WHO EQAP for the detection of influenza A virus subtype by PCR Wilina Lim Centre for Health...
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Transcript of WHO EQAP for the detection of influenza A virus subtype by PCR Wilina Lim Centre for Health...
WHO EQAP for the detection of WHO EQAP for the detection of influenza A virus subtype by PCRinfluenza A virus subtype by PCR
Wilina LimWilina Lim
Centre for Health ProtectionCentre for Health Protection
Hong Kong SAR, ChinaHong Kong SAR, China
Aims/objectivesAims/objectives
To monitor quality and standards of To monitor quality and standards of performanceperformance
To facilitate information exchangeTo facilitate information exchange To identify problems with assaysTo identify problems with assays To help develop testing strategiesTo help develop testing strategies To provide mechanisms to remedy any To provide mechanisms to remedy any
deficiencies revealeddeficiencies revealed Requirement of lab accreditation in Requirement of lab accreditation in
accordance with international standard accordance with international standard such as ISO 15189such as ISO 15189
Benefits identified by NICs
To monitor feasibility to ship samples to
countries laboratory capability timeliness of reporting
1A. Invitation
2.2. Preparation of PanelsPreparation of Panels
4.4. Data CollectionData Collection
3.3. Panel DistributionPanel Distribution
5.5. Preliminary reportPreliminary report
6.6. Data Analysis Data Analysis
7.7. Final reportFinal report
QAP Process
QAP Process
QAP Process
QAP Process
1B. New Participants
The EQAP has beenaccredited in accordanceWith ISO 17043
Preparation of panelsPreparation of panels
Include different subtypes/cladesInclude different subtypes/cladesDried RNA of influenza AH5, H1, H3, H1v and influenza B virusesDried RNA of influenza AH5, H1, H3, H1v and influenza B virusesGamma-ray inactivated seasonal influenza samplesGamma-ray inactivated seasonal influenza samples
Verify sample contentVerify sample content
Verify sufficient homogeneityVerify sufficient homogeneitySamples in final packaged form selected and tested Samples in final packaged form selected and tested
Assure sufficient stabilityAssure sufficient stability
Test over a range of storage conditions prior to distributionTest over a range of storage conditions prior to distributionSamples were tested after 7 days of storage at 37Samples were tested after 7 days of storage at 37ooC using both C using both conventional and real-time PCR assays conventional and real-time PCR assays
Temperature surveyTemperature survey Monitor the temperature change during shipmentMonitor the temperature change during shipment Target participants Target participants
• With temperature record higher than 37℃ during sample With temperature record higher than 37℃ during sample dispatch perioddispatch period
• Panel 9: Jan-Mar 2011Panel 9: Jan-Mar 2011
RegionRegionNo. of logger No. of logger
sentsentNo. of logger No. of logger
returnedreturned
AFROAFRO 55 11
AMROAMRO 33 22
EMROEMRO 22 22
WPROWPRO 33 22
TotalTotal 1313 77
Temperature survey on EQAP panel 9 shipmentTemperature survey on EQAP panel 9 shipment
0
24
48
72
96
120
144
168
Lab A Lab B Lab C Lab D Lab E Lab F
Ship
men
t tim
e (h
r)
above 37℃
25℃-37℃
below 25℃
Longest duration with temperature higher than 37 was 6 hours℃
Lab D
0
10
20
30
40
50
60
Destination time
Tem
pera
ture
(℃
)
Duringshipment
Delivered
Temperature above 37℃ were recorded during the transit in daytime
Lab B
0
10
20
30
40
50
60
Destination time
Tem
pera
ture
(℃) During
shipmentdelivered
Panel contentsPanel contents
No. of samples in the panelNo. of samples in the panel
Panel 1Panel 1 Panel 2Panel 2 Panel 3Panel 3 Panel 4Panel 4 Panel 5Panel 5 Panel 6Panel 6 Panel 7Panel 7 Panel 8Panel 8 Panel 9Panel 9
20072007 20072007 20082008 20082008 20092009 20092009 20102010 20102010 20112011
Feb-MarFeb-Mar Aug-OctAug-Oct Jan-FebJan-Feb Jun-JulJun-Jul Jan-FebJan-Feb Jun-AugJun-Aug Jan-MarJan-Mar Jun-AugJun-Aug Jan-MarJan-Mar
