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Comparison of a commercial qualitative real-time RT-PCR kit with direct immunofluorescence assay (DFA) and cell culture for detection of influenza A and B in children

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Abstract

Background

Institutional pandemic planning prompted a study of the molecular detection of influenza virus from respiratory specimens in children, compared to conventional diagnostics.

Objective

To evaluate the performance of a commercial qualitative real-time RT-PCR kit (rRT-PCR), the artus™ Influenza LC RT-PCR (Qiagen).

Study design (methods)

Specimens were pre-selected to include a high percentage of positives by direct immunofluorescence assay (DFA) or culture. The sensitivity and specificity of the kit for detection of influenza A and B in children were determined against the gold standard, DFA and culture. Specimens yielding discordant results between artus™ and the gold standard were tested against a reference rRT-PCR assay (Centers for Disease Control) to create an “expanded gold standard”.

Results

When compared to DFA or cell culture, the sensitivity of the rRT-PCR artus™ kit was 96.2% and the specificity was 94%. It detected influenza RNA in 6.0% of clinical samples negative by DFA or culture. Using the expanded gold standard, the revised sensitivity was 98.7% (98.6% for influenza A and 97.6% for influenza B) and the specificity was 100%.

Conclusion

The artus™ Influenza LC RT-PCR kit is an effective alternative to virus isolation and DFA for the detection of influenza A and B in pediatric clinical specimens.

Introduction

Influenza types A and B are two of the most important causes of human respiratory infection. The possibility of an impending influenza pandemic increases the need to establish rapid, highly reliable diagnostic testing in laboratories around the world.

Prior to the advent of PCR, DFA and viral isolation were the most sensitive methods for the detection of influenza and other respiratory viruses (Bellau-Pujol et al., 2005, Weinberg et al., 2004). However, a significant number of specimens in patients with clinically compatible viral respiratory infection remain negative by DFA and viral culture, implying a failure to identify the causative virus in a percentage of cases (Ellis et al., 1997, Freymuth et al., 1995, Gilbert et al., 1996). Molecular methods, and in particular, real-time PCR demonstrate greater sensitivity than conventional PCR in detecting microbial agents (Dagher et al., 2004, Templeton et al., 2003).

The artus™ Influenza LC RT-PCR kit is a qualitative assay for both influenza A and B in a real-time format. A study of this kit has not been published. Our study was initiated within the context of the seasonal occurrence of influenza in a pediatric population.

Section snippets

Methods

Nasal swabs were collected from children (birth to 17 years) with suspected respiratory tract infection between January and March 2005 at The Hospital for Sick Children. Of 2070 specimens received, 441 were selected for study, to include about 1/3 positive for influenza A or B, in addition to those positive for other respiratory viruses and those negative for any virus (Table 1). Specimens were collected from the anterior nares using a sterile polyester tipped applicator (Puritan Medical

Results

Of 441 specimens tested, 159 were positive by DFA and/or culture, for influenza A or B (Table 1). 153/159 were positive by artus™ rRT-PCR (96.2% sensitivity). Of the six negative artus™ specimens, five were “DFA-positive/culture-negative” and one was “DFA-negative/culture-positive”. To confirm the PCR results all six RNA were tested with the CDC rRT-PCR assay. The only one of the six specimens confirmed to be positive by the reference PCR was an influenza A DFA-positive/culture-negative/artus™

Discussion

These results indicate the validity of using the artus™ Influenza LC RT-PCR kit for detection of influenza A and B RNA in clinical specimens obtained from a pediatric population. The sensitivity of the artus™ assay was comparable to other studies (Atmar et al., 1996, Steininger et al., 2002).

The chief features of this assay are its high sensitivity and specificity, and its rapidity, which allows all the steps from RNA extraction to amplification/detection to be completed in under 5 h, similar to

Acknowledgments

The authors would like to thank Dr. Stephen Lindstrom and Dr. Alexander Klimov, CDC, DHHS/CDC/CCID/NCIRD/ID/VSDB, Atlanta, Georgia, USA for authorizing the use of their real-time RT-PCR protocol.

The authors would also like to thank all staff members of the virology laboratory, Hospital for Sick Children, Toronto, Ontario, Canada, for their assistance.

We thank Qiagen Inc., for supporting this study by generously donating the artus™ Influenza LC RT-PCR kits.

References (11)

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