Objective: Respiratory tract infections account for more than 4 million deaths worldwide each year and are the greatest global killer among infectious diseases1. Viruses cause most respiratory tract infections, yet the specific infectious agent often remains unknown. In addition, community acquired pneumonia (CAP), can be a serious condition associated with significant morbidity and mortality. We aimed to produce a rapid real time multiplex PCR assay that could detect the presence of all major viral and bacterial causes of RTI from clinical specimens in less than 4 hours.
Methods: The following specimen sampling methods were evaluated for use with the assay: throat/nose swabs (dry and transport swabs), nasopharyngeal swabs, sputum, induced sputum and bronchial washings. Dry swabs were placed directly in sample extraction buffer, for transport medium or bronchial washings 150µl of liquid was placed in sample extraction buffer. If the sputum samples were primarily mucus an equal volume of lysis solution was added, mixed then 100µl transferred to fresh aliquot of lysis buffer. Samples were then heated at 90oC for 15 minutes.
Results: The sensitivity of all components of the multiplex assay was shown to be in the range of 5-20 copies of target. No cross reactivity was shown using a wide range of non-target bacteria and viruses commonly found in the respiratory tract. The results of testing over 300 patient samples as well as the prevalence of each viral target will be discussed.
Conclusion: The panels successfully detected the presence of specific viral/bacterial agents responsible for RTI from primary patient material in less than 4 hours. The test requires minimal hands on time (2-3 minutes) and can be implemented on a dedicated automated sample processing/PCR set up robot (GS1). Alternatively the assay can be carried out in clinical laboratories equipped with an automated sample extraction platform and real-time instrument without a further capital outlay for dedicated equipment.