RNA sampleRNA sample
H5 sample:H5 sample:
- clade 1- clade 1 22 11 11 22 22
- clade 2.1- clade 2.1 22 22 11 11 22
- clade 2.2- clade 2.2 22 22 11 11 22 22 22
- clade 2.3.2- clade 2.3.2 22 22 22 22 22
- clade 2.3.4- clade 2.3.4 22 22 11 11 22 22 22
H1 sampleH1 sample 11 11 11 11 22 11 11 11
H3 sampleH3 sample 11 11 11 11 11 11 11 11 11
H1pdm sampleH1pdm sample 22 22 22 22
Influenza B sampleInfluenza B sample 11 11 11
Negative sampleNegative sample 22 44 22 22 11 11 11 22
Inactivated virus sampleInactivated virus sample
H1 sampleH1 sample 11
H3 sampleH3 sample 11
TotalTotal 1010 1414 1010 1010 1010 1010 1010 1010 1212
Composition of panelsComposition of panels
WHOregion
No. of laboratories participated and reported results
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
2007 2007 2008 2008 2009 2009 2010 2010 2011
Feb-Mar Aug-Oct Jan-Feb Jun-Jul Jan-Feb Jun-Aug Jan-Mar Jun-Aug Jan-Mar
AFR 66 66 88 1313 1717 1919 21 22 24
AMR 55 1414 1616 2121 2323 2222 26 33 32
EMR 22 44 66 66 77 99 11 13 14
EUR 3434 3939 4343 4545 4545 5151 52 59 59
SEAR 33 44 55 55 66 66 6 8 8
WPR 1414 1616 1717 1919 1616 2121 23 23 23
All 6464 8383 9595 109109 114114 128128 139 158 160
0%
10%
20%
30%
40%
50%
60%
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
AFR AMR EMR EUR SEAR WPR
Proportion of laboratories participated and reported results
Response of Response of participantsparticipants
Problems encounteredProblems encountered
No PCR capacityNo PCR capacity No reagentsNo reagents Delay in obtaining import permitDelay in obtaining import permit Varying requirements at the customsVarying requirements at the customs Shipment detained at customs for Shipment detained at customs for
prolonged periodprolonged period
0%
5%
10%
15%
20%
25%
30%
35%
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
0
20
40
60
80
100
120
140
160
180
% of laboratories w ithout response
% of laboratories not w illing to participate
% of laboratories w ith import permit or logistical problems
No. of laboratories invited"
No. of laboratories not receiving samples
No. of laboratories% of laboratories
Reasons for laboratories not receiving Reasons for laboratories not receiving panels among total invitedpanels among total invited
Performance of participantsPerformance of participants
67%65%
74%77% 76%
79%
85% 86%
76%
160
95
139
128
114
109
83
64
158
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
160% of laboratories with all correct resultsNo. of laboratories invited
No. of participants% of laboratories
PanelNo. of participants
invited responded participated received reported all correct
Panel 1 99 44 44 33 33 2
Panel 2 99 55 55 44 44 2
Panel 3 1010 77 66 55 55 4
Panel 4 88 77 66 66 55 4
Panel 5 88 88 66 66 66 5
Panel 6 8 8 6 6 6 4
Panel 7 8 7 6 6 6 5
Panel 8 9 8 8 8 8 7
Panel 9 9 8 8 8 8 6
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
Performance of SEAR participantsPerformance of SEAR participants
% of all correct
PanelNo. of participants
invited responded participated received reported all correct
Panel 1 1919 1616 1616 1515 1414 8
Panel 2 2121 1717 1717 1717 1616 10
Panel 3 2020 1919 1919 1717 1717 13
Panel 4 2121 2121 1919 1919 1919 16
Panel 5 2121 2020 2020 1818 1616 14
Panel 6 22 22 22 22 21 16
Panel 7 23 23 23 23 23 19
Panel 8 24 24 24 24 23 22
Panel 9 24 24 24 24 23 21
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6 Panel 7 Panel 8 Panel 9
Performance of WPR participantsPerformance of WPR participants
% of all correct
Performance in Panel 8
Performance in Panel 9
Problems identified in EQAPProblems identified in EQAP
Inconsistent technical performanceInconsistent technical performance Positive control not used appropriatelyPositive control not used appropriately Lab contaminationLab contamination Misinterpretation of resultsMisinterpretation of results Primers and probes mismatchPrimers and probes mismatch Transcriptional errorTranscriptional error
