The implications of endemic IMP-4 carbapenemase for clinical laboratory susceptibility testing.

A local predominance of carbapenemase producing Enterobacteriaceae with low minimum inhibitory concentrations (MIC) to meropenem prompted a review of methods available for carbapenemase detection. We report on results using two selective media, temocillin discs, CarbaNP test, GeneXpert Carba-R assay and an in-house PCR assay.

Real-time PCR assay for detection of blaZ genes in Staphylococcus aureus clinical isolates.

The Clinical and Laboratory Standards Institute recommends 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.

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes.

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.

HPV status of oropharyngeal cancer by combination HPV DNA/p16 testing: biological relevance of discordant results.

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.

Human papillomavirus, smoking status and outcomes in tonsillar squamous cell carcinoma.

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. A total of 403 patients from nine centers 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 modeled using Cox regression. Smoking status was a significant predictor of overall survival (p = 0.04). There were nonstatistically 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.

The prognostic significance of hypoxia inducing factor 1-α in oropharyngeal cancer in relation to human papillomavirus status.

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 51 months 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.

Respiratory viral pathogens associated with lower respiratory tract disease among young children in the highlands of Papua New Guinea.

BACKGROUND: 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. OBJECTIVES: To determine the viruses associated with ALRI among children living in the PNG highlands using sensitive molecular detection techniques. STUDY DESIGN: 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. RESULTS: 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). CONCLUSIONS: The burden of infection with respiratory viruses remains significant in young children living in the PNG highlands.

Human papillomavirus and gene mutations in head and neck squamous carcinomas.

BACKGROUND: Human papillomavirus (HPV) infection is implicated as an aetiological factor in head and neck squamous carcinomas (HNSCC), especially in the tonsils of the oropharyngeal region. This study investigates the frequency of HPV infection, p16 and p53 tumour profile and mutations in epidermal growth factor receptor (EGFR), Kirsten RNA Associated Rat Sarcoma 2 Virus (KRAS) and B-Raf proto-oncogene serine/threonine protein kinase (BRAF) genes in tonsillar and non-tonsillar HNSCCs and correlates with clinical outcome and histopathological parameters in previously unstudied cohort of patients. METHODS: A retrospective clinical study was performed utilising the demographic data and pathological specimens from 60 out of 726 head and neck cancer patients. Smoking and alcohol history, tumour staging, treatment and outcomes were recorded. Histopathology and immunochemistry for p16 and p53 was performed and HPV DNA was detected with polymerase chain reaction. Genomic DNA from all cancers were analysed for somatic mutations of EGFR, BRAF and KRAS genes. RESULTS: 20 (33%) of 60 cases were tonsillar squamous carcinomas and 38 (66%) were non-tonsillar. 19 (95%) of the 20 tonsillar cancers and three (8%) of 38 non-tonsillar patients were patients who were HPV 16-positive. Nine (47%) of the 19 HPV 16-positive tonsillar cases were p16 positive. Gene mutations were rare. There was a statistically significant (P < 0.05) improved survival of patients with HPV positive tonsillar tumours, younger age and non-smokers. CONCLUSION: Although limited in numbers, this study reinforces the role of HPV infection in HNSCC and its association with a more favourable clinical course in younger non-smokers worldwide. Gene mutation frequencies were low in all cancers tested and routine testing not recommended.

High detection rates of nucleic acids of a wide range of respiratory viruses in the nasopharynx and the middle ear of children with a history of recurrent acute otitis media.

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.

Aeromonas aquariorum is widely distributed in clinical and environmental specimens and can be misidentified as Aeromonas hydrophila.

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.

Comparison of methods for AmpC ß-lactamase detection in Enterobacteriaceae.

AmpC ß-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 ß-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.

Use of cyclin D1 in conjunction with human papillomavirus status to predict outcome in oropharyngeal cancer.

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.

The detection of oseltamivir-resistant pandemic influenza A/H1N1 2009 viruses using a real-time RT-PCR assay.

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.

Relationships between epidermal growth factor receptor expression and human papillomavirus status as markers of prognosis in oropharyngeal cancer.

PURPOSE: 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). MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: HPV and EGFR are independent prognostic markers in oropharyngeal SCC. Combining testing for HPV and EGFR appears to provide additional prognostic information.

Squamous cell carcinoma of the oropharynx in Australian males induced by human papillomavirus vaccine targets.

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.

Duplex real-time reverse transcriptase PCR assays for rapid detection and identification of pandemic (H1N1) 2009 and seasonal influenza A/H1, A/H3, and B viruses.

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.

Prognostic significance of vascular endothelial growth factor in squamous cell carcinomas of the tonsil in relation to human papillomavirus status and epidermal growth factor receptor.

BACKGROUND: Angiogenesis markers, vascular endothelial growth factor (VEGF) and microvessel density (MVD) have been associated with prognosis in squamous cell carcinomas (SCCs) of the head and neck. Other prognostic variables such as human papillomavirus (HPV) and epidermal growth factor (EGFR) may also be involved in tumour angiogenesis. This study determined relationships between VEGF, MVD, EGFR, HPV, response to radiotherapy and clinical outcome in 85 tonsillar SCCs. METHODS: HPV status was determined by an HPV multiplex real-time polymerase chain reaction (PCR) assay/p16 immunohistochemistry. Expression of VEGF, CD31 (as marker of MVD) and EGFR was assessed by semiquantitative immunohistochemistry. RESULTS: Strong VEGF expressers were significantly more likely to have higher MVD than were weak expressers. There were no associations between VEGF or MVD and gender, patient age, TNM stage, EGFR expression or HPV status. Tumours with MVD of >15 per high-power field were significantly more likely to be poorly differentiated. There was a significant inverse relationship between EGFR and HPV status. HPV was a strong independent marker of loco-regional recurrence and death. VEGF and EGFR were risk factors for local recurrence and disease-specific death on univariate analysis but the associations weakened after adjustment for HPV. Among patients treated with radiotherapy, VEGF was associated with disease-specific death after adjusting for HPV and TMN stage. High-VEGF-expressing tumours positive for EGFR had a worse prognosis than all other groups combined after adjusting for HPV and TNM stage. CONCLUSIONS: HPV is a stronger prognostic marker than VEGF or EGFR in tonsillar SCCs. VEGF correlates with MVD in these tumours.

The molecular epidemiology of ocular Chlamydia trachomatis infections in Western Australia: implications for trachoma control.

We studied the serovar distribution of Chlamydia trachomatis in patients with clinical eye disease in Western Australia. Most disease occurred in indigenous communities and was caused by trachoma serovars Ba and C. Serovar Ba was genetically homogeneous throughout Western Australia and identical to strains previously described in indigenous communities in Northern Territory. This finding probably results from movement of these populations, and suggests that a widely coordinated, rather than local or regional, approach is needed to control trachoma in mobile populations. Serovar C strains within Western Australia were homogeneous but distinct from those in Northern Territory, possibly because of inherent differences in transmissibility or differences in population movements among communities carrying the different serovars. Genital serovars were occasional causes of eye diseases in infants, adolescents, and adults in trachoma-endemic areas. These serovars should be considered in the differential diagnosis of acute follicular conjunctivitis in these groups.