDNA, potentially derived from blood cells, predicted established bacteraemia. These findings deserve further investigation in different hospital settings, where cfDNA measurement could provide simple and quantifiable parameters for monitoring a disease progression.

Acceptability of OP/Na swabbing for SARS-CoV-2: a prospective observational cohort surveillance study in Western Australian schools.

OBJECTIVES: When the COVID-19 pandemic was declared, Governments responded with lockdown and isolation measures to combat viral spread, including the closure of many schools. More than a year later, widespread screening for SARS-CoV-2 is critical to allow schools and other institutions to remain open. Here, we describe the acceptability of a minimally invasive COVID-19 screening protocol trialled by the Western Australian Government to mitigate the risks of and boost public confidence in schools remaining open. To minimise discomfort, and optimise recruitment and tolerability in unaccompanied children, a combined throat and nasal (OP/Na) swab was chosen over the nasopharyngeal swab commonly used, despite slightly reduced test performance. DESIGN, SETTING AND PARTICIPANTS: Trialling of OP/Na swabbing took place as part of a prospective observational cohort surveillance study in 79 schools across Western Australia. Swabs were collected from 5903 asymptomatic students and 1036 asymptomatic staff in 40 schools monthly between June and September 2020. OUTCOME MEASURES: PCR testing was performed with a two-step diagnostic and independent confirmatory PCR for any diagnostic PCR positives. Concurrent surveys, collected online through the REDCap platform, evaluated participant experiences of in-school swabbing. RESULTS: 13 988 swabs were collected from students and staff. There were zero positive test results for SARS-CoV-2, including no false positives. Participants reported high acceptability: 71% of students reported no or minimal discomfort and most were willing to be reswabbed (4% refusal rate). CONCLUSIONS: OP/Na swabbing is acceptable and repeatable in schoolchildren as young as 4 years old and may combat noncompliance rates by significantly increasing the acceptability of testing. This kind of minimally-invasive testing will be key to the success of ongoing, voluntary mass screening as society adjusts to a new 'normal' in the face of COVID-19. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry-ACTRN12620000922976.

Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

Accurate and rapid diagnostic tests are a critical component for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of the overall control strategy for the current pandemic. Nucleic acid amplification tests are the gold standard for diagnosis of acute SARS-CoV-2 infection, and many real-time PCR diagnostic assays have been developed. Mutations that occur within the primer/probe binding regions of the SARS-CoV-2 genome can negatively impact the performance of diagnostic assays. Here, we report two single-point mutations in the N gene of SARS-CoV-2 associated with N gene target detection failures in the Cepheid Xpert Xpress SARS-CoV-2 assay, the first a C to T mutation at position 29197, found in five patients, and the second a C to T mutation at position 29200, found in eight patients. By sequencing the Xpert amplicons, we showed both mutations to be located within the amplified region of the Xpert N gene target. This report highlights the necessity for multiple genetic targets and the continual monitoring and evaluation of diagnostic assay performance. IMPORTANCE This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases. This report is the first to our knowledge which characterizes the amplified PCR products of the Xpert system, confirming the mutations associated with the gene target failure. The mutations identified have previously been reported.

High-redundancy draft sequencing of 15 clinical and environmental Burkholderia strains.

The Gram-negative Burkholderia genus includes several species of intracellular bacterial pathogens that pose substantial risk to humans. In this study, we have generated draft genome sequences of 15 strains of B. oklahomensis, B. pseudomallei, B. thailandensis, and B. ubonensis to an average sequence read coverage of 25- to 40-fold.

Multiplex amplified nominal tandem-repeat analysis (MANTRA), a rapid method for genotyping Mycobacterium tuberculosis by use of multiplex PCR and a microfluidic laboratory chip.

A variable-number tandem-repeat genotyping method for Mycobacterium tuberculosis was converted to run in a multiplex PCR format on a 12-well microfluidic laboratory chip. Epidemiologically and genotypically distinct isolate clusters of M. tuberculosis were identified. This rapid genotyping method has potential application in smaller clinical laboratories and public health field investigations.

Inactivation of virulent Burkholderia pseudomallei by sunlight.

