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1.  Acanthamoeba Encephalitis: Isolation of Genotype T1 in Mycobacterial Liquid Culture Medium 
Journal of Clinical Microbiology  2014;53(2):735-739.
We report a case of Acanthamoeba encephalitis diagnosed from an antemortem brain biopsy specimen, where the organism was first isolated in mycobacterial liquid medium and first identified by using a sequence generated by a commercial panfungal sequencing assay. We correlate susceptibility results with clinical outcome.
PMCID: PMC4298535  PMID: 25502534
2.  Rapid Time to Results and High Sensitivity of the CarbaNP Test on Early Cultures 
Journal of Clinical Microbiology  2014;52(11):4023-4026.
For rapid results, we adapted the CarbaNP test by using 5-hour-old cultures; the sensitivity and specificity for detection of carbapenemase production were 100% for non-OXA-producing organisms. The sensitivity from early cultures was 94% for detection of carbapenemase production in Enterobacteriaceae strains. Utilizing younger cultures allows the test to be incorporated into a single day's workflow, facilitating timely patient care and antimicrobial stewardship.
PMCID: PMC4313238  PMID: 25143572
3.  Genetic and Molecular Predictors of High Vancomycin MIC in Staphylococcus aureus Bacteremia Isolates 
Journal of Clinical Microbiology  2014;52(9):3384-3393.
An elevated vancomycin MIC is associated with poor outcomes in Staphylococcus aureus bacteremia (SAB) and is reported in patients with methicillin-susceptible S. aureus (MSSA) bacteremia in the absence of vancomycin treatment. Here, using DNA microarray and phenotype analysis, we investigated the genetic predictors and accessory gene regulator (agr) function and their relationship with elevated vancomycin MIC using blood culture isolates from a multicenter binational cohort of patients with SAB. Specific clonal complexes were associated with elevated (clonal complex 8 [CC8] [P < 0.001]) or low (CC22 [P < 0.001], CC88 [P < 0.001], and CC188 [P = 0.002]) vancomycin MIC. agr dysfunction (P = 0.014) or agr genotype II (P = 0.043) were also associated with an elevated vancomycin MIC. Specific resistance and virulence genes were also linked to an elevated vancomycin MIC, including blaZ (P = 0.002), sea (P < 0.001), clfA (P < 0.001), splA (P = 0.001), and the arginine catabolic mobile element (ACME) locus (P = 0.02). These data suggest that inherent organism characteristics may explain the link between elevated vancomycin MICs and poor outcomes in patients with SAB, regardless of the antibiotic treatment received. A consideration of clonal specificity should be included in future research when attempting to ascertain treatment effects or clinical outcomes.
PMCID: PMC4313166  PMID: 25031442
4.  Utility of EBUS-TBNA for diagnosis of mediastinal tuberculous lymphadenitis: a multicentre Australian experience 
Journal of Thoracic Disease  2015;7(3):439-448.
Endobronchial ultrasound (EBUS) transbronchial needle aspiration (TBNA) is an important diagnostic procedure for the interrogation of mediastinal lymph nodes. There is limited data describing the accuracy & safety of this technique for the diagnosis of tuberculous mediastinal lymphadenitis.
A multi-centre retrospective study of all EBUS-guided TBNA procedures that referred samples for mycobacteriology was performed. Results were correlated with post-procedural diagnoses after a period of surveillance and cross-checked against relevant statewide tuberculosis (TB) registries, and sensitivity and specificity was calculated. In addition, nucleic acid amplification techniques (NAAT) were assessed, and sensitivity and specificity calculated using positive mycobacterial culture as the reference gold standard.
One hundred and fifty-nine patients underwent EBUS-TBNA and had tissue referred for mycobacterial culture, of which 158 were included in the final analysis. Thirty-nine were ultimately diagnosed with TB (25%). Sensitivity of EBUS-TBNA for microbiologically confirmed tuberculous mediastinal lymphadenitis was 62% (24/39 cases). Specificity was 100%. Negative predictive value (NPV) and diagnostic accuracy for microbiologic diagnosis was 89% [95% confidence intervals (CI), 82-93%] and 91% (95% CI, 84-94%) respectively. For a composite clinicopathologic diagnosis of TB NPV and accuracy were 98% (95% CI, 93-99%) and 98% (95% CI, 95-99%) respectively. Sensitivity for NAAT was 38% (95% CI, 18-65%).
