WHO EQAP for the detection of
influenza A virus subtype by PCR
Centre for Health Protection
Hong Kong SAR, China
Aims/objectives




To monitor quality and standards of
performance
To facilitate information exchange
To identify problems with assays
To provide mechanisms to remedy
any deficiencies revealed
Benefits to participants

Able to
• compare performance
• provide evidence of quality
• minimize errors
• identify training if needed
Methodology

Targets
• mostly NICs and some non-NICs

Materials
• dried RNA of H5, H1, H3, H1v viruses

Schedule
• twice a year

Data analysis
• questionnaires on



testing strategy
test methodology
good laboratory practice (GLP)
Distribution of panels and response
of participants
No. of laboratories
WHO
Region
Invited
Received samples
Reported results
P1
P2
P3
P4
P5
P1
P2
P3
P4
P5
P1
P2
P3
P4
P5
AFR
10
11
12
19
19
6
8
9
16
17
6
6
8
13
17
AMR
26
28
28
27
27
6
16
19
22
24
5
14
16
21
23
EMR
8
9
9
10
10
2
5
6
7
8
2
4
6
6
7
EUR
50
51
53
52
57
35
40
43
45
48
34
39
43
45
45
SEAR
9
9
10
8
8
3
4
5
6
6
3
4
5
5
6
WPR
19
21
20
21
21
15
17
17
19
18
14
16
17
19
16
Total
122
129
132
137
142
67
90
99
115
121
64
83
95
109
114
Response of participants
AFRO, AMRO, EMRO, EURO
SEARO, WPRO
No. invited
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
102
108
119
99
113
94
28
30
30
29
29
30
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
Panel 6
No. received
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
90
69
No. reported
97
104
77
49
18
21
22
25
24
28
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
Panel 6
120
110
100
90
80
70
60
50
40
30
20
10
0
63
85
92
73
83
47
17
20
22
24
22
22
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
Panel 6
Reasons for laboratories not
receiving panels
No. (%) of laboratories
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
(N=122)
(N=129)
(N=132)
(N=137)
(N=142)
No response
38 (31)
26 (20)
20 (15)
11 (8)
9 (6)
Unwilling to participate
5 (4)
7 (5)
7 (5)
10 (7)
9 (6)
6 (5)
6 (5)
1 (1)
3 (2)
Problem
Import permit or logistical problems 12 (10)
Composition of panels
No. of samples in the panel
Panel Contents
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
Panel 6
2007
2007
2008
2008
2009
2009
Feb-Mar
Aug-Oct
Jan-Feb
Jun-Jul
Jan-Feb
Jun-Aug
2
1
1
2
H5 sample:
- clade 1
- clade 2.1
2
2
1
1

- clade 2.2
2
2
1
1

2
2
2
- clade 2.3.2
2
2
1
1
2

H1 sample
1
1
1
1
2

H3 sample
1
1
1
1
1

- clade 2.3.4

H1v sample
Negative sample
2
4
2
2
1
Total
10
14
10
10
10
10
Method of detection
(% of participants)
H5 PCR
80
60
Conventional
40
Real-time
20
Panel 2
Panel 3
H1 PCR
80
Panel 4
60
40
40
20
20
Panel 3
H3 PCR
80
60
Panel 2
Panel 5
Panel 4
Panel 5
Panel 2
Panel 3
Panel 4
Panel 5
Nucleic acid amplification tests

Most were developed in-house
• the primes/probes:



most commonly adapted from other
researchers
minority were own designed
Minority were commercial kits
Assessment criteria


The performance of individual laboratories was
assessed by adding up the number of correct
results.
Incorrect responses:
• failing to detect H5 samples and/or reporting the results
as non-H5 subtype
• failing to detect H1 samples and/or reporting the results
as non-H1 subtype
• failing to detect H3 samples and/or reporting the results
as non-H3 subtype
• failing to report correct influenza A test results for
H1/H3 samples if H1/H3 subtyping was not performed
• reporting positive results for a sample that did not
contain any viral RNA
Performance of laboratories
No. (%) of laboratories
Performance
Panel 1
Panel 2
Panel 3
(N=64)
(N=83)
(N=95) (N=109) (N=114)
43 (67)
54 (65)
70 (74)
84 (77)
87 (76)
All but 1 sample correct
6 (9)
14 (17)
10 (11)
11 (10)
12 (11)
50–89% of samples correct
9 (14)
12 (14)
11 (12)
10 (9)
14 (12)
<50% of samples correct
6 (9)
3 (4)
4 (4)
4 (4)
1 (1)
All samples correct
Panel 4
Panel 5
Testing errors made by laboratories
No. (%) of laboratories
Comparison factors
Incorrect H5 results
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
(N=64)
(N=83)
(N=95)
(N=109)
(N=114)
15
(23)
17 (20)
17 (18)
13 (12)
21 (18)
(14)
5
2
6
1
False-positive results 9
(6)
(2)
(6)
(1)
Overall performance
Panel 1
100
Panel 2
Panel 3
Panel 4
Panel 5
90
80
70
60
50
40
30
20
10
AFR
AMR
EMR
EUR
SEAR
WPR
Total
H5 performance
Panel 1
Panel 2
Panel 3
Panel 4
Panel 5
100
90
80
70
60
50
40
30
20
10
AFR
AMR
EMR
EUR
SEAR
WPR
Total
Performance
AFRO, AMRO, EMRO, EURO
SEARO, WPRO
% of H5 all correct
% of all correct
95
95
85
83
86
82
77
75
86
85
88
86
82
75
75
71
65
65
59
60
55
55
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6
Panel 1 Panel 2 Panel 3 Panel 4 Panel 5 Panel 6
Problems identified in EQAP




