Gerald B Harnett

Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia

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Publications (32)116.86 Total impact

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    ABSTRACT: The Clinical and Laboratory Standards Institute recommend consideration of blaZ gene testing for cases of serious Staphylococcus aureus infection. Conventional PCR methods have demonstrated superior sensitivity and specificity to phenotypic tests. To our knowledge, this is the first description of real-time PCR detection of the blaZ gene.
    Journal of clinical microbiology 01/2014; · 4.16 Impact Factor
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    ABSTRACT: Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents' and children's mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14 K lytic enzyme is derived from a microbial source.
    Experimental and Applied Acarology 06/2013; · 1.85 Impact Factor
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    ABSTRACT: OBJECTIVES: This study examines the prognostic significance of hypoxia inducing factor-1α (HIF-1α) expression in relation to human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (SCC). MATERIALS AND METHODS: Clinical details on 233 oropharyngeal SCCs were extracted from institutional databases. Recurrence in any form or death from any cause was recorded for a median of 51months after diagnosis. HIF-1α expression was evaluated by semiquantitative immunohistochemistry and HPV status was determined by HPV E6-targeted multiplex real-time PCR and p16 immunohistochemistry. Determinants of recurrence and mortality hazards were modeled using Cox regression with censoring at dates of last follow-up. RESULTS: The HIF-1α positivity rate was 58.8%. HIF-1α positivity was associated with higher T category (T3/T4 vs. T1/T2, 64.2% vs. 48.4%, p=0.001) and lower grade (Grade 1-2 vs. 3, 62% vs. 46.9%, p=0.001). There was no significant association between HIF-1α expression and HPV status. After adjustment for clinico-pathological variables, HPV status but not HIF-1α was a strong predictor of outcome. The combination of HPV and HIF-1α was not a prognostic variable but the worst outcomes were seen in those with HPV negative and HIF-1α positive cancers. There was no statistically significant evidence of an interaction between HPV and HIF-1α. CONCLUSIONS: The degree of hypoxia as measured by HIF-1α expression does not differ between HPV positive and HPV negative cancers. The role of hypoxia in HPV negative oropharyngeal cancer warrants further investigation.
    Oral Oncology 12/2012; · 2.70 Impact Factor
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    ABSTRACT: BACKGROUND AND PURPOSE: Human papillomavirus (HPV) causes up to 70 % of oropharyngeal cancers (OSCC). HPV positive OSCC has a more favorable outcome, thus HPV status is being used to guide treatment and predict outcome. Combination HPV DNA/p16(ink4) (p16) testing is commonly used for HPV status, but there are no standardized methods, scoring or interpretative criteria. The significance of discordant (HPV DNA positive/p16 negative and HPV DNA negative/p16 positive) cancers is controversial. In this study, 647 OSCCs from 10 Australian centers were tested for HPV DNA/p16 expression. Our aims are to determine p16 distribution by HPV DNA status to inform decisions on p16 scoring and to assess clinical significance of discordant cancers. METHODS: HPV DNA was identified using a multiplex tandem HPV E6 polymerase chain reaction (PCR) assay and p16 expression by semiquantitative immunohistochemistry. RESULTS: p16 distribution was essentially bimodal (42 % of cancers had ≥70 % positive staining, 52 % <5 % positive, 6 % between 5 and 70 %). Cancers with 5 to <50 % staining had similar characteristics to the p16 negative group, and cancers with 50 to <70 % staining were consistent with the ≥70 % group. Using a p16 cut-point of 50 %, there were 25 % HPV DNA positive/p16 negative cancers and 1 % HPV DNA negative/p16 positive cancers. HPV DNA positive/p16 negative cancers had outcomes similar to HPV DNA negative/p16 negative cancers. CONCLUSIONS: 50 % is a reasonable cut-point for p16; HPV DNA positive/p16 negative OSCCs may be treated as HPV negative for clinical purposes; HPV DNA/p16 testing may add no prognostic information over p16 alone.
