Journal of Clinical Virology 42 (2008) 190–193

Short communication

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
Farhad Gharabaghi a,∗ , Raymond Tellier a,b , Rose Cheung a , Carol Collins a ,
George Broukhanski c , Steven J. Drews b,c , Susan E. Richardson a,b
aDivision of Microbiology, Department of Paediatric Laboratory Medicine, Hospital for Sick Children,
555 University Avenue, Toronto, Ontario, M5G 1X8 Canada
b University of Toronto, Toronto, Ontario, Canada
c Molecular Diagnostics, Ontario Public Health Laboratories, 81 Resources Road, Toronto, Ontario, M9P 3T1 Canada
Received 31 May 2007; received in revised form 12 January 2008; accepted 31 January 2008

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 artusTM 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 artusTM 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 artusTM 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 artusTM 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.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Influenza; DFA; Culture; artusTM rRT-PCR; Children

1. Introduction other respiratory viruses (Bellau-Pujol et al., 2005; Weinberg

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
∗ Corresponding author. Tel.: +1 416 813 7654x4612;
fax: +1 416 813 6257.
E-mail address: farhad.gharabaghi@sickkids.ca (F. Gharabaghi).
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 fail-
ure 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 demon-
strate greater sensitivity than conventional PCR in detecting
microbial agents (Dagher et al., 2004; Templeton et al.,
2003).
The artusTM 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 initi-