Positive control not used appropriately
Lab contamination
Misinterpretation of results
Primers and probes mismatch
The primers/probes sequences of the H5 gene used by participants
Prime r/Probe name
H5 + 1456
H5 - 1685
H5 + 1637
Prime r/Probe
Se que nce 5' to 3'
Primer
ACG T AT GAC T AT CCA CAA T AC T CA G
Primer
AGA CCA GCT ACC AT G AT T GC
Probe
T CA ACA GT G GCG AGT T CC CT A GCA
Le ngth
25
20
24
Position
1512-1536
1663-1644
1617-1640
Dir
F
R
F
Journal
J Clin Microbiol 2002 40 3256-3260
J Clin Microbiol 2002 40 3256-3260
J Clin Microbiol 2002 40 3256-3260
H5Forward
H5Reverse
H5probe
Primer
Primer
Probe
GCC GAA T GA T GC MAT MAA YT
CGC ACC CAT T GG AGT T T G AC
CAT T GC T CC AGA AWA T
20
20
16
758-777
908-889
797-812
F
R
F
J Clin Microbiol 2007 45 1535-1543
J Clin Microbiol 2007 45 1535-1543
J Clin Microbiol 2007 45 1535-1543
H5-943
H5-1300
Primer
Primer
GCC ACT CCA CAA T AT ACA CCC
CAA AT T CT C T AT CCT CCT T T C CAA
21
24
923-943
1285-1263
F
R
Lancet 1998 351 467-471
Lancet 1998 351 467-471
HA-1144
H5-1735R
Primer
Primer
GGA AT G AT A GAT GGN T GG T AY GG
GT G T T T T T A AYT AMA AT C T GR ACT MA
23
26
1092-1114
1746-1721
F
R
WHO (2002)
WHO (2002)
H5-1
H5-3
Primer
Primer
GCC AT T CCA CAA CAT ACA CCC
CT C CCC T GC T CA T T G CT A T G
21
20
923-943
1141-1122
F
R
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
H5-266F
H5-1615F
H5-347R
H5-1695R
H5-290P
H5-1634P
Primer
Primer
Primer
Primer
Probe
Probe
T GC CGG AAT GGT CT T ACA T AG T G
GT G GCG AGC T CC CT A GCA
T CT T CA T AG T CA T T G AAA T CC CCT G
T CT GCA T T G T AA CGA CCC AT T G
AGA AGG CCA AT C CAG T CA AT G ACC T CT GT T A
T GG CAA T CA T GG T AG CT G GT C T AT CCT T AT GG
23
18
25
22
31
32
274-296
1623-1640
355-331
1703-1682
298-328
1642-1673
F
F
R
R
F
F
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
WHO (2007), CHP, Hong Kong SAR
H5-248-270F
H5-671-647R
Primer
Primer
GT G ACG AAT T CA T CA AT G T RC CG
CT C T GG T T T AGT GT T GAT GT Y CCA A
23
25
256-278
679-655
F
R
WHO (2007), NIID, Japan
WHO (2007), NIID, Japan
H5HA-205-227v2-For
H5HA-326-302v2-Rev
H5-Probe-239-RVa
H5-Probe-239-RVb
Primer
Primer
Probe
Probe
CGA T CT AGA YGG GGT GAA RCC T C
CCT T CT CCA CT A T GT ANG ACC AT T C
AGC CAY CCA GCT ACR CT A CA
AGC CAT CCC GCA ACA CT A CA
23
25
20
20
182-204
303-279
238-219
238-219
F
R
R
R
WHO (2007), NIID, Japan
WHO (2007), NIID, Japan
WHO (2007), NIID, Japan
WHO (2007), NIID, Japan
RF 1151
RF 1152
RF 1153
Primer
Primer
Probe
GGA ACT T AC CAA AT A CT G T CA AT T T AT T CA
CCA T AA AGA T AG ACC AGC T AC CAT GA
T T G CCA GT G CT A GGG AAC T CG CCA C
30
26
25
1590-1619
1673-1648
1647-1623
F
R
R
WHO (2007), EMC, the Netherlands
WHO (2007), EMC, the Netherlands
WHO (2007), EMC, the Netherlands
PCR7ModMMForward
PCR7Reverse
PCR7degen
Primer
Primer
Probe
GCC GAA T GA T GC MAT MAA YT
CGC ACC CAT T GG AGT T T G AC
CAT T GC T CC AGA AWA T
20
20
16
758-777
908-889
797-812
F
R
F
HPA VSOP41
HPA VSOP41
HPA VSOP41
H5VietFor
H5VietRev
H5VietProbe
Primer
Primer
Probe
GGA T GG CAG GGA AT G GT A GA
T CT AT T GCC T T T T GA GT G GAT T CT T
T GG GT A CCA CCA T AG CAA YGA GCA GG
20
25
26
1083-1102
1183-1159
1112-1137
F
R
F
HPA VSOP46
HPA VSOP46
HPA VSOP46
The sequences of the H5 primers/probes were obtained from PubMed and WHO website.
The positions of the oligonucleotides are based on the HA gene of A/HongKong/156/1997(H5N1), GenBank accession number AF046088.
Dir, direction; F, forward; R, reverse
The most widely adopted H5 PCR
primers/probes
No. of laboratories
Panel
reported
results
performed
H5 subtyping
adopted CDC
primers/probes
with incorrect
H5 resultsa
N
N
%b
N
%c
N
%d
Panel 2
83
81
98
17
21
3
18
Panel 3
95
94
99
29
31
4
14
Panel 4
109
108
99
44
41
2
5
Panel 5
114
114
100
47
41
5
11
a
Incorrect results were due to either false-positive, false negative or any non-H5 results reported in H5 samples.
b
Percentages are based on the number of laboratories reported results.
c
Percentages are based on the number of laboratories performed H5 subtyping.
d
Percentages are based on the number of laboratories adopted CDC primers/probes.
Factors affecting H5 performance
Panel
Panel 2
Panel 3
Panel 4
Panel 5
aP
Technical factora
Real-time assay Commercial kit
0.002
0.678
0.006
0.565
0.006
0.637
0.188
0.348
values were calculated by Yates-corrected chi-square test.
TAT > 28 days
Not applicable
0.002
0.945
0.022
H1v performance in Panel 6
% of H1v all correct
100
95
AFRO
AMRO
90
EMRO
EURO
85
SEARO
WPRO
80
Panel 6
One participant each in AFRO, AMRO and EMRO reported incorrect H1v subtyping results.
H1v primers/probes adopted by 91
participants in Panel 6
No.*
%
Centres for Disease Control and Prevention
71
78
Institut Pasteur, Paris, France
5
5
Robert Koch-Institute
4
4
Health Protection Agency, United Kingdom
3
3
Other 19 sources
19
1
Source
* More than one set of primers/probes were used by 10 participants, the total number is larger than 91.
GLP Survey



