A woman who recently traveled to Thailand came to a local emergency department with a fever and papular rash. She was tested for measles, malaria, and dengue. Positive finding for IgM antibody against dengue and a failure to seroconvert for IgG against dengue for multiple blood samples suggested an alternate flavivirus etiology. Amplification of a conserved region of the non-structural protein 5 gene of the genus Flavivirus yielded a polymerase chain reaction product with a matching sequence of 99% identity with Zika virus. A urine sample and a nasopharygeal swab specimen obtained for the measles investigation were also positive for this virus by reverse transcription polymerase chain reaction. Subsequently, the urine sample yielded a Zika virus isolate in cell culture. This case report describes a number of novel clinical and laboratory findings, the first documentation of this virus in Canada, and the second documentation from this region in Thailand.

Detection of respiratory viruses using sensitive real-time nucleic acid amplification tests (NATs) is invaluable for patient and outbreak management. However, the wide range of potential respiratory virus pathogens makes testing using individual real-time NATs expensive and laborious. The objective of this study was to compare the detection of respiratory virus targets using the Luminex xTAG respiratory viral panel ( The sensitivity and specificity of respiratory virus detection have improved considerably with the advent of nucleic acid amplification tests (NATs). There have been several recent reviews of the use of NATs for detection of respiratory viruses that outline the methodologies, advantages, and future directions (4, 5, 8). The fluorogenic probe-based real-time assays are particularly useful for detection of a small number of targets. However, multiplexing real-time NATs can be technologically challenging and can result in a loss of sensitivity. The reagents for these assays are expensive, and setup for individual targets is time-consuming. Multiplex assays for amplification and detection of a panel of respiratory viruses using suspension microarrays may provide a practical solution. Currently, up to 100 different spectrally distinct fluorescence-labeled beads are available for multiplex target detection on the Luminex suspension microarray platform (3). A different nucleic acid probe can be conjugated to each bead, and a mixture of beads is used for detection and differentiation of products amplified in a multiplex PCR. The technology is flexible since more probes can be added or replaced by mixing individual beads, making it ideal for complex analyses, such as those needed for respiratory virus detection.The development of kits for the detection of a panel of respiratory viruses using suspension microarrays has been described (1, 2, 9, 11-13, 16). These assays show good (and in some cases enhanced) performance compared with traditional antigen and culture methods for respiratory virus detection. Based on these studies, it was thought that these assays may be a suitable replacement for in-house NATs with good sensitivity, specificity, and savings in cost and time. Array approaches also have the added benefit of broad viral detection compared with our in-house NAT panel. The Luminex xTAG respiratory viral panel (RVP) used in this evaluation allows detection of a panel of 20 respiratory virus targets with differentiation between 19 of these (enteroviruses and rhinoviruses are detected by the generic picornavirus probe) (14). Here we report the results of a prospective and retrospective evaluation of the RVP assay compared with a panel of in-house real-time NATs.