False negative results due to probe False negative results due to probe mis-matchesmis-matches
An example of a H5 real-time PCR An example of a H5 real-time PCR primer/probe set primer/probe set
Forward primer: 1 bp mis-matchForward primer: 1 bp mis-match Probe: 2 bp mis-matchesProbe: 2 bp mis-matches Reverse primer: noneReverse primer: none
GLP sGLP surveyurvey
2007 survey composed of 73 questions2007 survey composed of 73 questions 2008 survey composed of 25 questions2008 survey composed of 25 questions 2010 survey composed of 32 questions2010 survey composed of 32 questions Questions on the following seven categories:Questions on the following seven categories:
• personnelpersonnel• quality managementsquality managements• design, equipment and consumablesdesign, equipment and consumables• pre-analytical procedurespre-analytical procedures• analytical proceduresanalytical procedures• post-analytical procedurespost-analytical procedures• reporting and record keepingreporting and record keeping• safetysafety
Molecular diagnosis (PCR) & Good laboratory practice (GLP)Molecular diagnosis (PCR) & Good laboratory practice (GLP)
Laboratories returning completed GLP survey formsLaboratories returning completed GLP survey forms
2007200764 ( 96%)64 ( 96%)
2008200894(82%)94(82%)
20102010142 (89%)142 (89%)
Good laboratory practices
More than 80% of laboratories
Separate work room for molecular diagnosis (99%)
Separate set of equipment and consumables in each working area (94%)
Equipment maintenance programme (87%)
Control materials for molecular diagnosis (100%)
Standard operating procedures (94%)
Separate work room for molecular diagnosis (99%)
Separate set of equipment and consumables in each working area (94%)
Equipment maintenance programme (87%)
Control materials for molecular diagnosis (100%)
Standard operating procedures (94%)
Less than 80% of laboratories
Evaluation of the reagents used for molecular tests (67%)
Evaluation of the sensitivity/specificity of the molecular tests (59%)
Internal audit programme (54%)
Accredited by international/national scheme (34%)
Countercheck results (78%)
Evaluation of the reagents used for molecular tests (67%)
Evaluation of the sensitivity/specificity of the molecular tests (59%)
Internal audit programme (54%)
Accredited by international/national scheme (34%)
Countercheck results (78%)
Data from GLP survey 2010
GLP and EQAP performance
Compare GLP with EQAP results
• Group A (laboratories returned correct answers for all 10 samples)
• Group B (laboratories returned less than 10 corrects answers)
Laboratories with less good performance (Group B)
Lab did not return all correct results tends to meet less quality parameters; significantly more likely (p < 0.05) not having- audits of personnel
- separate rooms for all steps involving PCR
- programme to monitor equipment
- more samples in recent representative month
- SOP on preparation of in-house controls
- established test turn-around-time
Reagents/tests evaluation, internal audit and accreditation Reagents/tests evaluation, internal audit and accreditation in laboratories of different WHO regionsin laboratories of different WHO regions
67 6569 67 65
7568
59
35
59
33
6975
70
54 55
38
50
65
100
44
34
0
24
8
56
25
39
0
20
40
60
80
100
120
All region AFR AMR EMR EUR SEAR WPR
% o
f la
bora
tori
es
Reagents evaluation Tests evaluation Internal audit Accreditation
Data from GLP survey 2010
The way forwardThe way forward
ReviewReview- materials for simulated specimens/scope- materials for simulated specimens/scope- type/subtype/clade to include in the panel- type/subtype/clade to include in the panel- frequency of shipment- frequency of shipment
Enhance performance through trainingEnhance performance through training Accreditation by recognized authorityAccreditation by recognized authority
http://www.wpro.who.int/sites/htl/documents/Laboratory+Quality+Standards+and+Implementation.htm
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