Transcript Présentation PowerPoint - 2016 HIV Diagnostics Conference

Vironostika® HIV-1 Plus O Microelisa System
• Main Features
• Assay configuration and procedures for S/P and DBS
• Solid Phase Antigens
• Comparison to the current licensed Vironostika® HIV-1
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Main Features
• Vironostika® HIV-1 Plus O Mircoelisa System is an ELISA for the
qualitative detection of antibodies to HIV-1 including Group O, in
human specimens collected as serum, plasma, or dried blood
spots on filter paper.
• It is not intended for use in screening blood donors.
• Solid phase contains HIV-1 viral lysate, viral envelope and Group
O peptide.
• The system contains Sample Addition Monitoring, Color Coded
Reagents, liquid controls and Substrate, and an improved ease of
use.
• The assay is designed to improve the overall sensitivity while
maintaining the specificity as compared to the current licensed
Vironostika® HIV-1.
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Kit Components
3
Overview of Vironostika® HIV-1 Plus O
Microelisa System
WASH
WASH
STOP
Solid phase:
Anti-HIV in Sample
•
• Sample diluent contains
sample addition monitor
Viral Lysate
• Viral env
•Group O
Peptide
Incubation 37°C
Goat anti-human Ig/HRP
conjugate
• One-step preparation
Incubation 37°C
Chromogenic
substrate (ABTS)
NaF
• Ready to use format
Incubation 15-30°C
Read (405nm)
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Assay Procedures for Serum/Plasma (S/P) and
Dried Blood Spots (DBS)
S/P
10µl SAMPLE
200µl DILSIM III
(1:21)
60 min, 37°C
Wash
DBS
25µl ELUTE
125µl DILSIM III
(1:6)
Conj
150µl
Wash
10-13 min, RT
[S]
150µl
STOP
150µl
READ
At 405 nm
Elute with 150µl
of DBS Elution
Medium
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Sample Addition Monitoring
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Solid Phase Antigen
• HIV-1 Viral Lysate
• Prepared from H9/HIV-1IIIB
• Same viral lysate as in the current Vironostika HIV-1
• Group O peptide
• RRETLLQNQQLLSLWGCKGKLVCYT
• Synthetic peptide of 23 amino acids with Arg-Arg added to the
N-terminal
• The sequence derived from ANT70 gp41 regions (Journal of
Virology, 1990,1207-1216)
• Conjugated to BSA for plate coating
• Native HIV-1 gp160
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Development of Native HIV-1 gp160
• Advanced Bioscience Laboratories developed a cell culture
system that secreted gp160 which contained the gp41 moiety
(minus a small truncation at the C-terminus).
• The native gp160 is highly soluble and exists with carbohydrate
moiety.
Kalyanaraman et al., AIDS Res. Hum. Retroviruses, 1990;
Kalyanaraman et al., Virology, 1990.
• Shown to be an excellent diagnostic antigen.
Nair et al., J. Cln. Microbiol., 1994
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Immunodominant Domains in HIV-1 env
Proteins
gp160
296-331
504-518
579-601
gp120
296-331
504-518
gp41
579-601
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Serum EIA Reactivity of HIV-1 gp160
1.2
1.0
False Positive
Postive
A450
0.8
0.6
Very Weak
Positive
0.4
Medium Positive
0.2
0.0
1000
1000
100
100
10
10
11
gp160 (ng/well)
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The Vironostika® HIV-1 Plus O Trial
Summary
• 18 geographically diverse sites participated.
• 4 kit lots evaluated.
• 36,000+ tests performed (Serum/Plasma + DBS).
