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.

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.

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.

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.

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.

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.

Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus.

BACKGROUND: Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70. OBJECTIVES: We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus. STUDY DESIGN: Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results. RESULTS: The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products. CONCLUSION: The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.

The impact of respiratory syncytial virus infection on endothelin receptor function and release in sheep bronchial explants.

We investigated the impact of respiratory syncytial virus (RSV) infection, an important asthma precipitant, on endothelin receptor function and release in sheep bronchial explants. RSV infection was confirmed using polymerase chain reaction and immunohistochemistry. Since sheep airway smooth muscle contains only endothelin-A receptors, sarafotoxin (Stx) S6c did not cause airway contraction. In contrast, sarafotoxin S6c (300 nM) caused contraction in RSV-infected bronchial explants (8 +/- 3% carbachol Emax). However, we could not detect airway smooth muscle endothelin-B receptors in explants using autoradiography. RSV infection per se did not alter the release of immunoreactive endothelin from sheep bronchial explants (control = 11.6 +/- 0.9 pg versus RSV = 12.1 +/- 0.9 pg). Interestingly, dexamethasone (1 microM) alone increased endothelin release in both control (17.9 +/- 2.0 pg) and RSV-infected tissue (18.3 +/- 3.1 pg). The combined presence of protease-activated receptor-2 (PAR-2) ligand (100 microM) and dexamethasone (1 microM) also increased endothelin release from control tissue (17.3 +/- 1.4 pg), but endothelin release was suppressed by PAR-2 ligand in RSV-infected tissue (10.3 +/- 0.8 pg), probably because PAR-2 expression was increased by RSV. In summary, the novel expression of endothelin-B receptors triggered by RSV might be relevant to RSV-associated asthma. Furthermore, activation of airway PAR-2 may be protective in asthma where endothelin levels are elevated in part via endothelin release suppression.

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.

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.

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.

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.

Human metapneumovirus, Australia, 2001-2004.

We examined 10,025 respiratory samples collected for 4 years (2001-2004) and found a 7.1% average annual incidence of human metapneumovirus. The epidemic peak of infection was late winter to spring, and genotyping showed a change in predominant viral genotype in 3 of the 4 years.

Use of the P gene to genotype human metapneumovirus identifies 4 viral subtypes.

This study, conducted during 2001-2003, undertook the screening of patients with acute infectious respiratory-tract disease. A random selection of positive specimens was used for sequencing studies of the human metapneumovirus (hMPV) nucleoprotein gene and the phosphoprotein (P) gene. Australian and international sequences were compared, and a global classification scheme was developed. The hMPV P gene was an ideal molecular target for the detection and genotyping of hMPV. The region contained conserved sequences for reverse-transcriptase-polymerase chain-reaction primers and adequate variability to permit the accurate genotyping of the virus into 2 main lineages and 4 sublineages. Establishing viral identity is essential for the linking of genotype and disease severity.