    Annals of Surgical Oncology 12/2012; · 4.12 Impact Factor
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    ABSTRACT: It is now clear that the two separate entitles of tonsillar cancer, HPV induced and non-HPV induced (smoking induced), have significantly different presenting stage and outcomes. A significant proportion of patients with human papillomavirus positive tonsillar cancer have had exposure to smoking. We examined the combined effect of human papillomavirus and smoking on the outcomes and determined whether smoking can modify the beneficial effect of human papillomavirus. 403 patients from 9 centres were followed up for recurrence or death for a median of 38 months. Determinants of the rate of loco-regional recurrence, death from tonsillar cancer and overall survival were modelled using Cox regression. Smoking status was a significant predictor of overall survival (p=0.04). There were non-statistically significant trends favoring never smokers for loco-regional recurrence and disease specific survival. In addition, there was no statistically significant interactions between smoking and human papillomavirus (p-values for the interaction were 0.26 for loco-regional recurrence, 0.97 for disease specific survival and 0.73 for overall survival). The effect of smoking on loco-regional recurrence and disease specific survival outcomes was not statistically significant, nor was there significant evidence that the effect of smoking status on these outcomes was modified by HPV status. Irrespective of HPV status however, smokers did have poorer overall survival than never-smokers; presumably due to effects of smoking that are unrelated to the primary cancer. © 2012 Wiley Periodicals, Inc.
    International Journal of Cancer 11/2012; · 6.20 Impact Factor
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    ABSTRACT: Background Despite the association with more advanced nodal stage, patients with human papillomavirus (HPV) positive oropharyngeal cancers have better outcomes. We examined whether the HPV can modify the effect of known prognostic factors in tonsillar cancer.Patients and methodsA total of 489 patients from 10 centres were followed up for recurrence or death for a median of 3.2 years. Determinants of the rate of locoregional recurrence, death from tonsillar cancer and overall survival were modelled using Cox regression.ResultsThe prognostic value of T and N stages were modified by HPV as indicated by statistically significant interaction terms. After adjusting for age, gender and treatment, T stage appeared relevant only for HPV-positive cancers (where a higher T stage was associated with worse outcomes). There was some evidence that N stage was a more relevant prognostic factor for HPV-negative than -positive cancers. There was no evidence that the HPV modifies the effect of age, gender or grade on outcomes.Conclusions This study suggests that the prognostic significance of the conventional staging system in tonsillar cancer is modified by HPV.
    Annals of Oncology 08/2012; · 7.38 Impact Factor
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    ABSTRACT: Acute lower respiratory tract infections (ALRI) commonly result in fatal outcomes in the young children of Papua New Guinea (PNG). However, comprehensive studies of the viral aetiology of ALRI have not been conducted in PNG for almost 30 years. To determine the viruses associated with ALRI among children living in the PNG highlands using sensitive molecular detection techniques. Pernasal swabs were collected routinely between 1 week and 18 months of age and also during episodes of ALRI, as part of a neonatal pneumococcal conjugate vaccine trial. A tandem multiplex real-time PCR assay was used to test for a comprehensive range of respiratory viruses in samples collected from 221 young children. Picornavirus typing was supported by DNA sequence analysis. Recognized pathogenic respiratory viruses were detected in 198/273 (73%) samples collected from children with no evidence of ALRI and 69/80 (86%) samples collected during ALRI episodes. Human rhinoviruses (HRV) species A, B and C were detected in 152 (56%) samples from non-ALRI children and 50 (63%) samples collected during ALRI episodes. Partial structural region sequences for two new species C rhinoviruses were added to the GenBank database. ALRI was associated with detection of adenovirus species B (p<0.01) or C (p<0.05), influenza A (p<0.0001) or respiratory syncytial virus (p<0.0001). Multiple viruses were detected more often during ALRI episodes (49%) than when children displayed no symptoms of ALRI (18%) (p<0.0001). The burden of infection with respiratory viruses remains significant in young children living in the PNG highlands.
    Journal of clinical virology: the official publication of the Pan American Society for Clinical Virology 05/2012; 54(3):235-9. · 3.12 Impact Factor
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    ABSTRACT: Both bacteria and viruses play a role in the development of acute otitis media, however, the importance of specific viruses is unclear. In this study molecular methods were used to determine the presence of nucleic acids of human rhinoviruses (HRV; types A, B, and C), respiratory syncytial viruses (RSV; types A and B), bocavirus (HBoV), adenovirus, enterovirus, coronaviruses (229E, HKU1, NL63, and OC43), influenza viruses (types A, B, and C), parainfluenza viruses (types 1, 2, 3, 4A, and 4B), human metapneumovirus, and polyomaviruses (KI and WU) in the nasopharynx of children between 6 and 36 months of age either with (n = 180) or without (n = 66) a history of recurrent acute otitis media and in 238 middle ear effusion samples collected from 143 children with recurrent acute otitis media. The co-detection of these viruses with Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis was analyzed. HRV (58.3% vs. 42.4%), HBoV (52.2% vs. 19.7%), polyomaviruses (36.1% vs. 15.2%), parainfluenza viruses (29.4% vs. 9.1%), adenovirus (25.0% vs. 6.1%), and RSV (27.8% vs. 9.1%) were detected significantly more often in the nasopharynx of children with a history of recurrent acute otitis media compared to healthy children. HRV was predominant in the middle ear and detected in middle ear effusion of 46% of children. Since respiratory viruses were detected frequently in the nasopharynx of both children with and without a history of recurrent acute otitis media, the etiological role of specific viruses in recurrent acute otitis media remains uncertain, however, anti-viral therapies may be beneficial in future treatment and prevention strategies for acute otitis media.
