Search

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 β 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.

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.

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.

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.

ObjectivesWhen 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 participantsTrialling 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 measuresPCR 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.Results13 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).ConclusionsOP/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 numberAustralian New Zealand Clinical Trials Registry—ACTRN12620000922976.

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.

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.

Burkholderia pseudomallei can cause healthcare-associated infections outside its recognized tropical zone., Melioidosis usually occurs after environmental exposure to Burkholderia pseudomallei in the tropics. A cluster of 5 cutaneous melioidosis cases occurred in suburban southwest Australia after an earlier case in January 2012. We collected environmental samples at the first patient’s home in January 2012 and from a nearby health center in December 2013 after 2 new cases occurred in the same postal district. We isolated genotypically identical B. pseudomallei from the first patient and 5 other patients in the district. Environmental sampling implicated an opened bottle of saline wound irrigation fluid containing >106 B. pseudomallei/mL. The bottle included instructions to discard within 24 hours of opening. No further cases of B. pseudomallei infection occurred after removing the contaminated bottle. This cutaneous melioidosis cluster demonstrates that B. pseudomallei can survive and disseminate in widely used medical fluids beyond its known geographic distribution, highlighting a need to use these products according to manufacturers’ instructions.

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.

A 35-year-old, G4 P2-1 Australian woman presented with a seven-day history of shoulder pain, fevers, and chills 2 weeks after a vacation during the wet season in Phuket, Thailand. Unexpectedly, she was found to be 16 weeks pregnant. On examination, a fever of 38.3°C and tachycardia of 120 beats per minute were noted. A chest X-ray revealed signs of right upper lobe pneumonia. C-reactive protein (CRP) was elevated to 280 mg/L. Three consecutive blood cultures yielded growth of Burkholderia pseudomallei, confirming a diagnosis of bacteremic melioidosis with pneumonia. The patient resided in a nonendemic area for melioidosis in Perth, Western Australia. It was assumed that exposure to the organism occurred in Phuket. Initially reported susceptibilities for the isolate according to the Clinical and Laboratory Standards Institute (CLSI) guidelines were as follows: imipenem, ceftazidime, doxycycline, amoxicillin/clavulanic acid—all susceptible; meropenem minimum inhibitory concentration (MIC) 2 mg/L (no CLSI guidelines); and trimethoprim/sulfamethoxazole—resistant (MIC > 32 mg/L). Intravenous (IV) meropenem 1 g 8 hourly was commenced, which was subsequently modified to ceftazidime 6 g/day at day 3 and administered as continuous infusion for 30 days. Symptoms resolved and CRP normalized to 1.6 mg/L at day 20 of IV therapy. Following cessation of ceftazidime, oral amoxicillin/clavulanic acid 875/125 mg twice daily (BD) was instituted with a plan to continue for 3 months as eradication therapy. At 19 weeks and 4 days gestation, mild cerebral ventriculomegaly was noted on formal fetal anatomy scan. This persisted at the 23-week ultrasound which confirmed normal fetal growth, generous amniotic fluid volume, and a long closed cervix. Six days after cessation of IV antibiotics, the patient developed fevers, night sweats, suprapubic pain, and threatened premature labor. The gestational age, calculated from the 16-week ultrasound scan, was 23 weeks and 4 days (±7 days), on the cusp of viability. A repeat ultrasound confirmed fetal ventriculomegaly (12 mm atrial width), polyhydramnios, and cervical shortening. Contractions settled initially with nifedipine tocolysis. Maternal corticosteroids were administered to enhance fetal maturation. The CRP was elevated at 131 mg/L. After obtaining blood, vaginal, and urine isolates for culture, 1 g 8 hourly administration of meropenem was commenced. Twelve hours later, uterine contractions returned and premature labor progressed. Magnesium sulfate was administered for fetal neuroprotection. Two doses of meropenem were given to the mother before the infant was born. A live 750 g female infant was delivered vaginally with Apgar scores of 4, 7, and 8 at 1, 5 and 10 minutes, respectively. The infant was intubated, admitted to the neonatal intensive care unit, and commenced on meropenem and gentamicin therapy. Maternal blood, vaginal, urine, and