Enhanced Etiological Diagnosis Of Respiratory Virus Outbreaks

1
Enhanced Etiological Diagnosis Of Respiratory
Virus Outbreaks Using Nucleic Acid Amplification
Testing Against An Expanded Range Of Targets
Sallene Wong1, Bonita E. Lee2,3, Kanti
Pabbaraju1, Kara L. Tokaryk1, Anita Wong1, Kevin
Ho1 and Julie D. Fox1,4 1Provincial Laboratory
for Public Health (ProvLab), Calgary, 2ProvLab,
Edmonton, 3Department of Pediatrics, University
of Alberta, Edmonton, 4Microbiology Infectious
Diseases, University of Calgary, Alberta, Canada
PASCV 2008, M-38 s.wong_at_provlab.ab.ca j.fox_at_provl
ab.ab.ca
STUDY BACKGROUND AND AIMS
Undiagnosed outbreak investigation by RVP
Phylogenetic analysis of picornaviruses
Use of nucleic acid amplification tests (NATs)
has enhanced our ability to provide an
etiological diagnosis in respiratory virus
outbreaks. The aim of this study was to evaluate
the performance of NATs on epidemiologically
linked cases of respiratory infection and to
assess the utility of the Luminex xTag?
Respiratory Viral Panel (RVP) assay to enhance
outbreak investigations.

• Human rhinovirus serotypes circulating during
  2006-2007 in Alberta were typed by sequencing.
• Sequences with greater than 95 identity to
  published sequences are included.
• Human rhinoviruses detected included
• Group A 1B, 12, 23, 29, 41, 43, 51, 58, 59 and
  94.
• Group B 6.
• Unclassified W10, W13, W21, W23, W28, QPM, and
  Antwerp rhinovirus 98/99 isolate 9812826.
• Identical rhinovirus serotypes were detected in
  all samples from an outbreak except 2 different
  serotypes were found in 2 individual outbreaks.

A
Unclassified
MATERIALS AND METHODS
Etiology of outbreaks by RVP
Samples Nasopharyngeal (NP) and throat swab
specimens from 243 suspected respiratory virus
outbreaks were submitted to the ProvLab for
diagnostic investigation in 2006/2007 (sample
n1093). Diagnostic algorithm In our routine
testing algorithm NP samples are first subjected
to direct fluorescent antigen (DFA) testing for
influenza virus (IFV)A, IFVB, parainfluenza
(PIV)1-3 and respiratory syncytial virus (RSV).
If DFA positive results are obtained no further
testing of the outbreak is undertaken.
DFA-negative NP samples from undiagnosed
outbreaks and all throat swab specimens from
outbreaks are then screened for a panel of
viruses by NATs (1). Nucleic acid extraction and
NATs Nucleic acid was extracted using the
easyMAG automated extractor and reagents
(bioMérieux). Individual real-time NATs were
directed against IFVA, IFVB, (PIV) 1-4, RSV,
human metapneumovirus (hMPV) and respiratory
adenoviruses (ADVs) (2). Luminex xTag? RVP assay
(Luminex Molecular Diagnostic Inc.) was performed
according to the manufacturers instructions (3)
on samples for which no positive results had been
obtained by DFA/in house NATs. Sequencing of
picornavirus positive samples Primers in the VP1
and 5NCR regions of picornaviruses (4) were used
for amplification of positive samples. Products
were purified and sequenced using the BigDye?
Terminator v1.1 Cycle Sequencing Kit (Applied
Biosystems). Sequences were analyzed using
Sequencing Analysis Software v5.1.1 (ABI) and
aligned using ClustalW. Phylogenetic trees were
constructed using the MegAlign module from
Lasergene 6 (DNAstar).
1st January 2006 - 31st December 2007 Total
number of outbreaks tested 63
Etiological agents detected by RVP in outbreaks
without a diagnosis by DFA/in house NAT
A
Unclassified
A
Unclassified
B
2006/2007 OUTBREAK DIAGNOSIS SUMMARY
Comparison of the partial 5NCR sequences for
rhinoviruses from respiratory samples. Prototype
sequences from GenBank are also included.
CONCLUSIONS

• The Luminex xTag? RVP assay allows for
  efficient, multiplex detection of a broader range
  of respiratory viral targets than our routine NAT
  panel.
• The added identification of picornaviruses and
  coronaviruses, which are not included in our
  current algorithm, will greatly improve our
  etiological diagnosis of respiratory virus
  outbreaks.
• The serotyping of rhinoviruses based on VP1 and
  5NCR was identical where both sequences were
  available.
• The same serotypes of human rhinovirus belong to
  individual outbreaks were observed.
• A total of 195 outbreaks in 2006/2007 had an
  etiological diagnosis using DFA/in house NATs.
  The detection rate increased from 67 to 80
  after addition of RVP testing.

• Low outbreaks activity during the summer months
  in 2006.
• An improvement from 83 to 91 in diagnosis
  using RVP in addition to routine testing.
• HKU1 and OC43 detected by RVP in Feb-06.
• Picornavirus detected in 9 outbreaks by RVP.

2006
Number of outbreaks
RESULTS

• Outbreaks without an etiological diagnosis
  occurred predominantly outside of the main
  respiratory virus season (especially summer and
  autumn of 2007).
• An improvement from 37 to 60 in diagnosis
  using RVP in addition to routine testing.
• Picornavirus (3), OC43 (2) and NL63 (2) detected
  by RVP in outbreaks during the period of Jan-07
  to Mar-07.
• During the months of May to October,
  picornavirus outbreaks were detected by RVP.

Month-year
Outbreak investigations by DFA/NATs
2007
REFERENCES

• Fox, J. D. 2007. Respiratory virus surveillance
  and outbreak investigation. J.Clin.Virol. 40
  Suppl 1S24-S30.
• Lee, B. E., J. L. Robinson, V. Khurana, X. L.
  Pang, J. K. Preiksaitis, and J. D. Fox. 2006.
  Enhanced identification of viral and atypical
  bacterial pathogens in lower respiratory tract
  samples with nucleic acid amplification tests.
  J.Med.Virol. 78702-710.
• Krunic, N., T. D. Yager, D. Himsworth, F.
  Merante, S. Yaghoubian, and R. Janeczko. 2007.
  xTAG RVP assay analytical and clinical
  performance. J.Clin.Virol. 40 Suppl 1S39-S46.
• Lee, W. M., C. Kiesner, T. Pappas, I. Lee, K.
  Grindle, T. Jartti, B. Jakiela, R. F. Lemanske,
  P. A. Shult, and J. E. Gern. 2007. A diverse
  group of previously unrecognized human
  rhinoviruses are common causes of respiratory
  illnesses in infants. PLoS.ONE. 2e966.

Number of outbreaks
Etiology of outbreaks by DFA/NATs
Etiological agents detected by DFA/NATs
Month-year
ACKNOWLEDGEMENTS
Virology and molecular diagnostic laboratory
technologists and assistants in PLPH (Calgary and
Edmonton) undertook routine specimen extraction
and testing by NASBA and RT-PCR.
1st January 2006 - 31st December 2007 Total
number of outbreaks tested 243