    Journal of Medical Virology 11/2011; 83(11):2008-17. · 2.37 Impact Factor
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    Journal of clinical microbiology 09/2011; 49(9):3444-5. · 4.16 Impact Factor
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    ABSTRACT: Genotypic characterization of 215 Aeromonas strains (143 clinical, 52 environmental, and 20 reference strains) showed that Aeromonas aquariorum (60 strains, 30.4%) was the most frequently isolated species in clinical and water samples and could be misidentified as Aeromonas hydrophila by phenotypic methods.
    Journal of clinical microbiology 06/2011; 49(8):3006-8. · 4.16 Impact Factor
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    ABSTRACT: Background / Purpose: Human rhinoviruses (HRV) are the most common cause of respiratory infections worldwide. Recent advances in the molecular detection of respiratory viruses led to the identification of a new HRV group, HRV-C, which has been associated with more frequent and severe lower respiratory infections.As part of the Kalgoorlie Otitis Media Research Project (KOMRP), nasopharyngeal aspirates (NPAs) were to be collected routinely on seven occasions from 100 healthy Aboriginal and 180 healthy non-Aboriginal children before two years of age. HRV was the most frequently identified virus (19.6%) and was identified more often in specimens from Aboriginal than non-Aboriginal children.We now describe the prevalence of HRV groups and potential risk factors for HRV carriage in this population. This is the first study to describe the epidemiology of HRV groups among Aboriginal children. Main conclusion: HRV-A was more common than HRV-C among healthy children. HRV-C was associated with mild upper respiratory symptoms in both Aboriginal and non-Aboriginal children while HRV-A was not. Furthermore, HRV group carriage was associated with ethnicity, age, seasonality, bacterial carriage, exclusive breastfeeding, gestational smoking and maternal socio-economic factors.
    Thoracic Society of Australia and New Zealand Annual Scientific Meeting 2011; 04/2011
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    ABSTRACT: There is increasing use of multiple molecular markers to predict prognosis in human cancer. Our aim was to examine the prognostic significance of cyclin D1 and retinoblastoma (pRb) expression in association with human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma. Clinical records and specimens of 226 patients with follow-up from 1 to 235 months postdiagnosis were retrieved. Tumor HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 semiquantitative immunohistochemistry and cyclin D1 and pRb expression by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modeled using Cox regression with censoring at dates of last follow-up. The HPV-positivity rate was 37% (91% type 16). HPV was a predictor of recurrence, an event (recurrence or death) and death after adjustment for clinicopathological variables. There were inverse relationships between HPV status and cyclin D1 and pRb. On univariate analysis, cyclin D1 predicted locoregional recurrence, event and death and pRb predicted event and death. Within the HPV-positive group, after adjusting for clinicopathological factors, patients with cyclin D1-positive cancers had up to a eightfold increased risk of poor outcome relative to those with cyclin D1-negative tumors. However, within the HPV-negative group, there was only a very small adjusted increased risk. A combination of pRb and HPV did not provide additional prognostic information. Our data provide the first evidence that a combination of HPV and cyclin D1 provides more prognostic information in oropharyngeal cancer than HPV alone. If findings are confirmed, treatment based on HPV and cyclin D1 may improve outcomes.