1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.jcv.2008.01.013
 F. Gharabaghi et al. / Journal of Clinical Virology 42 (2008) 190–193
Table 1
Distribution of specimens selected for analysis by the artusTM Influenza LC RT-PCR kit
Influenza
DFA−/culture−
Negative for all respiratory viruses 235
Influenza A 0 1
Influenza B 0 17
Positive for other respiratory virusesa 47
Total 282
a DFA and/or culture positive for respiratory viruses other than influenza (total n = 47) included adenovirus (n = 8), respiratory syncytial virus (n = 24),
parainfluenza virus 1 (n = 13) and parainfluenza virus 3 (n = 2).
ated within the context of the seasonal occurrence of influenza
in a pediatric population.
2. 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 Chil-
dren. 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 col-
lected from the anterior nares using a sterile polyester tipped
applicator (Puritan Medical Products, Guilford, ME) and
transferred into sterile viral transport medium (phosphate-
buffered saline (PBS), 0.5% gelatin and antibiotics). All
specimens were anonymized and de-linked from all personal
health identifiers; thus research ethics board approval was not
required.
Specimens were vortexed and a 100 ␮l aliquot was
removed and frozen at −80 ◦ C for later analysis by PCR.
The remainder was then centrifuged at 14,000 rpm for 3 min,
and the supernatant returned to the specimen vial for isola-
tion in cell culture. The pellet was resuspended in 50–100 ␮l
PBS. Five microliter aliquots were spotted on a multi-well
glass slide, air dried and then fixed with cold acetone. Each
slide had one well for each FITC or rhodamine-labeled mon-
oclonal antibody to the following targets: influenza A/B,
respiratory syncytial virus (RSV), adenovirus (ADV), and
parainfluenzavirus 1, 2, 3. The reagents for specific detec-
tion of various viruses were SimulFluor® Flu A/Flu B,
SimulFluor® RSV/para 3, para 1 MAB-FITC, para 2 MAB-
FITC and ADV MAB-FITC (Chemicon, Temecula, CA). The
rest of the protocol was completed according to the manu-
facturer’s recommendations.
The remaining resuspended cells were pooled with the
supernatant for inoculation into RhMK (Diagnostic Hybrids,
Athens, OH) and MDCK (ATCC, Manassas, VA) cell
cultures. After removing the medium, each culture was inoc-
ulated with 100–200 ␮l of the specimen and incubated at
37 ◦ C for at least 1 h. One milliliter of Eagle minimum essen-
tial medium containing 1% foetal bovine serum (Viromed,
Minnetonka, MN) was added and cultures were incubated
191
Influenza Influenza Influenza Total
DFA−/culture+ DFA+/culture− DFA+/culture+
0 0 0 235
47 26 74
8 60 85
0 0 0 47
18 55 86 441
at 37 ◦ C. After detecting cytopathic effect (CPE), the pres-
ence and type of virus was confirmed by DFA. If CPE
was not observed the RhMK cells were passed to a fresh
RhMK tube upon which a hemadsorption test was performed.
Hemadsorption-positive cultures confirmed by DFA were
reported as positive for influenza A or B.
Frozen aliquots were thawed once 5 months later for RNA
extraction using the biorobot M48 workstation (Qiagen, Mis-
sissauga, ON, Canada) and the MagAttract Virus Mini M48
kit (Qiagen). Extracted RNA was stored at −80 ◦ C before
being subjected to amplification by the artusTM Influenza
LC RT-PCR kit (Qiagen, Mississauga, ON, Canada). This
kit is also available in the United States and most Euro-
pean countries and does not differentiate influenza A from
B. The kit is labeled RUO (Research Use Only) for Canada,
ASR (Analyte Specific Reagent) for the United States and CE
marking for European countries. It includes one master solu-
tion, one magnesium salt solution, one positive control, one
negative control (water) and one proprietary internal control.
The assay required 5 ␮l of RNA for amplification and 15 ␮l
of master mix. Amplification was carried out according to
the manufacturer’s instructions.
Specimens were recorded as positive when an unambigu-
ous amplification curve was observed with a threshold cycle
(Ct ) of 40 or less.
For analysis of discordant results (DFA- positive or
culture-positive/artusTM PCR-negative and DFA- negative
or culture-negative/artusTM PCR-positive), the RNA sam-
ples were sent to the Section of Molecular Diagnostics,
Ontario Public Health Laboratories and rRT-PCR carried out
using the Centers for Disease Control and Prevention (CDC)
RT-PCR protocol for influenza A and influenza B “CDC
real-time RT-PCR protocol for detection and characteriza-
tion of influenza” (Ref. #: I-007-05, Personal communication
Dr. Stephen Lindstrom). This protocol is available to US
State and Canadian Provincial Public Health laboratories
as part of a materials transfer agreement with the CDC
(http://www.cdc.gov/od/ads/techtran/index.htm). The rRT-
PCR and detection was carried out in an optical 96 well plate
using an ABI 7900HT Sequence Detection System. Posi-
tive specimens were identified as those with a Ct of 40 or
less.
Specimens were classified based on the criteria listed in
Table 1. The specificity of the real-time RT-PCR assay was
192 F. Gharabaghi et al. / Journal of Clinical Virology 42 (2008) 190–193
Table 2
The artusTM Influenza LC RT-PCR assay sensitivity and specificity for the detection of influenza A and B compared to an expanded gold standard of DFA,
viral culture and CDC rRT-PCR assay for discordant results
Influenza A Influenza B
DFA Culture DFA + culture DFA
Sensitivity (%) 98.6 100 98.6 98.5
Specificity (%) 100 100 100 100
assessed by testing RNA purified from 47 specimens previ-
ously reported positive by DFA and/or culture for adenovirus,
parainfluenza viruses and RSV.
3. 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 artusTM rRT-PCR (96.2% sen-
sitivity). Of the six negative artusTM 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/artusTM PCR-negative specimen. Thus, 3/4 DFA-
positive/culture-negative/artusTM and CDC rRT-PCR neg-
atives should be re-classified as DFA false positives and
artusTM true negatives. Therefore, the re-calculated sensi-
tivity of the artusTM rRT-PCR assay using the expanded
Fig. 1. Comparison of influenza artusTM Influenza LC RT-PCR assay with
conventional screening methods (DFA and cell culture). In each field of
the Venn diagram the number of specimens positive for influenza A or
B is given, the distribution between influenza A and B is also included
in parentheses. *One specimen out of five was positive using CDC rRT-
PCR assay. **Although the artusTM assay does not identify the influenza
virus as A or B, these specimens were also tested and typed with the
CDC rRT-PCR assay. N = 221 negatives by DFA, culture and artusTM
rRT-PCR.
Influenza A and B
Culture DFA + culture DFA Culture DFA + culture
98.7 97.6 98.6 99.0 98.7
100 100 100 100 100
gold standard (DFA, culture and CDC rRT-PCR) was 98.7%
(153/155) (Table 2). The sensitivity for influenza A and B
individually was 98.6% and 97.6%, respectively, using the
same expanded gold standard.
Of 235 specimens that were negative by DFA and cul-
ture for all respiratory viruses (including influenza), 14 were
influenza rRT-PCR positive (6.0%) in both the artusTM and
CDC assays indicating that they were true positives. Fig. 1
summarizes the results for all assays. Furthermore cross reac-
tivity with the other respiratory viruses such as adenovirus,
respiratory syncytial virus, parainfluenza virus 1 and 3 was
not observed, which clearly showed that the artusTM kit’s
components were specific to influenza A and B with a speci-
ficity of 100%.
4. Discussion
These results indicate the validity of using the artusTM
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 artusTM assay was com-
parable 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 com-
pleted in under 5 h, similar to assays used for influenza and
RSV in other studies (Boivin et al., 2004; Schaefer et al.,
2003).
Complementing this assay with an rRT-PCR specific for
the prevailing pandemic strain, and achieving this in a timely
fashion, will be important for pandemic preparedness.
Acknowledgments
The authors would like to thank Dr. Stephen Lind-
strom 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 gener-
ously donating the artusTM Influenza LC RT-PCR kits.
 F. Gharabaghi et al. / Journal of Clinical Virology 42 (2008) 190–193
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