enhanced etiological diagnosis of respiratory virus outbreaks using nucleic acid amplification...
TRANSCRIPT
0
5
10
15
20
25
Jan
07
Feb
07
Mar
07
Apr 0
7
May
07
Jun
07
Jul 0
7
Aug 0
7
Sep 0
7
Oct 0
7
Nov 0
7
Dec 0
7
0
5
10
15
20
25
30
35
40
45
Jan
06
Feb
06
Mar
06
Apr 0
6
May
06
Jun
06
Jul 0
6
Aug 0
6
Sep 0
6
Oct 0
6
Nov 0
6
Dec 0
6
IFVA only
IFVB only
IFVA & IFVB
IFVA & other viruses
IFVB & other viruses
RSV
PIV
hMPV
ADV
Non-IFV mixed infections
Outbreaks without etiological diagnosis
(DFA/NAT)
IFVA , 2
OC43, 3
NL63, 2
HKU1, 1
PIV and PIV+OC43, 1
picornavirus, 22
Outbreaks without etiological diagnosis
(RVP), 32
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
STUDY BACKGROUND AND AIMSUse 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.
Virology and molecular diagnostic laboratory technologists and assistants in PLPH (Calgary and Edmonton) undertook routine specimen extraction and testing by NASBA and RT-PCR.
PASCV 2008, [email protected]
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 n=1093). 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 manufacturer’s 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 5’NCR 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).
Undiagnosed outbreak investigation by RVP Phylogenetic analysis of picornaviruses
Outbreak investigations by DFA/NATs
ACKNOWLEDGEMENTS
REFERENCES
CONCLUSIONS
2006/2007 OUTBREAK DIAGNOSIS SUMMARY
MATERIALS AND METHODS
Etiology of outbreaks by DFA/NATs
1st January 2006 - 31st December 2007Total number of outbreaks tested= 243
1st January 2006 - 31st December 2007Total number of outbreaks tested= 63
Etiology of outbreaks by RVP
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 5’NCR 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. Year Outbreaks with
etiological diagnosisOutbreaks without etiological diagnosis
Total outbreaks tested
% positive
2006 134 27 161 83%
2007 30 52 82 37%
Total 164 79 243 67%
Year Outbreaks with etiological diagnosis
Outbreaks without etiological diagnosis
Total outbreaks tested
% positive
2006 12 11 23 52%
2007 19 21 40 48%
Total 31 32 63 49%
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.
1. Fox, J. D. 2007. Respiratory virus surveillance and outbreak investigation. J.Clin.Virol. 40 Suppl 1:S24-S30.
2. 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. 78:702-710.
3. 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 1:S39-S46.
4. 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. 2:e966.
Positive targets Number of samples tested positive
IFVA 2
PIV4 2
PIV4 + OC43 1
OC43 3
NL63 5
HKU1 1
picornavirus 49
Comparison of the partial 5’NCR sequences for rhinoviruses from respiratory samples. Prototype sequences from GenBank are also included.
A
A
Unclassified
B
Unclassified
A
Unclassified
Month-year
RESULTSMonth-year
2006
Nucleotide Substitutions (x100) 0
40.0
5 10 15 20 25 30 35 40
07RM294-1 07RM294-2 07RM294-3
07RC328-1 07RM40-1 07RM40-2 EU096016 Human rhinovirus 29
07RM107-1 07RC311-1
EU126722 Human rhinovirus 59 06RC347-1
EU126686 Human rhinovirus 23 06RC363-3 06RC363-1 06RC363-2
EU126778 Human rhinovirus W23 07RC189-1
EU126769 Human rhinovirus W10 06RC25-1
06RC25-2 06RC25-3 EU126714 Human rhinovirus 51
06RC3-1 07RC335-1 07RC335-2
EU126706 Human rhinovirus 43 07RC363-1 07RC363-2 EU126704 Human rhinovirus 41
06RC292-2 06RC292-3
06RC292-1 EU126664 Human rhinovirus 1B EU126663 Human rhinovirus 1A
07RM296-2 07RM296-1 EU126756 Human rhinovirus 94
EU126782 Human rhinovirus W28 06RM57-1 06RC25-1
06RC323-1 07RM50-1 EU126675 Human rhinovirus 12
07RC396-1 EF186077 Human rhinovirus QPM EU126779 Human rhinovirus W24 07RC363-1 EU126721 Human rhinovirus 58
06RC169-1 06RC169-2
EU126777 Human rhinovirus W21 EU126772 Human rhinovirus W13
07RM255-1 EU126786 Human rhinovirus W35
DQ316287 Antwerp rhinovirus 98/99 isolate 98128526 07RM248-1
07RC338-1 07RC338-2 EU126669 Human rhinovirus 6
Num
ber
of o
utbr
eaks
Num
ber
of o
utbr
eaks
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.
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.
Etiological agents detected by RVP in outbreaks without a diagnosis by
DFA/in house NAT
Etiological agents detected by DFA/NATs
Outbreaks without diagnosis
Outbreaks without a diagnosis by DFA/in house NATs for which etiological diagnosis was provided by RVP
Outbreaks with etiological diagnosis by DFA/NATs
2007
Identified agent Number of positive outbreaksIFVA only 82IFVB only 10IFVA & IFVB 4IFVA & other viruses 27IFVB & other viruses 8RSV 5PIV 12hMPV 10ADV 0Non-Flu mixed infections 6Outbreaks without etiological diagnosis 79