placental cultures all subsequently yielded growth of B. pseudomallei. The following antimicrobials were reported to be susceptible by CLSI guidelines: ceftazidime MIC 2 mg/L, trimethoprim/sulfamethoxazole MIC 0.12 mg/L, doxycycline MIC 2 mg/L, and amoxycillin/clavulanic acid 2 mg/L. Meropenem MIC was 1 mg/L (no interpretative CLSI guidelines). Review of susceptibility testing and repeat testing of a stored initial isolate established that the initially reported MIC > 32 to trimethoprim/sulfamethoxazole was erroneous because of misinterpretation of trailing end points. Placental histology confirmed the presence of acute chorioamnionitis with features of an early fetal inflammatory reaction. Postpartum the mother continued to experience fevers up to 38.9°C. Doxycycline 100 mg BD was added until the fevers ceased. Three days later, repeat blood cultures were negative (day 5 of meropenem therapy). The infant received human donor breast milk until the maternal milk was confirmed to be culture negative for B. pseudomallei. Cultures from the infant yielded B. pseudomallei from a gastric aspirate only. Blood cultures and an endotracheal aspirate yielded no pathogens. Polymerase chain reaction (PCR) for B. pseudomallei was negative on both blood and endotracheal tube secretions. A CRP rise to 26 mg/L at day 1 of life prompted the performance of a lumbar puncture. Cerebrospinal fluid parameters were not suggestive of infection and yielded no bacterial growth. Given the presence of maternal chorioamnionitis, perinatal exposure to infected vaginal secretions, and extreme prematurity of the infant, the infant was treated pre-emptively with 17 days of meropenem at 20 mg/kg 12 hourly from birth. When gut function allowed oral administration, trimethoprim/sulfamethoxazole initially at 4/20 mg/kg BD and then escalating to 6/30 mg/kg BD at 6 weeks of age (supplemented with folic acid) was prescribed to complete a further 3 months of therapy. The mother completed a 6-week course of meropenem 3 g/day via outpatient parenteral therapy. Her intermittent night sweats resolved. The CRP measurement normalized to 10 mg/L at day 22 post reinstitution of IV antibiotics. A CT scan of chest, abdomen, and pelvis did not reveal any ongoing foci of infection. After clarification of susceptibilities, oral trimethoprim/sulfamethoxazole was commenced at a dose of 320/1600 mg BD with folic acid supplementation. A cutaneous delayed-type hypersensitivity reaction prompted a change in maternal therapy to doxycycline at day 12, during which time breast milk was discarded (with the infant receiving donated breast milk) until completion of 3 months of oral therapy. The infant’s clinical course was consistent with that of extreme prematurity complicated by suspected sepsis, including prolonged respiratory support until 36 weeks corrected gestation and administration of 2 courses of postnatal steroids, multiple blood products, and nutritional support. Neonatal MRI demonstrated brain morphology consistent with extreme prematurity, grade 2 intraventricular hemorrhage, and features of periventricular leukomalacia. The infant was discharged from the NICU at day 135 of life. Neurodevelopmental assessment at 12 months corrected gestational age confirmed normal developmental progress. Burkholderia pseudomallei infection did not recur in the mother or infant.

Francisella species are Gram-negative, nonmotile, pleomorphic coccobacilli, facultative intracellular fastidious bacteria. We report the isolation of a Francisella-like species from a blood culture collected from a 44-year-old bacteraemic patient in Perth, Western Australia. The organism was identified to species level by 16S rRNA sequencing and by fatty acid methyl esters analysis. The strain genotypically resembled Francisella hispaniensis, a species previously isolated from human blood in Spain.

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 .

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.

An investigation into the risk of occupationally acquired melioidosis at a mine site in northern Australia found that 45 (13%) of 345 staff had serologic evidence of exposure and 14 (4%) had recent exposure to Burkholderia pseudomallei or closely related bacteria. There was only one culture-confirmed case of melioidosis in mine staff during the study period. The lack of overt infection directly attributable to work activities despite detectable B. pseudomallei on site, the absence of an association between positive serology and occupational activity on site, or duration of employment in the mining industry stand against a significant occupationally acquired infection risk on this industrial site. Workplace exposure to a dust-generating tropical environment in the melioidosis-endemic north of Australia did not appear to pose a measurable increase in infection risk. The effect of long-term climatic trends on this potential biologic threat requires further study.