    International Journal of Cancer 04/2011; 128(7):1532-45. · 6.20 Impact Factor
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    ABSTRACT: AmpC β-lactamases (Bla(AmpC)) are an emerging group of antimicrobial resistance determinants. The lack of an agreed Bla(AmpC) detection method hinders investigation of their epidemiology and understanding of their clinical significance. This study compared the sensitivity and specificity of phenotypic methods of Bla(AmpC) detection in a collection of 246 Enterobacteriaceae with a diverse range of β-lactam resistance profiles. The Bla(AmpC) screening methods evaluated were based on cephamycin, ceftazidime and cefepime susceptibility. These were compared with Bla(AmpC) screening using conventional ESBL detection methods. The confirmatory methods evaluated were biologically based assays, inhibitor-based assays, an AmpC Etest and a rapid chromogenic assay. A multiplex nucleic acid amplification test and the three-dimensional enzyme extraction assay were used as reference methods. Bla(AmpC) activity was present in 74 isolates. The majority of the enzymes were plasmid-encoded and belonged to the CMY, DHA and EBC families. The screening methods had sensitivities between 47 and 99 % and specificities of 45-95 %. The performance of confirmatory tests varied widely, ranging in sensitivity from 19 % to 97 % and in specificity from 88 % to 100 %. Only the Tris-EDTA and MAST ID D68C disc tests had a sensitivity and a specificity above 90 %. Further investigation is needed to establish the most suitable enzyme substrates, inhibitor types, inhibitor concentrations and interpretative cut-offs in order to refine the inhibitor-based methods. A simple disc-based protocol using cefoxitin non-susceptibility as a screening tool, followed by the Tris-EDTA method for confirmation, detects Bla(AmpC) activity with 95 % sensitivity and 98 % specificity.
    Journal of Medical Microbiology 03/2011; 60(Pt 6):715-21. · 2.30 Impact Factor
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    ABSTRACT: A real-time reverse transcription PCR (rRT-PCR) assay was designed and evaluated for the detection of the point mutation in the influenza A N1 neuraminidase gene that results in a tyrosine to histidine substitution at amino acid position 275 (H275Y) causing resistance to oseltamivir, an antiviral neuraminidase inhibitor. The rRT-PCR assays detected the presence or absence of the H275Y mutation in 387/388 (99.7%) of clinical samples containing the pandemic influenza A/H1N1 2009 virus. The H275Y mutation was not detected in any of the community patient samples (0/132) but was detected in four hospitalized patients who had been treated with oseltamivir for several days. The sensitive rRT-PCR assays may be performed directly on patient specimens, can detect resistant virus at low levels, and therefore may provide early warning of developing resistance within individual patients or the wider population.
    Journal of virological methods 10/2010; 169(1):47-51. · 2.13 Impact Factor
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    ABSTRACT: This study examines the prognostic significance of human papillomavirus (HPV) in patients with locally advanced oropharyngeal squamous cell carcinoma (SCC) treated primarily with surgery or definitive radiotherapy. One hundred and ninety-eight patients with Stage 3/4 SCC were followed up for recurrence in any form or death from any cause for between 1 and 235 months after diagnosis. HPV status was determined using HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox's regression with censoring at follow-up dates. Forty-two per cent of cancers were HPV-positive (87% type 16). HPV predicted loco-regional control, event-free survival and overall survival in multivariable analysis. Within the surgery with adjuvant radiotherapy (n=110), definitive radiotherapy-alone (n=24) and definitive radiotherapy with chemotherapy (n=47) groups, patients with HPV-positive cancers were one-third or less as likely to have loco-regional recurrence, an event or to die of any cause as those with HPV-negative cancers after adjusting for age, gender, tumour grade, AJCC stage and primary site. The 14 patients treated with surgery alone were considered too few for multivariable analysis. HPV status predicts better outcome in oropharyngeal cancer treated with surgery plus adjuvant radiotherapy as well as with definitive radiation therapy±chemotherapy.
    British Journal of Cancer 10/2010; 103(10):1510-7. · 5.08 Impact Factor
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    ABSTRACT: This study examines the prognostic significance of epidermal growth factor receptor (EGFR) expression in relation to human papillomavirus (HPV) status in oropharyngeal squamous cell carcinoma (SCC). Pathological diagnosis of 270 oropharyngeal SCCs was verified by the study pathologist; clinical details were extracted from institutional databases. Recurrence in any form or death from any cause was recorded for a median of 2.5 (range: 0-19.3) years after diagnosis. HPV status was determined by HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry; EGFR expression was evaluated by semiquantitative immunohistochemistry. Determinants of recurrence and mortality hazards were modelled using Cox regression with censoring at dates of last follow-up. Thirty-seven percent of cancers were HPV-positive (91% type 16). HPV was a predictor of loco-regional recurrence, event-free and overall survival after adjustment for clinicopathological variables and EGFR. Patients with EGFR-positive cancers were 5-fold more likely to have loco-regional failure relative to those with EGFR-negative cancers. Patients with HPV-negative/EGFR-positive cancers had an adjusted 13-fold increased risk of having a loco-regional failure, an almost 4-fold increased risk of having an event and more than a 4-fold increased risk of dying of any cause relative to those with HPV-positive/EGFR-negative cancers. There was weak evidence that the effects of EGFR on outcome were limited to patients with HPV-negative cancers. HPV and EGFR are independent prognostic markers in oropharyngeal SCC. Combining testing for HPV and EGFR appears to provide additional prognostic information.
