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Recent Studies Prop Up Multiplex PCR Testing for Respiratory Viruses

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By Ben Butkus

Multiplex PCR respiratory virus assays perform as well or better than traditional fluorescent antibody tagging and cell culture methods at detecting respiratory viruses in clinical specimens, according to a pair of recently disclosed scientific studies.

The research results add to a growing body of evidence supporting the use of multiplex PCR respiratory virus panels, such as those offered by EraGen, Seegene, Luminex, and Qiagen, to aid in diagnosing respiratory viruses in a clinical setting.

In the first study, published last week online in the Journal of Clinical Microbiology, Researchers from Washington University School of Medicine compared the PLx Multi-Code respiratory virus panel, manufactured by Madison, Wis.-based EraGen, with both conventional virologic testing methods and laboratory-developed PCR assays for single viruses.

"Multiplex respiratory virus panels are very exciting," Gregory Storch, a professor of pediatrics and corresponding author on the paper, told PCR Insider this week. Such panels, Storch added, are "a huge development in diagnostic microbiology. We've been very interested in them from the onset."

In their study, the researchers tested a set of 410 archived respiratory specimens, mostly nasopharyngeal swabs, including 210 that had been deemed positive for a variety of common viruses by conventional testing methods.

The researchers tested the specimens using conventional fluorescent antibody staining plus fibroblast viral cultures; with home-brewed PCR assays for specific viruses; and with the PLx Multi-Code panel.

The Multi-Code panel uses multiplex PCR to amplify target sequences and interrogate the resulting amplicons via target-specific extension and solid-phase hybridization and detection. The assay uses EraGen's proprietary isoC and isoG, probe-free bases that are specific to each other, but do not pair with natural nucleic acid bases, enabling specificity superior to that of other nucleic acid-based assays.

The researchers read the EraGen assays using a Luminex 100 instrument and proprietary software. They performed the tests at EraGen's laboratories in Madison using nucleic acid extracts prepared from specimens at Washington University.

In all, the PLx-RVP panel detected 292 viruses in 264 specimens, compared to 215 in 210 specimens by conventional testing; representing a 35.8 percent increase in detected viruses and 25.7 percent increase in positive specimens.

According to the researchers, the increased detection yield was primarily a result of the EraGen test's superior ability to detect rhinoviruses, as well as its ability to detect metapneumovirus and human coronaviruses, which were not detected using conventional methods.

The sensitivity of the PLx respiratory virus panel ranged from 83 percent to 100 percent; while specificity ranged from 91 percent to 100 percent, depending on the virus detected.

In addition, the researchers performed "homebrewed" reference PCR assays on all specimens that were positive either by the PLx panel or conventional testing, and also on a sample of approximately 25 percent of specimens that were negative for any viruses by both methods. The researchers found that their reference PCR assays agreed with the PLx assay panel results 97.9 percent of the time.

"We're very impressed with the assay; it performed very, very well," Storch said. "We developed what we consider to be very high-quality homebrewed PCR assays for all of the respiratory viruses.

"Then we compared the [EraGen test] with our conventional procedures of fluorescent antibodies and culture; and all of the positives from either test, and a sampling of the negatives, were all tested with our panel of PCR assays, and we found very excellent concordance," Storch added.

Storch also noted that the EraGen assay panel "outperformed" the laboratory-developed PCR test for rhinovirus; but did fail to detect some parainfluenza viruses that were detected using a homebrewed test, "so it was a little weak there," Storch said. "But I think [EraGen] has since tried to improve that."

EraGen's multiplex PCR assay panels, including the PLx respiratory virus assay panel, are currently under development. EraGen's assays are no longer being developed for use on Luminex's platforms after the companies nixed a longstanding licensing agreement and partnership last year.

Instead, EraGen has partnered with other bead-based instrument platforms for multiplex assays, including Illumina's BeadXpress (see related story, this issue).

Seegene, Qiagen, Luminex

In the second study, presented in a poster at last month's Pan American Society for Clinical Virology meeting in Daytona Beach, Fla., researchers from Toronto's Hospital for Sick Children compared the effectiveness of three commercial multiplex endpoint PCR-based respiratory assay panels with direct fluorescent antibody testing and viral culture.

"Because of their sensitivity and specificity, nucleic acid amplification assays are emerging more and more as the preferred methods of diagnostic testing," the researchers wrote in their poster. "These assays offer significant advantages over antigen detection methods and in most cases conventional culture methods."

The researchers selected 750 nasopharyngeal swabs from children with respiratory symptoms, and tested extracted nucleic acid from the samples using Qiagen's ResPlex II Panel v2.0, Seegene's Seeplex RV15, and Luminex's xTag RVP. Both the Qiagen and Luminex assay panels were tested on a Luminex 100 or 200 instrument platform; while the Seegene test used the Lab901 ScreenTape or Shimadzu MultiNA high-throughput electrophoresis platforms.

They compared the results of the multiplex PCR tests with that of direct immunofluorescence assay and viral culture methods, and found that all three multiplex PCR tests, for most viruses, offered greater sensitivity than and comparable specificity to both immunofluorescence and viral culture.

In particular, all three multiplex PCR tests demonstrated 100 percent sensitivity for influenza B virus (compared to about 75 percent for both immunofluorescence and culture methods); while Seegene's test demonstrated 100 percent sensitivity in detecting several viruses.

The researchers underscored the fact that the Seeplex assay showed a 2.6-fold increase in sensitivity (from 38.1 percent to 100 percent) over immunofluorescence in detecting adenovirus; and that both the Seeplex and xTag assays demonstrated a 2.2-fold increase in sensitivity (from 43.3 percent to 97.4 percent) over immunofluorescence in detecting human metapneumovirus.

The researchers also compared three multiplex PCR platforms — Resplex II Panel Plus, Seeplex Influenza A/B subtyping, and Astra Diagnostics' Influenza Screen & Type RT kit 1.0 — to immunofluorescence for detecting H1N1 influenza A (2009) from 75 collected nasopharyngeal swabs, 49 of which had been positively diagnosed as having H1N1 influenza. All three assays outperformed immunofluorescence, with the Astra test achieving 100 percent sensitivity.

The results of the study were particularly encouraging for Seegene, as the Seeplex test had a higher sensitivity than Luminex's xTag RVP for all but one target virus in the multi-virus portion of the study. Seeplex's Seegene diagnostic kits are not currently approved for use in the US, but are licensed for use in Canada; while Luminex's xTag RVP is cleared by the US Food and Drug Administration.

Seegene founder and CEO Jong-Yoon Chun said in a statement that the study results were "extremely gratifying."

Qiagen's Resplex II v2.0 test, while demonstrating better sensitivity than traditional methods, showed markedly less sensitivity than the other multiplex PCR assays. In May, Qiagen said that it will distribute a next-generation version of the respiratory virus panel developed by Celera (PCR Insider, 2/11/2010). Qiagen's RVP is also not cleared for use by the FDA.

Overall, the researchers concluded that multiplex PCR testing "offers rapid, comprehensive viral respiratory detection in a single test with high sensitivity and specificity. Moreover, newer targets can be added to multiplex assays which, combined with high-throughput analysis capabilities, make these assays extremely versatile, sensitive, cost-effective, and time-saving diagnostic tools."

Other companies developing multiplex molecular tests for respiratory viruses include Osmetech and Autogenomics.

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