This study describes the isolation and characterization of several Burkholderia species from soil in northern Australia. Phenotypic and molecular tests indicate that these isolates belong to the species Burkholderia thailandensis and Burkholderia ubonensis. These observations significantly extend our knowledge of the geographic distribution of these 2 species. Evidence of these species in Australia has implications for bacterial identification in clinical laboratories, diagnostic serology tests, and environmental biodiversity studies.

Limited experience and a lack of validated diagnostic reagents make Burkholderia pseudomallei , the cause of melioidosis, difficult to recognize in the diagnostic microbiology laboratory. We compared three methods of confirming the identity of presumptive B. pseudomallei strains using a collection of Burkholderia species drawn from diverse geographic, clinical, and environmental sources. The 95 isolates studied included 71 B. pseudomallei and 3 B. thailandensis isolates. The API 20NE method identified only 37% of the B. pseudomallei isolates. The agglutinating antibody test identified 82% at first the attempt and 90% including results of a repeat test with previously negative isolates. Gas-liquid chromatography analysis of bacterial fatty acid methyl esters (GLC-FAME) identified 98% of the B. pseudomallei isolates. The agglutination test produced four false positive results, one B. cepacia , one B. multivorans , and two B. thailandensis . API produced three false positive results, one positive B. cepacia and two positive B. thailandensis . GLC-FAME analysis was positive for one B. cepacia isolate. On the basis of these results, the most robust B. pseudomallei discovery pathway combines the previously recommended isolate screening tests (Gram stain, oxidase test, gentamicin and polymyxin susceptibility) with monoclonal antibody agglutination on primary culture, followed by a repeat after 24 h incubation on agglutination-negative isolates and GLC-FAME analysis. Incorporation of PCR-based identification within this schema may improve percentages of recognition further but requires more detailed evaluation.

Here, we present the draft genome sequences of 80 isolates of Burkholderia pseudomallei . The isolates represent clinical cases of melioidosis and environmental isolates from regions in Australia and Papua New Guinea where B. pseudomallei is endemic. The genomes provide further context for the diversity of the pathogen.

The invasive Burkholderia species, B. pseudomallei and B. mallei, cause a remarkably wide range of disease outcomes, varying from subacute, localized inflammation to severe organ system damage and overwhelming septicaemia. Significant advances in understanding the molecular basis of B. pseudomallei and B. mallei pathogenesis have followed the publication of the first fully annotated genomes. Transcriptomic and proteomic insights have started to find their place in a multiscale appreciation of invasive Burkholderia infection. These advances have had practical consequences in the clinical laboratory. A range of nucleic acid amplification tests are currently in use and proteome analysis by MALDI-TOF mass spectrometer is now available for rapid clinical laboratory identification of suspected isolates. We expect the gathering pace of integrated systems biology to shed further light on the molecular biology of invasive Burkholderia infection, but its role in clinical laboratory practice has yet to be determined.

Burkholderia pseudomallei, the bacterial cause of the potentially fatal infection known as melioidosis, has a facultative intracellular lifestyle. The intracellular presence of B. pseudomallei in various eukaryotes including arbuscular mycorrhizal fungus (AMF) spores can be demonstrated in vitro. AMF spores were isolated from soils in a melioidosis-endemic area. B. pseudomallei and other Burkholderia spp. DNA was detected in these AMF spore samples, confirming an AMF spore-Burkholderia spp. association in soils which did not yield Burkholderia spp. by culture. This association may explain the environmental persistence, difficulty of recovery and dispersal of Burkholderia spp. in specific environments.

Melioidosis became a notifiable disease in Western Australia (WA) 2 years after the West Kimberley melioidosis outbreak. Two cases of melioidosis caused by the outbreak genotype of Burkholderia pseudomallei (National Collection of Type Cultures [NCTC] 13177) occurred in 1998 and 1999 in persons who visited the outbreak location at the time. No other infections caused by the outbreak strain have been recorded in WA since that time, despite an average of four culture-positive cases per year. Sporadic cases of melioidosis often follow tropical storms and cyclones during summer, and they have been detected outside the endemic area when cyclones travel far inland. In 2007, environmental isolates resembling NCTC 13177 were found 500 km east of the outbreak location after unusually severe weather. Recent whole-genome analysis places NCTC 13177 genetically close to other Australian isolates. Additional biogeographic and ecological studies are needed to establish the relative importance of environmental cofactors in disease pathogenesis.

