Diagnostics Rhinovirus


offered as part of a multiplex respiratory virus panel. Due to the complexity of in-house assay development, verification, and continuous validation, most laboratories that offer respiratory virus detection panels use commercial kits. Several published validation studies of these commercial kits compared the results of the panel with those of culture and/or FA detection,64–67


with alternate molecular assays used


to confirm samples with discrepant results or samples with positive results for viruses not detected by culture and/or FA.68–71


More rigorous


validation of these panels for RhV detection is needed, including testing all specimens by both the panel and another, well-validated molecular assay capable of detecting all RhV subtypes, including the new type C.


PLx MultiCode Respiratory Virus Panel (Eragen),64,68,70,73 and xTAG Respiratory Virus Panel (Luminex),69,74


which detect 21, 17, and


20 respiratory pathogen targets, respectively. These three assays use two-step, multiplex RT-PCR amplification and hybridization to Luminex fluorescent particles for amplicon identification. All include an internal extraction/inhibition control and/or amplification/assay performance control. The amplicons are biotin labeled in a multiplex target-specific primer extension step, in which the primers contain both target-specific sequences and tag sequences that hybridize to complementary antitag oligonucleotides that are bound to microspheres embedded with different proportions of fluorescent dyes. The Eragen assay employs a proprietary novel base pair for target-specific labeling and room temperature hybridization to the microspheres. In one study evaluating the Eragen panel, the rate of RhV detection was lower than usually found in the population.68


The Luminex


assay is currently the only RhV assay approved by the US Food and Drug Administration (FDA). However, the assay detects respiratory picornaviruses as a group and not rhinoviruses specifically. Luminex also has a streamlined version, the RVP Fast assay, that detects 12 respiratory viruses. Detection of picornaviruses by this assay compared with realtime RT-PCR had 89.6% sensitivity and 94.5% specificity.74


Another commercial multiplex respiratory virus detection panel that includes RhV is the Infiniti (AutoGenomics) system, which identifies 23 respiratory viruses. Following multiplex RT and PCR, automated primer extension is performed to label amplicons with a fluorescent dye and tag them with a specific sequence, followed by microarray hybridization to immobilized anti-tags. The sensitivity of the assay was 96.6% compared with realtime RT-PCR for detection of picornaviruses.75


The


Seeplex RV12 Kit (Seegene) identifies 12 respiratory viruses, including RhV A subtypes only, using two PCR dual-priming oligonucleotide primer mixes.76


that


The Respifinder DC Two-Step Kit (PathoFinder) is a multiplex ligation-dependent probe amplification method77,65


detects 15 respiratory viruses in one reaction. Following conventional PCR, amplicons are detected using a set of two probes that ligate in the presence of target-specific complementary sequences. Probes have a target sequence, a common sequence, and a stuffer sequence to provide a unique length. Ligated probes are amplified using the common sequences. The amplicons from both the Seegene and Pathofinder assays were identified by size after slab gel or capillary electrophoresis. In one study, the Respifinder assay detected39 of 42 RhV-positive specimens compared with 37 positive by a realtime RT-PCR assay.77


78


Several commercial kits that provide detection of RhV include Resplex II (Qiagen),72


Quantitative Rhinoviruse Realtime Reverse Transcription-polymerase Chain Reaction Assays Some RhV realtime PCR assays have provided quantitative results.55,56,78 Absolute quantification of viral copies by realtime PCR requires use of a standard with known viral quantity. Standards that are prepared using cell culture, with 50% tissue culture infectious dose units or plaque-forming units, may not be reliable for quantifying the number of viral particles detected by PCR because one method measures infectious virus and the other measures viral RNA copies. Standards that are DNA plasmids containing the PCR target will not reflect the efficiency of the RT step. A one-step RT-PCR using a well-quantified RNA standard will provide the most accurate quantitative results. However, even this method may be unreliable for quantification of all RhV. Due to the sequence diversity among subtypes, the amplification and detection efficiency of a single primer and probe set will vary from RhV subtype to subtype. Therefore, a standard curve generated using RNA from one RhV genotype may not provide accurate quantification of a different genotype.


Optimizing and Validating Molecular Detection Optimal detection of RhV depends not only on the assay used but also on other factors such as specimen quality and nucleic acid extraction. Nasal wash, aspirate, or nasopharyngeal swab samples are the preferred specimens. RhV RNA has also been detected in blood and pericardial samples.2


Stringent specimen collection, transport, and


storage are less important for detection of RhV by molecular methods than by culture. Detection by NAAT usually requires extraction or purification of RNA prior to RT and amplification. Methods should be used that remove inhibitors of amplification from the specimen while optimizing the concentration of RNA. Amplification assay primer and probe design is critical. Mismatches in primer and probe binding sequences reduce the sensitivity and specificity of RT-PCR assays. Although the RhV 5´ NCR sequences are conserved among genotypes, the regions of complete homology are small. Various primer and probe design techniques have been described to overcome this problem including the use of minor groove binder molecules on the 3´ end of the TaqMan probe,57


locked nucleic acid bases and other modified bases,53 oligonucleotides76,77


sites of base mismatch.


The analytical and clinical sensitivities of RhV NAAT are difficult to determine when estimated by comparison with growth of the virus in cell culture, which detects only viruses that are viable in that cell line and, being less sensitive than RT-PCR,4,5,8,10–13,44,53,59


may overestimate the


sensitivity of a molecular assay when used as the comparator assay. Comparison of an RhV NAAT with another NAAT that uses a different primer and probe set allows a better comparison of how the assay performs. One important performance parameter of RhV molecular assays is the amount of cross-reactivity of the primers and probe with enterovirus RNA. Inclusion of an internal control (human or spiked RNA) to monitor for adequate RNA extraction and for the presence of inhibitors of amplification is useful.


RNA viruses are prone to change over time and RhV primer and probe sequences may need to be updated periodically to include new variants


US RESPIRATORY DISEASE


degenerate bases in the primers and probes,51,53,57 and dual-priming


to increase annealing temperatures and binding at


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