2007 survey composed of 73 questions
2008 survey composed of 25 questions
Both surveys composed questions on the
following seven categories:
•
•
•
•
•
•
•
•
personnel
quality managements
design, equipment and consumables
pre-analytical procedures
analytical procedures
post-analytical procedures
reporting and record keeping
safety
Quality management and facility design
GLP in
Parameter
2007
N
n
2008
%
N
n
%
Quality management
- Laboratory has a continuous improvement programme
84 68 81
92 85 92
- Laboratory is accredited by national/international laboratory
accreditation organizations
85 53 62
92 29 32
- sample preparation
84 67 80
94 93 99
- reagent preparation
84 80 95
94 92 98
- amplification and product detection
84 83 99
94 92 98
84 70 83
94 64 68
Facility design
- Laboratory has separate rooms for:
- Laboratory has documented policy requiring a unidirectional
workflow
Examination procedures and postexamination procedures
GLP in
Parameter
2007
2008
N
n
%
N
n
%
- viral RNA extraction
84
80
95
92 90
98
- preparation of in-house controls
77
49
64
92 48
63
- positive control
82
78
95
93 91
98
- negative control
79
74
94
94 93
99
- the date of testing
85
84
99
91 91
100
- the reagent expiry dates
84
59
70
91 59
65
85
59
69
94 67
71
Examination procedures
- Laboratory has SOP for the following individual testing procedure:
- Controls included when performing molecular diagnostic tests:
Post-examination procedures
- Laboratory has worksheet to record:
- Laboratory has another staff to countercheck the test results
Factors affecting performance
based on GLP analysis

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
- 100 samples in recent representative month
- SOP on preparation of in-house controls
- established test turn-around-time
Way forward



Regularly review results of EQAP
Identify problems related to test
protocols
Enhance performance through
training
Thank You