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The advent of PCR has transformed the utility of the virus diagnostic laboratory. In comparison to traditional gel based PCR assays, real time PCR offers increased sensitivity and specificity in a rapid format. Over the past 4 years, we have introduced a number of qualitative and quantitative real time PCR assays into our routine testing service. During this period, we have gained substantial experience relating to the development and implementation of real-time assays. Furthermore, we have developed strategies that have allowed us to increase our sample throughput while maintaining or even reducing turn around times. The issues resulting from this experience (some of it bad) are discussed in detail with the aim of informing laboratories that are only just beginning to investigate the potential of this technology.

Influenza A infection was detected in the majority of patients with ILI; picornavirus infection was also shown to be an important cause of illness. PCR is a rapid and sensitive method for respiratory virus surveillance. Serology is slow, insensitive and difficult to interpret at low titres.

Acute respiratory tract infections are a major cause of morbidity and mortality worldwide and exert a considerable economic burden on healthcare systems. Acute respiratory tract infections of the upper and lower respiratory tract are caused by a wide variety of viral and bacterial pathogens, which require comprehensive laboratory investigations. Conventional serological and immunofluorescence-based diagnostic methods for acute respiratory tract infections lack sensitivity when compared to polymerase chain reaction (PCR)-based approaches and the development of new diagnostic methodologies is required, to provide accurate, sensitive and rapid diagnoses. In the present study, a PCR-based low density oligonucleotide microarray was developed for the detection of 16 viral and two atypical bacterial pathogens. The performance of this DNA microarray-based analysis exhibited comparable sensitivities and specificities to multiplex real-time reverse transcription polymerase chain reactions (rtPCRs) confirming the potential diagnostic utility of the method. In contrast to routine multiplex PCR, the microarray incorporates an intrinsic redundancy as multiple and non-identical probes per target on the array allow direct intra-assay confirmation of positives. This study demonstrates that microarray technology provides a viable alternative to conventional serological-based approaches and multiplex PCR for pathogen identification in acute respiratory tract infections.

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