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Introduction. Diagnosis of acute respiratory infections (ARIs) can be facilitated by the Panther Fusion (PF) automatic, random access PCR system for the detection of influenzavirus A (Flu A) and B (Flu B), parainfluenzavirus (Paraflu), respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinovirus (RV) and human adenovirus (AdV) in nasopharyngeal swabs. Aim. To evaluate the performance of PF in comparison with established methods, including subsets of (1) lower respiratory tract (LRT) specimens and (2) upper respiratory tract (URT) hygiene screening specimens of patients without ARI symptoms. Methodology. The performance characteristics of PF were compared with bioMérieux R-Gene and laboratory-developed PCR tests (LDTs). Overall, 1544 specimens with 6658 individual diagnostic requests were analysed. Results. The overall concordances of PF and LDTs for Flu A, Flu B and AdV were 98.4, 99.9 and 96.1%, respectively; by re-testing of discrepant specimens concordances increased to 99.4, 99.9 and 98.0%, respectively. Initial concordances of PF and R-Gene assays for RSV, Paraflu, hMPV and RV were 98.4, 96.3, 99.3 and 96.0%, respectively, and retest concordances were 99.7, 97.9, 99.9 and 98.9%, respectively. No differences to the overall performance were found for the subgroups of LRT and hygiene screening specimens. PCR cycle threshold (Ct) values correlated very well between methods, indicating that a semi-quantitative diagnostic approach using Ct values (e.g. highly vs. weakly positive) could augment the diagnostic information. Conclusion. PF performed similar to R-Gene and LDTs not only for its intended use but also for LRT and hygiene screening specimens with shorter hands-on and turnaround times.

Although infections with the novel pandemic 2009 influenza A (H1N1) virus (A/H1N1/2009) appeared to be relatively mild during the first summer of circulation ('off season'), there has been significant morbidity and hospitalization and several fatal cases. Thus, rapid detection of A/H1N1/2009 is crucial for efficient treatment and infection control measures. In contrast to seasonal influenza, where point-of-care (POC) rapid antigen tests and direct fluorescent antibody (DFA) staining ensure rapid detection, diagnosis of A/H1N1/2009 has so far been based on RT-PCR. This study retrospectively compared the performance of the Quidel QuickVue POC test, DFA staining and virus isolation with that of RT-PCR for A/H1N1/2009 detection in 526 respiratory specimens collected during the first wave of the outbreak from May to September 2009. A/H1N1/2009 was detected in 9.1% (48/526) of samples. One hundred and thirty-seven of the A/H1N1/2009 PCR-negative samples were additionally tested using a RealAccurate Respiratory RT-PCR panel, revealing other respiratory viruses (mainly entero/rhino- and adenoviruses) in 42.3% (58/137). All methods analysed detected A/H1N1/2009 with excellent specificity but different sensitivities (POC test: 18.2%; DFA staining: 38.7%; virus isolation: 45.7%). Therefore, the POC test was not suitable for diagnosis, detecting A/H1N1/2009 only if present in high concentrations (corresponding median Ct value=19.0; range=16.5-21.4). DFA staining was also able to detect A/H1N1/2009 in specimens with a lower virus concentration (median Ct value=24.0; range=16.5-29.8). Virus isolation, which was positive after a median time of 7.5 days, was too time-consuming. In summary, DFA staining is superior to POC testing and may be appropriate for patients expected to have a rather high level of virus replication. Nevertheless, in DFA-negative specimens, A/H1N1/2009 should be excluded by RT-PCR.

We have assessed two approaches to sequencing complete human cytomegalovirus (HCMV) genomes (236 kbp) in DNA extracted from infected cell cultures (strains 3157, HAN13, HAN20 and HAN38) or clinical specimens (strains JP and 3301). The first approach involved amplifying genomes from the DNA samples as overlapping PCR products, sequencing these by the Sanger method, acquiring reads from a capillary instrument and assembling these using the Staden programs. The second approach involved generating sequence data from the DNA samples by using an Illumina Genome Analyzer (IGA), processing the filtered reads by reference-independent (de novo) assembly, utilizing the resulting sequence to direct reference-dependent assembly of the same data and finishing by limited PCR sequencing. Both approaches were successful. In particular, the investigation demonstrated the utility of IGA data for efficiently sequencing genomes from clinical samples containing as little as 3 % HCMV DNA. Analysis of the genome sequences obtained showed that each of the strains grown in cell culture was a mutant. Certain of the mutations were shared among strains from independent clinical sources, thus suggesting that they may have arisen in a common ancestor during natural infection. Moreover, one of the strains (JP) sequenced directly from a clinical specimen was mutated in two genes, one of which encodes a proposed immune-evasion function, viral interleukin-10. These observations imply that HCMV mutants exist in human infections.