Microbiology and Infectious Disease / MULTIPLEX PCR DETECTION OF RESPIRATORY VIRUSES

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients. Respiratory infections caused by influenza viruses (A and B), parainfluenza viruses (1, 2, and 3) and respiratory syncytial viruses (RSV; A and B) can result in severe lower respiratory tract infections that may require hospitalization.1 These viral agents account for about 80% to 90% of viral acute lower respiratory tract infections in infants and for 30% of all childhood deaths in developing countries.2,3 The results of these infections can range from mild respiratory illness to primary viral pneumonia and death. Infants, elderly people, and people with underlying health problems are at increased risk for complications that may result in hospitalization.4-10 Rapid differential diagnosis of these agents is necessary for monitoring infected patients, for the prevention of nosocomial spread, and to guide the choice of possible specific antiviral therapy.7,10 Moreover, Woo et al11 demonstrated that rapid viral diagnosis for respiratory infections at a tertiarycare university hospital reduces patients’ hospital stay and costs and reduces antibiotic use by up to 52%. The use of classic diagnostic techniques such as viral isolation and serologic testing often can result in a delay of several weeks before results are available, and these methods are therefore less useful for making therapeutic decisions.6,12 Several immunofluorescent, optical immunoassay, and enzyme-linked immunosorbent assays for direct detection of influenza and parainfluenza virus antigens are available commercially.13-15 However, these assays lack the sensitivity (50%-90%) for rapid diagnosis of these viral pathogens. In addition, direct antigen testing also may fail to detect emerging virus variants having altered amino acid sequences on envelope or outer capsid proteins.16,17 Finally, specimen integrity and the number of intact cells present in the specimens are crucial for reliable direct immunofluorescence assay results.18 Microbiology and Infectious Disease / ORIGINAL ARTICLE Am J Clin Pathol 2001;116:218-224 219 © American Society of Clinical Pathologists Rapid and sensitive molecular diagnostic techniques (reverse transcriptase–polymerase chain reaction [RT-PCR]) for the detection of one or several respiratory viruses in patient samples have been developed.2,19-23 These assays are more sensitive than the classic viral culture or viral antigen detection assay.1,2,19 Moreover, these assays allow rapid detection of microorganisms in patients infected with 2 or more viruses, which is more difficult by the classic diagnostic techniques.24 We evaluated the Hexaplex multiplex PCR assay (Prodesse, Waukesha, WI) for the direct detection of 7 respiratory viruses in 300 respiratory samples submitted to ARUP Laboratories, Salt Lake, City, UT, during the peak of the 1999-2000 respiratory virus season. This assay simultaneously detects influenza viruses A and B, RSV A and B, and parainfluenza viruses 1, 2, and 3. Materials and Methods