    European journal of cancer (Oxford, England: 1990) 07/2010; 46(11):2088-96. · 4.12 Impact Factor
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    ABSTRACT: Reports of a novel influenza virus type A (H1N1), now designated by the World Health Organization as pandemic (H1N1) 2009, emerged from the United States and Mexico in April 2009. The management of the pandemic in Australia required rapid and reliable testing of large numbers of specimens for the novel influenza strain and differentiation from seasonal influenza strains. A real-time reverse transcriptase PCR (RT-PCR) assay for the detection of pandemic (H1N1) 2009 was designed and used with existing real-time RT-PCR assays for seasonal influenza viruses A and B. MS2 coliphage was added to all samples and amplified as a quality control. Three duplex RT-PCR assays, each containing two primer pairs and corresponding 5' nuclease probes, were initially evaluated on control material and stored samples and showed high sensitivity and specificity. More than 11,000 clinical samples were then tested for influenza A and B matrix gene targets and specific hemagglutinin gene targets for seasonal influenza A/H1, A/H3, and pandemic A (H1N1) 2009. Minimum sensitivities and specificities were 98.8% and 100%, respectively, for pandemic (H1N1) 2009, 81.5% and 98.9% for seasonal A/H1, and 96.3% and 99.6% for A/H3. Automated sample extraction facilitated the rapid processing of samples so that the assays allowed accurate, rapid, and cost-effective screening of large numbers of clinical samples.
    Journal of clinical microbiology 03/2010; 48(3):862-6. · 4.16 Impact Factor
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    ABSTRACT: This study provides Australian data on the incidence of human papillomavirus (HPV)-related oropharyngeal cancer to aid the debate on extending the HPV vaccination programme to males. The HPV status for 302 oropharyngeal cancers diagnosed between 1987 and 2006 was determined by HPV E6-targeted multiplex real-time PCR/p16 immunohistochemistry. The overall HPV-positivity rate was 36% (94% types 16 and 18). HPV-related cancer increased from 19% (1987-1990) to 47% (2001-2005). HPV data used in conjunction with Australian cancer incidence data 2001-2005 showed that 1.56 cases of oropharyngeal cancer per 100,000 males per year were associated with HPV types targeted by the vaccine. Vaccinating males may substantially reduce the burden of oropharyngeal cancer in Australia.
    Vaccine 03/2010; 28(19):3269-72. · 3.77 Impact Factor
  • BMJ (online) 01/2010; 340:c2518. · 17.22 Impact Factor
  • Gerald B. Harnett, Glenys R. Chidlow
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    ABSTRACT: This protocol describes three singleplex real-time RT-PCR assays to detect the nucleoprotein genes of respiratory syncytial viruses types A and B (RSVA and RSVB) and the coat protein gene of MS2 RNA coliphage. MS2 is added to the lysis buffer of the extraction kit to serve as a control of RNA extraction, reverse transcription, the PCR reaction and removal of PCR inhibitors.
    01/2010;

Publication Stats

336 Citations
116.86 Total Impact Points

Institutions

  • 2012
    • Royal Prince Alfred Hospital
      • Department of Radiation Oncology
      Camperdown, New South Wales, Australia
  • 2011
    • The Queen Elizabeth Hospital
      Tarndarnya, South Australia, Australia
  • 2010
    • University of Western Australia
      Perth City, Western Australia, Australia
    • Western Australia Health
      Perth City, Western Australia, Australia
  • 2009–2010
    • PathWest Laboratory Medicine
      Perth City, Western Australia, Australia
    • Sydney Head and Neck Cancer Institute
      Sydney, New South Wales, Australia
  • 2008
    • Government of Western Australia
      Perth City, Western Australia, Australia
    • Curtin University Australia
      Bentley, Western Australia, Australia