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.

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.

The virulence factor genotypes of a large cohort of clinical, nosocomial environment and community environment isolates (184 in total) of Pseudomonas aeruginosa from Tasmania, Australia, were determined by PCR. The virulence factor genotype of the majority of isolates was highly conserved, with the exception of the virulence gene exoU, which demonstrated low prevalence (33 isolates; 18 %) in the population tested. Isolates collected from the environment of intensive therapy wards (intensive care unit and neurosurgical units) of the major tertiary referral hospital in Tasmania were found to be more likely (PPexoU than all other isolates. Adult cystic fibrosis isolates showed a decreased prevalence of the exoU gene (PP

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.

An investigation into the risk of occupationally acquired melioidosis at a mine site in northern Australia found that 45 (13%) of 345 staff had serologic evidence of exposure and 14 (4%) had recent exposure to Burkholderia pseudomallei or closely related bacteria. There was only one culture-confirmed case of melioidosis in mine staff during the study period. The lack of overt infection directly attributable to work activities despite detectable B. pseudomallei on site, the absence of an association between positive serology and occupational activity on site, or duration of employment in the mining industry stand against a significant occupationally acquired infection risk on this industrial site. Workplace exposure to a dust-generating tropical environment in the melioidosis-endemic north of Australia did not appear to pose a measurable increase in infection risk. The effect of long-term climatic trends on this potential biologic threat requires further study.

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.

This study describes the isolation and characterization of several Burkholderia species from soil in northern Australia. Phenotypic and molecular tests indicate that these isolates belong to the species Burkholderia thailandensis and Burkholderia ubonensis. These observations significantly extend our knowledge of the geographic distribution of these 2 species. Evidence of these species in Australia has implications for bacterial identification in clinical laboratories, diagnostic serology tests, and environmental biodiversity studies.

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. As técnicas de amplificação de DNA estão sendo cada vez mais utilizadas em laboratórios clínicos para a confirmação da identificação de bactérias que têm importância médica. Um método de identificação de Burkholderia pseudomallei baseado em PCR tem sido usado em nosso centro há 10 anos e foi utilizado para confirmar a identificação de bactérias isoladas de casos de melioidose no Ceará desde 2003. Este método particular tem sido usado como padrão ouro para métodos menos discriminatórios. Nesse estudo, avaliamos três métodos de identificação de B. pseudomallei baseados em PCR e usamos seqüenciamento de DNA para solucionar discrepâncias entre os resultados baseados em PCR e os métodos de identificação fenotípica. O estabelecido protocolo de PCR semi-nested para a região espacial 16-23s da B. pseudomallei produziu um consistente resultado negativo para um de nossos 100 isolados testados (BCC#99), mas identificou corretamente todos os outros 71 isolados de B. pseudomallei. Uma variação anômala da seqüência foi detectada na região interna do sítio de ligação do primer reverso para este método. Métodos de PCR foram desenvolvidos para a detecção de outros dois genes bacterianos metabólicos de B. pseudomallei. O protocolo de PCR IpxO convencional teve sensibilidade de 0,89 e especificidade de 1,0, enquanto que o PCR em tempo real mostrou-se ainda melhor, com sensibilidade de 1,0 e especificidade de 1,0. Este método identificou todos os isolados de B. pseudomallei, incluindo o isolado discrepante que teve o PCR negativo. O protocolo de PCR phaC detectou o gene de todos os B. pseudomallei e em todos exceto três isolados de B. cepacia, tornando este método de identificação de B. pseudomallei baseado em PCR inadequado. Esta experiência com métodos de identificação de B. pseudomallei baseados em PCR indica que devemos ter precaução quando estes forem utilizados sozinhos para identificação dessa bactéria e que eles necessitam ser interpretados em conjunto com métodos fenotípicos e moleculares alternativos, tais como seqüenciamento genético.

The facultative anaerobic Gram-negative species Escherichia albertii has been isolated from human faeces in gastrointestinal infection and from a range of wild bird species. Here we report the first case of a febrile infection associated with E. albertii bacteraemia in a 76-year-old woman with gastric dysplasia.