The goal of this study was to determine the sensitivity of virulent Burkholderia pseudomallei to natural sunlight. We describe solar dosimetry calibrated to integrate radiation between 295 and 305 nm and an exposure system that minimizes thermal effects on bacterial cells. Burkholderia pseudomallei cells were either exposed to sunlight in UV transparent dishes or maintained in the dark covered by opaque foil. The cells maintained in the dark remained at constant levels for the duration of all experiments. The exposed cells nearby were killed with a kinetic studied through 5 Log10 inactivation. We found that cells in stationary phase of growth were nearly two-fold more resistant to sunlight than cells in lag or exponential growth. A virulent strain of B. pseudomallei that produced mucoid colonies showed sensitivity to sunlight similar to both a virulent strain that produced nonmucoid colonies and a strain of B. thailandensis. The inactivation of B. pseudomallei by sunlight in different types of water of environmental relevance or inside amoebae was investigated. The sensitivity of virulent B. pseudomallei was calculated and its comparison with previous studies employing monochromatic germicidal light (254 nm) is discussed. This may be the first report in the open literature of the inactivation of a virulent biological threat agent by natural sunlight. These data should assist in estimating the risk for contracting melioidosis and in predicting the time period during which B. pseudomallei remains infectious after an accidental or intentional release in the environment.

Deployable laboratory response to emergence of melioidosis in central Sri Lanka.

A portable molecular diagnostic laboratory was used to provide molecular confirmation of suspected melioidosis cases seen at Peradeniya Hospital, central Sri Lanka. Soil supernatants from rice field and rubber plantation samples also produced PCR-positive results. These procedures could be used for melioidosis field work in other remote locations.

Case Report: Chorioamnionitis and Premature Delivery due to Burkholderia pseudomallei Infection in Pregnancy.

We report a case of placental infection leading to preterm delivery in a mother diagnosed with septicemia and pneumonia due to Burkholderia pseudomallei in pregnancy. Placental infection occurred despite prolonged ceftazidime therapy.

Clinical, Bacteriologic, and Geographic Stratification of Melioidosis Emerges from the Sri Lankan National Surveillance Program.

Melioidosis, a potentially fatal tropical infection, is said to be underdiagnosed in low-income countries. An increase in melioidosis cases in Sri Lanka allowed us to analyze the relationship among clinical outcome, bacteriology, epidemiology, and geography in the first 108 laboratory-confirmed cases of melioidosis from a nationwide surveillance program. The additional 76 cases of laboratory-confirmed melioidosis confirmed further associations between Burkholderia pseudomallei multilocus sequence typing (MLST) and infection phenotype; ST1137/unifocal bacteremic infection (χ2 = 3.86, P < 0.05), ST1136/multifocal infection without bacteremia (χ2 = 15.8, P < 0.001), and ST1132/unifocal nonbacteremic infection (χ2 = 6.34, P = 0.02). ST1137 infections were predominantly seen in the Western Province, whereas ST1132, 1135, and 1136 infections predominated in the Northwestern Province. Early participating centers in the surveillance program had a lower melioidosis-associated mortality than later participants (χ2 = 3.99, P < 0.05). The based upon related sequence types (eBURST) algorithm, a MLST clustering method that infers founding genotypes and patterns of descent for related isolates and clonal complexes in an unrooted tree, showed uneven distribution of sequence types (STs). There was spatial clustering of the commonest STs (ST1132, 1136, and 1137) in the Western, Northwestern, and Central provinces. The recent increase in melioidosis in Sri Lanka uncovered by laboratory-enhanced surveillance is likely to be the result of a combination of improved laboratory detection, increased clinician awareness, recruitment of clinical centers, and small outbreaks. Further development of the surveillance program into a national genotyping-supported melioidosis registry will improve melioidosis diagnosis, treatment, and prevention where underdiagnosis and mortality rates remain high.

The Role of Climate in the Epidemiology of Melioidosis.

PURPOSE OF REVIEW: Melioidosis epidemiology is susceptible to climate change through direct and indirect effects on human encounter with the causative agent, Burkholderia pseudomallei. This review describes the current depth of knowledge and recent advances in the understanding of this relationship and applies it to observations of melioidosis in Western Australia. RECENT FINDINGS: High maximum rainfall and dense cloud cover have been shown to predict environmental presence of B. pseudomallei and cases of melioidosis, probably through correspondingly high moisture levels in B. pseudomallei-receptive soils. Increased melioidosis cases have been observed following storms in Taiwan and cyclones in the Australian Northern Territory and strengthen the association between melioidosis and extreme weather events. Indirect weather effects contribute to bacterial exposure through mechanisms such as increasing B. pseudomallei output from water seeps after heavy rain or localised flooding. Climate and weather have been directly implicated in dissemination of B. pseudomallei and cases of melioidosis in several notable events in Western Australia. Over a 10-year surveillance period, the cases that lay in the path of a tropical cyclone co-located with cyclone systems that repeatedly crossed the Western Australian coast. Cyclone-associated cases were caused by different B. pseudomallei MLST genotypes, arguing against airborne dissemination from a common source. SUMMARY: Predicted increases in temperature, changes in global precipitation patterns and an increased incidence of extreme weather events are expected to change melioidosis epidemiology. Further studies of the physical geographic drivers of melioidosis will deepen understanding of the impact of climate on melioidosis.

