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ENHANCED DETECTION OF RESPIRATORY VIRUSES USING
THE LUMINEX xTAGTM RVP ASSAY R. Manji1, F.
Zhang1, M. Lotlikar2, L. Falk2, M. Kowerska2, M.
Bornfreund2, S. Arora2, and C. C. Ginocchio 1,
2 1. North Shore-LIJ Health System Laboratories,
Lake Success, NY and 2. North Shore University
Hospital, Manhasset, NY
CVS May 2008 M 37
INTRODUCTION
RESULTS Percentage of Samples by Specific Virus
Identified by DFA, R-Mix and RVP
RVP DETECTION BY UNIVERSAL ARRAY
The reliable and timely identification of
respiratory viral infections is of tremendous
importance for diagnosis, patient management
(rational use of antibiotics and antiviral
agents), infection control, and for epidemiology
and surveillance studies. This study evaluated
the investigational xTAGTM RVP assay (Luminex
Molecular Diagnostics, Austin, TX) for the
detection of 18 of the most common strains and
subtypes of respiratory viruses, including
respiratory syncytial virus (RSV) A and B,
influenza A (H1, H3), influenza B, parainfluenza
1, 2, 3, 4, adenovirus, coronavirus (229E, OC43,
NL63, HKU1), human metapneumovirus (hMPV), and
enterovirus/rhinovirus (E/R). Aims of the
Study To compare the rates of respiratory virus
detection by direct immunofluorescence (DFA),
R-Mix culture and xTAG RVP assay.
Figure 4
Figure 3
MATERIALS AND METHODS
LUMINEX xMAP DETECTION PLATFORM
NT not tested, E/R enterovirus/rhinovirus
group Percentage () is based on total samples
(n 354). Mixed infections were found in 20
samples (5.65) including 2 E/RRSV 10
E/RInfluA 2 FluAhMPV 1 FluARSV 2 FluANL63
1 RSVHKU-1 1 hMPVNL63 1 NL63229E.

• Samples tested included
• Flocked nasopharyngeal swabs (Copan), placed in
  viral transport media, were collected during the
  2007-2008 winter season from 354 patients (ages
  19-59 yr) with symptoms of respiratory infection.
  
• Specimen processing and nucleic acid (NA)
  extraction
• Clinical respiratory samples (200 ml) and an
  internal control consisting of MS2 bacteriophage
  were added to 2.0 ml NucliSENS lysis buffer
  (bioMerieux, Durham, NC).
• Lysis buffer was vortexed and held at room
  temperature for 15-30 min to allow for cell and
  viral lysis.
• NA extraction was performed using the easyMAG
  instrument (bioMerieux) according to the
  manufacturers directions (Figure 1).
• Samples were tested with the RVP assay, according
  to an in-house validated protocol.
• Results were compared to standard laboratory
  testing, which included DFA and R-Mix culture
  (Diagnostic Hybrids, Athens, OH).

Figure 5
CONCLUSIONS

1. xTagTM RVP assay can efficiently detect the major
   respiratory viruses, including mixed infections.
2. Overall detection rate was significantly higher
   for RVP as compared to DFA and culture.
3. The assay can subtype influenza A strains (H1,
   H3), which is critical for surveillance of
   circulating strains.
4. The addition of an internal control provided a
   way to monitor the entire process including
   extraction, reverse transcription, amplification
   and detection.
5. This assay will provide insight on respiratory
   pathogens (ex coronavirus and hMPV) that are not
   routinely cultured in most laboratories, the
   prevalence of mixed viral infections, and their
   role in disease severity and outcome.

• Samples are then placed in a Luminex xMAP
  instrument where beads are read and analyzed by
  lasers.
• The lasers identify the color of the bead
  (specific to a virus or subtype) and the presence
  or absence of the labeled primer.
• If a particular virus is present at sufficient
  titer, it will be identified by the associated
  software as a positive.

Figure 1 easyMAG Extraction Instrument
REFERENCES
RVP ASSAY RT-PCR and TSPE
RESULTS Overall Percentage of Samples with a
Virus(es) Identified by DFA, R-Mix and RVP
Figure 2 Target Specific Primer Extension (TSPE)

• Mahoney J. et al. Development of a respiratory
  virus panel test for detection of twenty human
  respiratory viruses by use of multiplex PCR and a
  fluid microbead-based assay. J Clin Microbiol.
  2007 Sep45(9)2965-70.
• Merante F. et al. Principles of the xTAG
  respiratory viral panel assay (RVP Assay). J
  Clin Virol. 2007 Nov40 Suppl 1S31-5.

• No positive number of samples positive for a
  respiratory virus(es).
• Viruses tested by DFA panel included adenovirus,
  hMPV, influenza A and B, parainfluenza 1, 2, 3,
  and RSV.
• R-Mix culture screened for adenovirus, influenza
  A and B, parainfluenza 1, 2, 3, and RSV.