EBUS-TBNA is a safe and well tolerated procedure in the assessment of patients with suspected isolated mediastinal lymphadenitis and demonstrates good sensitivity for a microbiologic diagnosis of isolated mediastinal lymphadenitis. When culture and histological results are combined with high clinical suspicion, EBUS-TBNA demonstrates excellent diagnostic accuracy and NPV for the diagnosis of mediastinal TB lymphadenitis. We suggest EBUS-TBNA should be considered the procedure of choice for patients in whom TB is suspected.
PMCID: PMC4387413  PMID: 25922723
Endobronchial ultrasound (EBUS); minimally invasive; bronchoscopy; tuberculosis (TB); mycobacteria; culture; PCR
5.  Streptococcus pyogenes aortic aneurysm infection: forgotten but not gone 
Infectious Disease Reports  2013;5(2):e11.
Historically, Streptococcus pyogenes was a common cause of endocarditis and infected aortic aneurysm. Today, endovascular infections due to this organism have become exceedingly rare. We report the first case of aortic aneurysm infection due to S. pyogenes treated with initial endoluminal repair, review previous reports and discuss current treatment options.
PMCID: PMC3892607  PMID: 24470961
Streptococcus pyogenes; aortic aneurysm infection; mycotic aneurysm
6.  Vancomycin AUC/MIC Ratio and 30-Day Mortality in Patients with Staphylococcus aureus Bacteremia 
A ratio of the vancomycin area under the concentration-time curve to the MIC (AUC/MIC) of ≥400 has been associated with clinical success when treating Staphylococcus aureus pneumonia, and this target was recommended by recently published vancomycin therapeutic monitoring consensus guidelines for treating all serious S. aureus infections. Here, vancomycin serum trough levels and vancomycin AUC/MIC were evaluated in a “real-world” context by following a cohort of 182 patients with S. aureus bacteremia (SAB) and analyzing these parameters within the critical first 96 h of vancomycin therapy. The median vancomycin trough level at this time point was 19.5 mg/liter. There was a significant difference in vancomycin AUC/MIC when using broth microdilution (BMD) compared with Etest MIC (medians of 436.1 and 271.5, respectively; P < 0.001). Obtaining the recommended vancomycin target AUC/MIC of ≥400 using BMD was not associated with lower 30-day all-cause or attributable mortality from SAB (P = 0.132 and P = 0.273, respectively). However, an alternative vancomycin AUC/MIC of >373, derived using classification and regression tree analysis, was associated with reduced mortality (P = 0.043) and remained significant in a multivariable model. This study demonstrated that we obtained vancomycin trough levels in the target therapeutic range early during the course of therapy and that obtaining a higher vancomycin AUC/MIC (in this case, >373) within 96 h was associated with reduced mortality. The MIC test method has a significant impact on vancomycin AUC/MIC estimation. Clinicians should be aware that the current target AUC/MIC of ≥400 was derived using the reference BMD method, so adjustments to this target need to be made when calculating AUC/MIC ratio using other MIC testing methods.
PMCID: PMC3623342  PMID: 23335735
7.  Influenza Vaccine Effectiveness against Hospitalisation with Confirmed Influenza in the 2010–11 Seasons: A Test-negative Observational Study 
PLoS ONE  2013;8(7):e68760.
Immunisation programs are designed to reduce serious morbidity and mortality from influenza, but most evidence supporting the effectiveness of this intervention has focused on disease in the community or in primary care settings. We aimed to examine the effectiveness of influenza vaccination against hospitalisation with confirmed influenza. We compared influenza vaccination status in patients hospitalised with PCR-confirmed influenza with patients hospitalised with influenza-negative respiratory infections in an Australian sentinel surveillance system. Vaccine effectiveness was estimated from the odds ratio of vaccination in cases and controls. We performed both simple multivariate regression and a stratified analysis based on propensity score of vaccination. Vaccination status was ascertained in 333 of 598 patients with confirmed influenza and 785 of 1384 test-negative patients. Overall estimated crude vaccine effectiveness was 57% (41%, 68%). After adjusting for age, chronic comorbidities and pregnancy status, the estimated vaccine effectiveness was 37% (95% CI: 12%, 55%). In an analysis accounting for a propensity score for vaccination, the estimated vaccine effectiveness was 48.3% (95% CI: 30.0, 61.8%). Influenza vaccination was moderately protective against hospitalisation with influenza in the 2010 and 2011 seasons.
