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SUMMARY Clinical laboratories historically diagnose seven or eight respiratory virus infections using a combination of techniques including enzyme immunoassay, direct fluorescent antibody staining, cell culture, and nucleic acid amplification tests. With the discovery of six new respiratory viruses since 2000, laboratories are faced with the challenge of detecting up to 19 different viruses that cause acute respiratory disease of both the upper and lower respiratory tracts. The application of nucleic acid amplification technology, particularly multiplex PCR coupled with fluidic or fixed microarrays, provides an important new approach for the detection of multiple respiratory viruses in a single test. These multiplex amplification tests provide a sensitive and comprehensive approach for the diagnosis of respiratory tract infections in individual hospitalized patients and the identification of the etiological agent in outbreaks of respiratory tract infection in the community. This review describes the molecular methods used to detect respiratory viruses and discusses the contribution that molecular testing, especially multiplex PCR, has made to our ability to detect respiratory viruses and to increase our understanding of the roles of various viral agents in acute respiratory disease.

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

The appearance of eight new respiratory viruses, including the SARS coronavirus in 2003 and swine-origin influenza A/H1N1 in 2009, in the human population in the past nine years has tested the ability of virology laboratories to develop diagnostic tests to identify these viruses. Nucleic acid based amplification tests (NATs) for respiratory viruses were first introduced two decades ago and today are utilized for the detection of both conventional and emerging viruses. These tests are more sensitive than other diagnostic approaches, including virus isolation in cell culture, shell vial culture (SVC), antigen detection by direct fluorescent antibody (DFA) staining, and rapid enzyme immunoassay (EIA), and now form the backbone of clinical virology laboratory testing around the world. NATs not only provide fast, accurate and sensitive detection of respiratory viruses in clinical specimens but also have increased our understanding of the epidemiology of both new emerging viruses such as the pandemic H1N1 influenza virus of 2009, and conventional viruses such as the common cold viruses, including rhinovirus and coronavirus. Multiplex polymerase chain reaction (PCR) assays introduced in the last five years detect up to 19 different viruses in a single test. Several multiplex PCR tests are now commercially available and tests are working their way into clinical laboratories. The final chapter in the evolution of respiratory virus diagnostics has been the addition of allelic discrimination and detection of single nucleotide polymorphisms associated with antiviral resistance. These assays are now being multiplexed with primary detection and subtyping assays, especially in the case of influenza virus. These resistance assays, together with viral load assays, will enable clinical laboratories to provide physicians with new and important information for optimal treatment of respiratory virus infections.

Anal cytology and HPV detection have high sensitivity but low specificity for detecting AIN 2+. HIV-positive men who have sex with men have a high prevalence of AIN 2+ and require high-resolution anoscopy for optimal detection of high-grade anal dysplasia.

Two different severe acute respiratory syndrome (SARS) vaccine strategies were evaluated for their ability to protect against live SARS coronavirus (CoV) challenge in a murine model of infection. A whole killed (inactivated by b-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection. (Marra et al., 2003;Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates . 0008-1579 G 2006 SGM Printed in Great BritainCurrently, there is no effective treatment for SARS. Prevention through contact-reduction or transmission-blocking measures has been the only means available to modify the devastating impact of this illness. Prevention through vaccination would be an attractive alternative that is less reliant on individual case detection to be effective. No vaccines are currently licensed for any of the human CoVs, but effective vaccines have been produced for some animal CoVs, such as certain strains of Infectious bronchitis virus (poultry), Bovine coronavirus and Canine coronavirus (Cavanagh, 2003;Enjuanes et al., 1995;Pratelli et al., 2003;Saif, 2004;Takamura et al., 2002). Individuals convalescing from SARS develop high titres of neutralizing antibodies (Tan et al., 2004) and the appearance of antibodies coincides with the onset of resolution of SARS pneumonia Woo et al., 2004). Thus, there is some optimism that an effective vaccine against SARS-CoV may also be possible.Coronavirus spike (S) proteins have long been known to be a major determinant in coronavirus pathogenesis, given that this viral protein interacts with cellular receptors as well as con...

We performed a cost analysis study using decision tree modeling to determine whether the use of multiplex PCR testing for respiratory viruses (xTAG RVP test) is a more or less costly strategy than the status quo testing methods used for the diagnosis of respiratory virus infections in pediatric patients. The decision tree model was constructed by using four testing strategies for respiratory virus detection, viz., direct fluorescent-antibody staining (DFA) alone, DFA plus shell vial culture (SVC), the xTAG RVP test alone, or DFA plus the xTAG RVP test. A review of the charts of 661 pediatric patients was used to determine the length of hospital stay, the number of days in isolation, antibiotic usage, and all other medical procedures performed. The cost of hospitalization by diagnostic status was determined on the basis of the average cost per patient and the number of patients in each arm of the decision tree. The cost per case was the highest for DFA plus SVC at $3,914 (in Canadian dollars), and the lowest was for the xTAG RVP test alone at $3,623, while the costs of DFA alone ($3,911) and DFA plus RVP ($3,849) were intermediate. When all four diagnostic strategies were compared, the least costly strategy was the xTAG RVP test alone when the prevalence of infection was 11% or higher and DFA alone when the prevalence was under 11%. These data indicate a savings of $291 per case investigated if the strategy of using the xTAG RVP test alone was used to replace the status quo test of DFA plus SVC, resulting in a savings of $529,620 per year in direct costs for the four Hamilton, Ontario, Canada, hospitals on the basis of the testing of specimens from 1,820 pediatric inpatients. We conclude that the use of the xTAG RVP test is the least costly strategy for the diagnosis of respiratory virus infections in children and would generate a significant savings for hospitals.

SummaryThe ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoformspecific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.