PCR-based identification of Burkholderia pseudomallei.

DNA amplification techniques are being used increasingly in clinical laboratories to confirm the identity of medically important bacteria. A PCR-based identification method has been in use in our centre for 10 years for Burkholderia pseudomallei and was used to confirm the identity of bacteria isolated from cases of melioidosis in Ceará since 2003. This particular method has been used as a reference standard for less discriminatory methods. In this study we evaluated three PCR-based methods of B. pseudomallei identification and used DNA sequencing to resolve discrepancies between PCR-based results and phenotypic identification methods. The established semi-nested PCR protocol for B. pseudomallei 16-23s spacer region produced a consistent negative result for one of our 100 test isolates (BCC #99), but correctly identified all 71 other B. pseudomallei isolates tested. Anomalous sequence variation was detected at the inner, reverse primer binding site for this method. PCR methods were developed for detection of two other B. pseudomallei bacterial metabolic genes. The conventional lpxO PCR protocol had a sensitivity of 0.89 and a specificity of 1.00, while a real-time lpxO protocol performed even better with sensitivity and specificity of 1.00, and 1.00. This method identified all B. pseudomallei isolates including the PCR-negative discrepant isolate. The phaC PCR protocol detected the gene in all B. pseudomallei and all but three B. cepacia isolates, making this method unsuitable for PCR-based identification of B. pseudomallei. This experience with PCR-based B. pseudomallei identification methods indicates that single PCR targets should be used with caution for identification of these bacteria, and need to be interpreted alongside phenotypic and alternative molecular methods such as gene sequencing.

Sri Lankan National Melioidosis Surveillance Program Uncovers a Nationwide Distribution of Invasive Melioidosis.

The epidemiologic status of melioidosis in Sri Lanka was unclear from the few previous case reports. We established laboratory support for a case definition and started a nationwide case-finding study. Suspected Burkholderia pseudomallei isolates were collated, identified by polymerase chain reaction assay, referred for Matrix Assisted Laser Desorption Ionization-Time of Flight analysis and multilocus sequence typing (MLST), and named according to the international MLST database. Between 2006 and early 2014, there were 32 patients with culture-confirmed melioidosis with an increasing annual total and a falling fatality rate. Patients were predominantly from rural communities, diabetic, and male. The major clinical presentations were sepsis, pneumonia, soft tissue and joint infections, and other focal infection. Burkholderia pseudomallei isolates came from all parts of Sri Lanka except the Sabaragamuwa Province, the south central hill country, and parts of northern Sri Lanka. Bacterial isolates belonged to 18 multilocus sequence types, one of which (ST 1137) was associated with septicemia and a single-organ focus (Fisher's exact, P = 0.004). Melioidosis is an established endemic infection throughout Sri Lanka, and is caused by multiple genotypes of B. pseudomallei, which form a distinct geographic group based upon related sequence types (BURST) cluster at the junction of the southeast Asian and Australasian clades.

New Foci of Spotted Fever Group Rickettsiae Including Rickettsia honei in Western Australia.

We describe the first reported case of spotted fever group rickettsiosis in Western Australia, and two cases of probable Rickettsia honei from a new geographic focus. These findings highlight the need to raise awareness of ricksettsial infection among local clinicians as well as those treating visitors to this region, important for outdoor recreation.

Deployable Molecular Detection of Arboviruses in the Australian Outback.

The most common causes of human infection from the arboviruses that are endemic in Australia are the arthritogenic alphaviruses: Ross River virus (RRV) and Barmah Forest virus (BFV). The most serious infections are caused by the neurotropic flaviviruses, Murray Valley encephalitis virus (MVEV) and the Kunjin subtype of West Nile virus. The greatest individual risk of arbovirus infection occurs in tropical/subtropical northern Australia because of the warm, wet summer conditions from December to June, where conventional arbovirus surveillance is difficult due to a combination of low population density, large distances between population centers, poor roads, and seasonal flooding. Furthermore, virus detection requires samples to be sent to Perth up to 2,000 km away for definitive analysis, causing delays of days to weeks before test results are available and public health interventions can be started. We deployed a portable molecular biology laboratory for remote field detection of endemic arboviruses in northern Queensland, then in tropical Western Australia and detected BFV, MVEV, and RRV RNA by polymerase chain reaction (PCR) assays of extracts from mosquitoes trapped in Queensland. We then used a field-portable compact real-time thermocycler for the samples collected in the Kimberley region of Western Australia. Real-time field PCR assays enabled concurrent endemic arbovirus distribution mapping in outback Queensland and Western Australia. Our deployable laboratory method provides a concept of operations for future remote area arbovirus surveillance.