PMCID: PMC3712933  PMID: 23874754
8.  Heterogeneous Vancomycin-Intermediate Susceptibility Phenotype in Bloodstream Methicillin-Resistant Staphylococcus aureus Isolates from an International Cohort of Patients with Infective Endocarditis: Prevalence, Genotype, and Clinical Significance 
The Journal of infectious diseases  2009;200(9):1355-1366.
The significance of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) is unknown. Using a multinational collection of isolates from methicillin-resistant S. aureus (MRSA) infective endocarditis (IE), we characterized IE patients with and without hVISA, and genotyped the infecting strains.
MRSA bloodstream isolates from 65 patients with definite IE from 8 countries underwent PCR for 31 virulence genes, pulsed-field gel electrophoresis, and multilocus sequence typing. hVISA was defined using population analysis profiling (PAP).
Nineteen (29.2%) of 65 MRSA IE isolates exhibited hVISA by PAP. Isolates from Oceania and Europe were more likely to exhibit hVISA than isolates from the United States (77.8% vs. 35.0% vs. 13.9%; P < .001). The prevalence of hVISA was higher among isolates with a vancomycin minimum inhibitory concentration of 2 mg/L (P = .026). hVISA-infected patients were more likely to have persistent bacteremia (68.4% vs. 37.0%; P = .029) and heart failure (47.4% vs. 19.6%; P = .033). Mortality of hVISA- and non-hVISA-infected patients did not differ (42.1% vs. 34.8%, P = .586). hVISA and non-hVISA isolates were genotypically similar.
In these analyses, hVISA occurred in over one-quarter of MRSA IE isolates, was associated with certain IE complications, and varied in frequency by geographic region.
PMCID: PMC3600359  PMID: 19811099
hVISA; Methicillin-resistant Staphylococcus aureus; endocarditis; genotype
9.  Methicillin-Susceptible Staphylococcus aureus Endocarditis Isolates Are Associated With Clonal Complex 30 Genotype and a Distinct Repertoire of Enterotoxins and Adhesins 
The Journal of Infectious Diseases  2011;204(5):704-713.
Background. Using multinational collections of methicillin-susceptible Staphylococcus aureus (MSSA) isolates from infective endocarditis (IE) and soft tissue infections (STIs), we sought to (1) validate the finding that S. aureus in clonal complex (CC) 30 is associated with hematogenous complications and (2) test the hypothesis that specific genetic characteristics in S. aureus are associated with infection severity.
Methods. IE and STI isolates from 2 cohorts were frequency matched by geographic origin. Isolates underwent spa typing to infer CC and multiplex polymerase chain reaction for presence of virulence genes.
Results. 114 isolate pairs were genotyped. IE isolates were more likely to be CC30 (19.5% vs 6.2%; P = .005) and to contain 3 adhesins (clfB, cna, map/eap; P < .0001 for all) and 5 enterotoxins (tst, sea, sed, see, and sei; P ≤ .005 for all). CC30 isolates were more likely to contain cna, tst, sea, see, seg, and chp (P < .05 for all).
Conclusions. MSSA IE isolates were significantly more likely to be CC30 and to possess a distinct repertoire of virulence genes than MSSA STI isolates from the same region. The genetic basis of this association requires further study.
PMCID: PMC3156104  PMID: 21844296
10.  Curtobacterium flaccumfaciens Septic Arthritis following Puncture with a Coxspur Hawthorn Thorn ▿ 
Journal of Clinical Microbiology  2011;49(7):2759-2760.
Curtobacterium species are recognized plant pathogens. We report the first well-documented case of Curtobacterium human infection, a child with septic arthritis following puncture with a Coxspur Hawthorn plant thorn. The organism isolated from synovial tissue and the plant thorn was identified as Curtobacterium flaccumfaciens by 16S rRNA gene sequence analysis.
PMCID: PMC3147839  PMID: 21562106
11.  Contamination of renal patients' hospital chart covers with vancomycin-­ resistant enterococci: Handle with care 
The Australasian Medical Journal  2011;4(10):538-541.
Vancomycin-­resistant enterococci (VRE) have been increasingly associated with patients with renal failure attending large metropolitan teaching hospitals. Monash Medical Center has been following guidelines issued by the Department of Human Services to reduce the spread of VRE, but unfortunately this has had limited impact, especially in the renal unit. In an attempt to investigate the causes of the sustained VRE prevalence in the renal unit, this study sought to determine if renal patient chart covers were contaminated with VRE and if there was any genetic similarity to patient VRE isolates.
