Common etiological viral agents of respiratory infections include adenoviruses (ADV), influenza types A and B (Flu A and B), parainfluenza types 1, 2, and 3 (PIV1, 2, 3),and respiratory syncytial virus (RSV). 1-4 These viruses are responsible for a spectrum of acute upper and lower respiratory tract disease. In children, the elderly and other immunocompromised groups, respiratory viruses can cause more serious clinical complications, such as croup, bronchiolitis, and pneumonia, which often require hospitalization. 5,6 Virus isolation by cell culture and direct immunofluorescent antibody assay (DFA) staining with monoclonal antibodies are two of the most commonly used laboratory techniques for detecting respiratory viruses. Both these methods have significant limitations in sensitivity and specificity. DFA detection is more rapid but less sensitive than viral culture, and results may be affected by specimen quality (ie, presence of intact, infected cells), virus type, and interpretation of a positive result, which is subjective and requires a great deal of technical skill. 2,7-10 DFA is also unable to detect minor variations in amino acid sequence on envelope or capsid proteins. 11 Viral culture is still considered the "gold standard" for respiratory virus detection, but is limited by a prolonged result turnaround time (ie, 2 days to 1 week) and is dependent on stringent specimen transport and storage conditions to preserve the infectivity of the virus. 9,[12][13][14][15] Although the combination of both these techniques can provide an increase in the number of positive results, a significant proportion of specimens still remain negative, despite clinical suspicion of viral infection. 8 Several studies have shown that polymerase chain reaction (PCR) amplification can resolve the intrinsic limitations associated with traditional diagnostic techniques by combining increased sensitivity, specificity, and rapid result turnaround time. 16,17 Also, PCR results are not dependent on infectious virus or viable cells. However, Supported by Royal Children's Hospital Foundation grants RA921-006 and I922-034 which were sponsored by the Woolworth's "Care for Kids" campaign.

The recent description of the respiratory pathogen human metapneumovirus (hMPV) has highlighted a deficiency in current diagnostic techniques for viral agents associated with acute lower respiratory tract infections. We describe two novel approaches to the detection of viral RNA by use of reverse transcriptase PCR (RT-PCR). The PCR products were identified after capture onto a solid-phase medium by hybridization with a sequence-specific, biotinylated oligonucleotide probe. The assay was applied to the screening of 329 nasopharyngeal aspirates sampled from patients suffering from respiratory tract disease. These samples were negative for other common microbial causes of respiratory tract disease. We were able to detect hMPV sequences in 32 (9.7%) samples collected from Australian patients during 2001. To further reduce result turnaround times we designed a fluorogenic TaqMan oligoprobe and combined it with the existing primers for use on the LightCycler platform. The real-time RT-PCR proved to be highly reproducible and detected hMPV in an additional 6 out of 62 samples (9.6%) tested during the comparison of the two diagnostic approaches. We found the real-time RT-PCR to be the test of choice for future investigation of samples for hMPV due to its speed, reproducibility, specificity, and sensitivity.

Despite recent reports of clonal strains of Pseudomonas aeruginosa in cystic fibrosis (CF) units, the need for routine microbiological surveillance remains contentious.Sputum was collected prospectively from productive patients attending the regional paediatric and adult CF units in Brisbane, Australia. All P. aeruginosa isolates were typed using pulsed-field gel electrophoresis. Spirometry, anthropometrics, hospitalisations and antibiotic sensitivity data were recorded.The first 100 sputum samples (first 50 patients at each clinic) harboured 163 isolates of P. aeruginosa. A total of 39 patients shared a common strain (pulsotype 2), 20 patients shared a strain with at least one other patient and 41 patients harboured unique strains. Eight patients shared a strain identical to a previously reported Australian transmissible strain (pulsotype 1). Compared with the unique strain group, patients harbouring pulsotype 2 were younger and had poorer lung function. Treatment requirements were similar in these two groups, as were the rates of multiresistance.In conclusion, 59% of patients harboured a clonal strain, supporting the need for routine microbiological surveillance. In contrast to previously described clonal strains, the dominant pulsotype was indistinguishable from nonclonal strains with respect to both colonial morphology and multiresistance. The clinical significance of clonal strains remains uncertain and requires longitudinal study.

In this study, the suitability of two repetitive-element-based PCR (rep-PCR) assays, enterobacterial repetitive intergenic consensus (ERIC)-PCR and BOX-PCR, to rapidly characterize Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis (CF) was examined. ERIC-PCR utilizes paired sequence-specific primers and BOX-PCR a single primer that target highly conserved repetitive elements in the P. aeruginosa genome. Using these rep-PCR assays, 163 P. aeruginosa isolates cultured from sputa collected from 50 patients attending an adult CF clinic and 50 children attending a paediatric CF clinic were typed. The results of the rep-PCR assays were compared to the results of PFGE. All three assays revealed the presence of six major clonal groups shared by multiple patients attending either of the CF clinics, with the dominant clonal group infecting 38 % of all patients. This dominant clonal group was not related to the dominant clonal group detected in Sydney or Melbourne (pulsotype 1), nor was it related to the dominant groups detected in the UK. In all, PFGE and rep-PCR identified 58 distinct clonal groups, with only three of these shared between the two clinics. The results of this study showed that both ERIC-PCR and BOX-PCR are rapid, highly discriminatory and reproducible assays that proved to be powerful surveillance screening tools for the typing of clinical P. aeruginosa isolates recovered from patients with CF. INTRODUCTIONPseudomonas aeruginosa is a major cause of chronic lung infection in children and adults with cystic fibrosis (CF) and results in significant morbidity and mortality (Hutchison & Govan, 1999). Epidemic, multi-resistant strains have been reported within CF clinics in Australia and the UK (Anthony et al., 2002;Armstrong et al., 2002;Jones et al., 2001; McCallum et al., 2001McCallum et al., , 2002. Strain typing by traditional phenotypic methods is an important part of epidemiological surveillance, but may lack discriminatory power and stability. Molecular techniques offer a considerable improvement, and can complement phenotypic data to obtain a better understanding of bacterial diversity (Olive, 1999).PFGE is commonly employed, and has achieved widespread recognition as the 'gold standard' for P. aeruginosa DNA typing (Bertrand et al., 2001;Breitenstein et al., 1997;Douglas et al., 2001;Grundmann et al., 1995;Spencker et al., 2000). However, this method is limited by technical complexity, expense and prolonged turnaround times for results (Olive, 1999 (Versalovic et al., 1991). Two such groups of repetitive elements are the enterobacterial repetitive intergenic consensus (ERIC) sequences common to Gram-negative enteric bacteria, and the BOX elements, originally detected in Streptococcus pneumoniae (Hulton et al., 1991;Martin et al., 1992;Tyler et al., 1997).To our knowledge, ERIC-and BOX-PCR have not previously been used to compare P. aeruginosa strains isolated from adult and paediatric patients with CF. In this study, we characterized 163 clinical isolates collected from 50 ch...

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