Nucleic acid tests are sensitive and specific and provide a rapid diagnosis, making them invaluable for patient and outbreak management. Multiplex PCR assays have additional advantages in providing an economical and comprehensive panel for many common respiratory viruses. Previous reports have shown the utility of the xTAG respiratory viral panel (RVP) assay manufactured by Luminex Molecular Diagnostics for this purpose. A newer generation of this kit, released in Canada in early 2010, is designed to simplify the procedure and reduce the turnaround time by about 24 h. The assay methodology and targets included in this version of the kit are different; consequently, the objective of this study was to compare the detection of a panel of respiratory viral targets using the older Luminex xTAG RVP (RVP Classic) assay with that using the newer xTAG RVP Fast assay. This study included 334 respiratory specimens that had been characterized for a variety of respiratory viral targets; all samples were tested by both versions of the RVP assay in parallel. Overall, the RVP Classic assay was more sensitive than the RVP Fast assay (88.6% and 77.5% sensitivities, respectively) for all the viral targets combined. Targets not detected by the RVP Fast assay included primarily influenza B virus, parainfluenza virus type 2, and human coronavirus 229E. A small number of samples positive for influenza A virus, respiratory syncytial virus B, human metapneumovirus, and parainfluenza virus type 1 were not detected by the RVP Classic assay and in general had low viral loads.The utility of the xTAG respiratory viral panel (RVP) (RVP Classic) assay, manufactured by Luminex Molecular Diagnostics for the detection of a panel of respiratory viruses, has been demonstrated. Previous reports have shown this assay to have comparable or superior sensitivity compared to those of direct fluorescent-antibody assay (DFA)-, culture-, and PCR-based methods (10,14,20). The main drawbacks of this assay were its lengthy protocol, resulting in longer turnaround times, and the need for a manipulation of the amplified product, which could be a potential clinical laboratory contamination risk. The RVP Classic assay was modified to have a simpler protocol, resulting in a shorter turnaround time, and was marketed as the xTAG RVP Fast assay. The targets detected have been slightly altered to allow the detection of influenza A virus (IFVA), with additional subtyping of positive specimens into subtypes H1 and H3; influenza B virus (IFVB); respiratory syncytial virus (RSV) types 1 and 2; human coronaviruses (hCoVs) NL63, 229E, OC43, and HKU1; parainfluenza viruses (PIV) types 1 to 4; human metapneumovirus (hMPV); picornaviruses (including enteroviruses [EV] and rhinoviruses [RVs]); and a range of adenovirus (ADV) types. This version of the kit can additionally detect the presence of human bocavirus (hBoV). In addition, RNA bacteriophage MS2 is used as an internal extraction/inhibition control, and DNA bacteriophage lambda is used as an amplification and assay perfor...

No abstract

No abstract

No abstract

Human adenoviruses (hAdVs) are associated with acute respiratory tract infections in pediatric populations and have been identified as a cause of outbreaks in institutional settings. Rapid diagnosis of hAdV infection is critical for appropriate and timely management. This study reports the design and validation of a sensitive and specific multiplex real-time PCR for the detection of a broad range of hAdV serotypes in respiratory samples. The assay targets the conserved region of the hAdV hexon gene and utilizes hydrolysis probes for the detection of amplified products. The assay was evaluated using retrospectively (n = 864) and prospectively (n = 11,451) collected samples from November 2005 to July 2006. Seasonality studies and analysis of outbreaks was conducted over a 2-year period from January 2005 to December 2006 (n = 33,067 samples). The assay gave a hAdV positive rate of 7.1% (n = 811) for specimens tested prospectively and was able to detect a broad range of hAdV serotypes with good sensitivity and specificity. A high rate of co-infection was noted (21.7%). Adenovirus infections were more prevalent in the young with a median age of 24 months for positive patients. Sequence analysis of hAdV positives showed that serotype 7 was the most prevalent followed by serotypes 2 and 3. Association of hAdVs with respiratory outbreaks was low at 2.3% (6 of 266 outbreaks tested) and no seasonal variation was observed for hAdV infections during the 2-year study period. This assay can improve the detection of hAdVs in respiratory samples and can be used to provide valuable epidemiological information.

For DNA viruses, genetic recombination, addition, and deletion represent important evolutionary mechanisms. Since these genetic alterations can lead to new, possibly severe pathogens, we applied a systems biology approach to study the pathogenicity of a novel human adenovirus with a naturally occurring deletion of the canonical penton base Arg-Gly-Asp (RGD) loop, thought to be critical to cellular entry by adenoviruses. Bioinformatic analysis revealed a new highly recombinant species D human adenovirus (HAdV-D60). A synthesis of in silico and laboratory approaches revealed a potential ocular tropism for the new virus. In vivo, inflammation induced by the virus was dramatically greater than that by adenovirus type 37, a major eye pathogen, possibly due to a novel alternate ligand, Tyr-Gly-Asp (YGD), on the penton base protein. The combination of bioinformatics and laboratory simulation may have important applications in the prediction of tissue tropism for newly discovered and emerging viruses.