Using convenience sampling, chart covers of patients colonised or infected with VRE were swabbed from July to September 2010 (n=46). Samples were also collected from chart covers of non-VRE patients. Molecular typing of all matching VRE patient and chart isolates was performed using pulsed field gel electrophoresis (PFGE) by the public health laboratory (Microbiological Diagnostic Unit, University of Melbourne).
None of the patients who were VRE negative (n=14) had contaminated chart covers. VRE was recovered from two drug chart covers (patient A and B) from the 31 VRE positive patients sampled. One patient (patient C) was misidentified as a VRE patient for two weeks and was subject to contact precautions while being dialysed, yet three chart types belonging to this patient were found to be contaminated with VRE.
The findings of this study demonstrate that it is possible for patients' hospital chart covers to be contaminated with VRE even though there was no genetic similarity to the current patient strain. In this regard, the study reveals that patient charts may have an important role in spreading VRE.
PMCID: PMC3562874  PMID: 23386864
Vancomycin-resistant enterococci; renal patients; patient chart covers
12.  The Dominant Australian Community-Acquired Methicillin-Resistant Staphylococcus aureus Clone ST93-IV [2B] Is Highly Virulent and Genetically Distinct 
PLoS ONE  2011;6(10):e25887.
Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 has spread rapidly across North America, and CA-MRSA is also increasing in Australia. However, the dominant Australian CA-MRSA strain, ST93-IV [2B] appears distantly related to USA300 despite strikingly similar clinical and epidemiological profiles. Here, we compared the virulence of a recent Australian ST93 isolate (JKD6159) to other MRSA, including USA300, and found that JKD6159 was the most virulent in a mouse skin infection model. We fully sequenced the genome of JKD6159 and confirmed that JKD6159 is a distinct clone with 7616 single nucleotide polymorphisms (SNPs) distinguishing this strain from all other S. aureus genomes. Despite its high virulence there were surprisingly few virulence determinants. However, genes encoding α-hemolysin, Panton-Valentine leukocidin (PVL) and α-type phenol soluble modulins were present. Genome comparisons revealed 32 additional CDS in JKD6159 but none appeared to encode new virulence factors, suggesting that this clone's enhanced pathogenicity could lie within subtler genome changes, such as SNPs within regulatory genes. To investigate the role of accessory genome elements in CA-MRSA epidemiology, we next sequenced three additional Australian non-ST93 CA-MRSA strains and compared them with JKD6159, 19 completed S. aureus genomes and 59 additional S. aureus genomes for which unassembled genome sequence data was publicly available (82 genomes in total). These comparisons showed that despite its distinctive genotype, JKD6159 and other CA-MRSA clones (including USA300) share a conserved repertoire of three notable accessory elements (SSCmecIV, PVL prophage, and pMW2). This study demonstrates that the genetically distinct ST93 CA-MRSA from Australia is highly virulent. Our comparisons of geographically and genetically diverse CA-MRSA genomes suggest that apparent convergent evolution in CA-MRSA may be better explained by the rapid dissemination of a highly conserved accessory genome from a common source.
PMCID: PMC3185049  PMID: 21991381
13.  Novel Use of Tryptose Sulfite Cycloserine Egg Yolk Agar for Isolation of Clostridium perfringens during an Outbreak of Necrotizing Enterocolitis in a Neonatal Unit▿  
Journal of Clinical Microbiology  2010;48(11):4263-4265.
Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.
PMCID: PMC3020804  PMID: 20826643
14.  Acquired bloodstream infection in the intensive care unit: incidence and attributable mortality 
Critical Care  2011;15(2):R100.
To estimate the incidence of intensive care unit (ICU)-acquired bloodstream infection (BSI) and its independent effect on hospital mortality.
We retrospectively studied acquisition of BSI during admissions of >72 hours to adult ICUs from two university-affiliated hospitals. We obtained demographics, illness severity and co-morbidity data from ICU databases and microbiological diagnoses from departmental electronic records. We assessed survival at hospital discharge or at 90 days if still hospitalized.
We identified 6339 ICU admissions, 330 of which were complicated by BSI (5.2%). Median time to first positive culture was 7 days (IQR 5-12). Overall mortality was 23.5%, 41.2% in patients with BSI and 22.5% in those without. Patients who developed BSI had higher illness severity at ICU admission (median APACHE III score: 79 vs. 68, P < 0.001). After controlling for illness severity and baseline demographics by Cox proportional-hazard model, BSI remained independently associated with risk of death (hazard ratio from diagnosis 2.89; 95% confidence interval 2.41-3.46; P < 0.001). However, only 5% of the deaths in this model could be attributed to acquired-BSI, equivalent to an absolute decrease in survival of 1% of the total population. When analyzed by microbiological classification, Candida, Staphylococcus aureus and gram-negative bacilli infections were independently associated with increased risk of death. In a sub-group analysis intravascular catheter associated BSI remained associated with significant risk of death (hazard ratio 2.64; 95% confidence interval 1.44-4.83; P = 0.002).
