False-negative results using Neisseria gonorrhoeae porA pseudogene PCR - a clinical gonococcal isolate with an N. meningitidis porA sequence, Australia, March 2011

The gonococcal porA pseudogene is a popular target for in-house Neisseria gonorrhoeae PCR methods. With this study we present two novel findings: the first case of an N. gonorrhoeae porA pseudogene PCR false-negative result caused by sequence variation, and in the same organism, the first description of a clinical N. gonorrhoeae strain harbouring an N. meningitidis porA sequence

A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

Background Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia. Methods Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources. Results We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples

Q fever seroprevalence in metropolitan samples is similar to rural/remote samples in Queensland, Australia

Q fever is a vaccine preventable disease; however, despite this, high notification numbers are still recorded annually in Australia. We investigated the seroprevalence of Coxiella burnetii, the Q fever agent, in a Queensland sample population. Notification data (N = 6425) from 1984-2008 were collated, identifying high risk areas of Q fever exposure. Of these 177 were recorded in children. Serum samples were collected from Queensland and screened using both an immunoflourescence assay at 1:10 dilution and a commercially available ELISA kit. Results were collated based on age, geographical location and sex. From 1988 Queensland samples screened, 103 were identified as Q fever IgG-positive, giving a seroprevalence of 5.2% (95% CI 4.3-6.2%). Seroprevalence in the rural/remote population was 5.3% (95% CI 4.6-6.6%), and the metropolitan Brisbane population, which is considered not at risk, was 5.0% (95% CI 3.7-6.7%). Sixty-three seropositive males and 40 females were identified, along with an increase in seropositivity with increasing age. The seropositivity of children was 1.3% (95% CI 0.7-2.3%) from 844 samples. We have shown that both metropolitan and paediatric populations which are considered low risk of Coxiella exposure have surprisingly high seropositivity. These emerging groups require further investigation and consideration for the introduction of preventive measures

Potential Animal and Environmental Sources of Q Fever Infection for Humans in Queensland

Q fever is a vaccine-preventable disease; despite this, high annual notification numbers are still recorded in Australia. We have previously shown seroprevalence in Queensland metropolitan regions is approaching that of rural areas. This study investigated the presence of nucleic acid from Coxiella burnetii, the agent responsible for Q fever, in a number of animal and environmental samples collected throughout Queensland, to identify potential sources of human infection. Samples were collected from 129 geographical locations and included urine, faeces and whole blood from 22 different animal species; 45 ticks were removed from two species, canines and possums; 151 soil samples; 72 atmospheric dust samples collected from two locations and 50 dust swabs collected from domestic vacuum cleaners. PCR testing was performed targeting the IS1111 and COM1 genes for the specific detection of C.burnetii DNA. There were 85 detections from 1318 animal samples, giving a detection rate for each sample type ranging from 2.1 to 6.8%. Equine samples produced a detection rate of 11.9%, whilst feline and canine samples showed detection rates of 7.8% and 5.2%, respectively. Native animals had varying detection rates: pooled urines from flying foxes had 7.8%, whilst koalas had 5.1%, and 6.7% of ticks screened were positive. The soil and dust samples showed the presence of C.burnetii DNA ranging from 2.0 to 6.9%, respectively. These data show that specimens from a variety of animal species and the general environment provide a number of potential sources for C.burnetii infections of humans living in Queensland. These previously unrecognized sources may account for the high seroprevalence rates seen in putative low-risk communities, including Q fever patients with no direct animal contact and those subjects living in a low-risk urban environment

Specific detection of enterovirus 71 directly from clinical specimens using real-time RT-PCR hybridization probe assay

10.1016/j.mcp.2005.11.003Molecular and Cellular Probes202135-14

Screening for H7N9 influenza A by matrix gene-based real-time reverse-transcription PCR

Rapid detection of novel influenza A strains, including H7N9, is pivotal to ensuring appropriate public health-based responses and real-time reverse-transcription polymerase chain reaction (RT-PCR) methods are used typically for this purpose. However, the utility of such methods can be undermined by ongoing sequence variations, particularly when targeting the variable influenza A haemagglutinin (HA) and neuraminidase (NA) genes. This may often be a source of frustration for clinical laboratories that are implementing methods in preparation for potential pandemics as primers and probe targets may need to be checked regularly and updated. In this study, screening methods were developed for H7N9 influenza A strains based on the highly-conserved influenza A matrix gene. Three assays were developed and evaluated in parallel, and included two methods which simply involved inclusion of a single H7N9 probe sequence into an established influenza A and B multiplex RT-PCR (FluAB-PCR). The detection limits of the methods were compared using ten-fold dilutions of H7N9 RNA, and the specificity of the methods were tested using 32 influenza A RT-PCR-positive samples and a panel of 18 influenza A isolates, including representives of seasonal H3N2, seasonal H1N1, pandemic H1N1, H5N1, H5N3, H9N2 and H7N7. The detection limits of the three methods were the same, and no cross-reactions were observed with sH3N2, sH1N1, pH1N1 or H5N1. However, cross-reactions were observed with H5N3, H9N2 and H7N7. Overall, the results show that the methods are useful for front-line screening for H7N9