Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.

The status of the cryptic bat species, Myotis mystacinus and Myotis brandtii in Ireland

The recent identification of Myotis brandtii in Ireland raised the possibility that many roosts previously identified as M. mystacinus had the potential of being misidentified M. brandtii. Thus, the distribution and population estimates for M. mystacinus may have been over-estimated, while M. brandtii may have been under-estimated. Results from an all Ireland genetic survey of known M. mystacinus maternity roosts confirm that no long term misidentification has taken place. All specimens caught and sampled were M. mystacinus. Additonally, no further records of M. brandtii were found during six nights of woodland trapping using the acoustic lure. While the status of M. mystacinus in Ireland is now listed as ‘least concern’ in the Irish Red List, M. brandtii is listed as ‘data deficient’ and cannot currently be considered a resident species

Molecular detection of CF lung pathogens:current status and future potential

Molecular diagnostic tests, based on the detection and identification of nucleic acids in human biological samples, are increasingly employed in the diagnosis of infectious diseases and may be of future benefit to CF microbiology services. Our growing understanding of the complex polymicrobial nature of CF airway infection has highlighted current and likely future shortcomings in standard diagnostic practices. Failure to detect fastidious or slow growing microbes and misidentification of newly emerging pathogens could potentially be addressed using culture-independent molecular technologies with high target specificity. This review considers existing molecular diagnostic tests in the context of the key requirements for an envisaged CF microbiology focussed assay. The issues of assay speed, throughput, detection of multiple pathogens, data interpretation and antimicrobial susceptibility testing are discussed.

Optimizing geometric accuracy of four dimensional CT scans acquired using the wall and couch mounted Varian Real-Time Position Management camera systems

Objective:

The aim of this study was to identify sources of anatomical misrepresentation due to the location of camera mounting, tumour motion velocity and image processing artefacts in order to optimise the 4DCT scan protocol and improve geometrical-temporal accuracy.

A phantom with an imaging insert was driven with a sinusoidal superior-inferior motion of varying amplitude and period for 4DCT scanning. The length of a high density cube within the insert was measured using treatment planning software to determine the accuracy of its spatial representation. Scan parameters were varied including the tube rotation period and the cine time between reconstructed images. A CT image quality phantom was used to measure various image quality signatures under the scan parameters tested.

No significant difference in spatial accuracy was found for 4DCT scans carried out using the wall mounted or couch mounted camera for sinusoidal target motion. Greater spatial accuracy was found for 4DCT scans carried out using a tube rotation speed of 0.5s rather than 1.0s. The reduction in image quality when using a faster rotation speed was not enough to require an increase in patient dose.

4DCT accuracy may be increased by optimising scan parameters, including choosing faster tube rotation speeds. Peak misidentification in the recorded breathing trace leads to spatial artefacts and this risk can be reduced by using a couch mounted infrared camera.

This study explicitly shows that 4DCT scan accuracy is improved by scanning with a faster CT tube rotation speed.

Identification of Pseudomonas aeruginosa by a duplex real-time polymerase chain reaction assay targeting the ecfX and the gyrB genes

Phenotypic identification of Gram-negative bacteria from respiratory specimens of patients with cystic fibrosis carries a high risk of misidentification. Molecular identification techniques that use single-gene targets are also susceptible to error, including cross-reaction issues with other Gram-negative organisms. In this study, we have designed a Pseudomonas aeruginosa duplex real-time polymerase chain reaction (PCR) (PAduplex) assay targeting the ecfX and the gyrB genes. The PAduplex was evaluated against a panel of 91 clinical and environmental isolates that were presumptively identified as P. aeruginosa. The results were compared with those obtained using a commercial biochemical identification kit and several other P. aeruginosa PCR assays. The results showed that the PAduplex assay is highly suitable for routine identification of P. aeruginosa isolates from clinical or environmental samples. The 2-target format provides simultaneous confirmation of P. aeruginosa identity where both the ecfX and gyrB PCR reactions are positive and may also reduce the potential for false negatives caused by sequence variation in primer or probe targets.