ICU-acquired BSI is associated with greater in-hospital mortality, but complicates only 5% of ICU admissions and its absolute effect on population mortality is limited. These findings have implications for the design and interpretation of clinical trials.
PMCID: PMC3219371  PMID: 21418635
15.  Report of oral clarithromycin desensitization 
PMCID: PMC2492920  PMID: 18460032
16.  Phylogenetic Analysis of Viridans Group Streptococci Causing Endocarditis ▿  
Journal of Clinical Microbiology  2008;46(9):3087-3090.
Identification of viridans group streptococci (VGS) to the species level is difficult because VGS exchange genetic material. We performed multilocus DNA target sequencing to assess phylogenetic concordance of VGS for a well-defined clinical syndrome. The hierarchy of sequence data was often discordant, underscoring the importance of establishing biological relevance for finer phylogenetic distinctions.
PMCID: PMC2546745  PMID: 18650347
17.  Genotypic Diversity of Coagulase-Negative Staphylococci Causing Endocarditis: a Global Perspective▿  
Journal of Clinical Microbiology  2008;46(5):1780-1784.
Coagulase-negative staphylococci (CNS) are important causes of infective endocarditis (IE), but their microbiological profiles are poorly described. We performed DNA target sequencing and susceptibility testing for 91 patients with definite CNS IE who were identified from the International Collaboration on Endocarditis—Microbiology, a large, multicenter, multinational consortium. A hierarchy of gene sequences demonstrated great genetic diversity within CNS from patients with definite endocarditis that represented diverse geographic regions. In particular, rpoB sequence data demonstrated unique genetic signatures with the potential to serve as an important tool for global surveillance.
PMCID: PMC2395089  PMID: 18367572
18.  Corynebacterium kutscheri Infection of Skin and Soft Tissue following Rat Bite▿  
Journal of Clinical Microbiology  2007;45(10):3468-3469.
Corynebacterium kutscheri is a common bacterium isolated from the oral cavity of healthy mice and rats. We report the first well-documented case of C. kutscheri human infection which followed a rat bite. The microorganism was identified by conventional biochemical tests and confirmed by 16S rRNA gene sequence analysis.
PMCID: PMC2045315  PMID: 17670928
19.  Fatal Case of Toxic Shock-Like Syndrome Due to Group C Streptococcus Associated with Superantigen Exotoxin 
Journal of Clinical Microbiology  2004;42(6):2866-2869.
Group C streptococci have been reported to cause invasive disease similar to that classically associated with group A streptococcus (GAS). We describe a fatal case of toxic shock-like syndrome due to Streptococcus equi subsp. zooepidemicus. The causative organism did not possess any known GAS superantigen exotoxin genes but did show evidence of superantigen production.
PMCID: PMC427866  PMID: 15184494
20.  Three Cases of Anaerobiospirillum succiniciproducens Bacteremia Confirmed by 16S rRNA Gene Sequencing 
Journal of Clinical Microbiology  1998;36(5):1209-1213.
We describe three cases of Anaerobiospirillum succiniciproducens bacteremia from Australia. We believe one of these cases represents the first report of A. succiniciproducens bacteremia in a human immunodeficiency virus (HIV)-infected individual. The other two patients had an underlying disorder (one patient had bleeding esophageal varices complicating alcohol liver disease and one patient had non-Hodgkin’s lymphoma). A motile, gram-negative, spiral anaerobe was isolated by culturing blood from all patients. Electron microscopy showed a curved bacterium with bipolar tufts of flagella resembling Anaerobiospirillum spp. Sequencing of the 16S rRNA genes of the isolates revealed no close relatives (organisms likely to be in the same genus) in the sequence databases, nor were any sequence data available for A. succiniciproducens. This report presents for the first time the 16S rRNA gene sequence of the type strain of A. succiniciproducens, strain ATCC 29305. Two of the three clinical isolates have sequences identical to that of the type strain, while the sequence of the other strain differs from that of the type strain at 4 nucleotides.
PMCID: PMC104801  PMID: 9574678

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