• Studies included:
• Reproducibility
• Sensitivity
• Known HIV-1 positive samples
• High risk populations
• Seroconversion panels
• Dilution panels
• Specificity
• Low risk populations
• Samples containing potentially interfering substances
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Specificity in Low-Risk Populations (n=6019)
5000
HIV-1 Plus O
4000
VirHIV
3000
2000
1000
0
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Known HIV-1 Specimens
Serum/Plasma
DBS
HIV-1 Clade A – 100% (10/10)
HIV-1 Clade B – 100% (12/12)
HIV-1 Clade B/D – 100% (1/1)
HIV-1 Clade C – 100% (10/10)
HIV-1 Clade C/E – 100% (1/1)
HIV-1 Clade D – 100% (10/10)
HIV-1 Clade E – 100% (7/7)
HIV-1 Clade E/A – 100% (3/3)
HIV-1 Clade E/C – 100% (1/1)
HIV-1 Clade E/F – 100% (1/1)
HIV-1 Clade F – 100% (10/10)
HIV-1 Clade G – 100% (4/4)
HIV-1 Clade H – 100% (2/2)
HIV-2 – 100% (20/20)
HIV-O – 100% (11/11)
HIV-1 Clade A – 100% (10/10)
HIV-1 Clade B – 100% (12/12)
HIV-1 Clade B/D – 100% (1/1)
HIV-1 Clade C – 100% (10/10)
HIV-1 Clade C/E – 100% (1/1)
HIV-1 Clade D – 100% (10/10)
HIV-1 Clade E – 100% (7/7)
HIV-1 Clade E/A – 100% (3/3)
HIV-1 Clade E/C – 100% (1/1)
HIV-1 Clade E/F – 100% (1/1)
HIV-1 Clade F – 100% (10/10)
HIV-1 Clade G – 100% (4/4)
HIV-1 Clade H – 100% (2/2)
HIV-2 – 100% (20/20)
HIV-O – 100% (11/11)
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Sensitivity
All serum/plasma specimens and dried blood spot specimens were
repeatedly reactive with Vironostika® HIV-1 Plus O Mircoelisa
System. Therefore, the sensitivity for both specimen types in this
study was 100% (95% CI: 99.64 – 100%).
Estimation of Clinical Sensitivity
Specimen
Type
Serum or
Plasma
Dried Blood
Spots
CD4+
Stratum
<200
200-499
>499
Total
<200
200-499
>499
Total
Number of
specimens
250
385
375
1,010
250
385
375
1,010
Number of
Initially
Reactive
250
385
375
1,010
250
385
375
1,010
Number of
Repeatedly
Reactive
250
385
375
1,010
250
385
375
1,010
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High-Risk Populations
Prison Inmates, STD, Hosp ER Pts, and HIV Outreach Clinic Pts
The specificity of the Vironostika® HIV-1 Plus O assay in this study
of high-risk populations was calculated to be 1,392/1,398 = 99.57%
(95% CI = 99.07% - 99.84%).
Specimen
Type
Serum or
Plasma
Dried Blood
Spots
Number of
Number of
Number of
Initially
Repeatedly
Western Blot
Population
specimens
Reactive
Reactive
Positive
1
2
3
4
Total
1
2
3
4
Total
251
513
500
250
1,514
0
0
500
250
750
16
13
73
28
130
N/A
N/A
68
27
95
14
13
68
27
122
N/A
N/A
68
27
95
8
13
68
27
116
N/A
N/A
68
27
95
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Seroconversion Panel Testing
Panel ID
Sample Collection Days
Vironostika
HIV-1 Plus O
Comparative Test1
924
8, 10, 26, 33, 35, 40
33
40
927
0, 28, 33, 35, 40
33
40
931
9, 15, 28, 33, 35, 42
28
33
932
0, 3, 13, 27, 34, 50, 78, 163, 194
34
NR2
940
0, 7, 11, 15, 18, 22, 25, 29
15
22
071
1, 3, 17, 22, 28
17
22
111
1, 2, 8, 16, 20, 22, 27
8
16
241
1, 7, 9, 15, 17, 22, 24
15
22
321
1, 8, 12, 15, 21
15
21
341
1, 7, 10, 21, 23, 28
21
28
351
1, 8, 11, 15
15
NR
361
1, 3, 9, 11, 16, 18
18
NR
1The
licensed Vironostika HIV-1 Microelisa System
2None
of the specimens in this panel was reactive
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Reactivity of H&L vs  Specific Conjugate
Seroconversion Panels
4.5
4
3.5
H&L
 Specific
S/CO
3
2.5
2
1.5
1
0.5
0
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Dilution Panel Testing
First dilutions with non-reactive test results for each dilution series.
SERUM
Sample ID
Clade Type
Vironostika HIV-1
Plus O System
Comparative Test 1
5805
HIV-1 Clade B
1:1,920
1:240
H629
HIV-1 Clade B
1:32,000
1:8,000
MD-O
HIV-1 Group O
1:25,600
1:400
302-1
HIV-1 Group O
1:1,600
NR 2
301-42
HIV-1 Clade A
1:12,800
1:1,600
302-18
HIV-1 Clade C
1:6,000
1:1,500
301-24
HIV-1 Clade D
1:48,000
1:3,000
302-23
HIV-1 Clade E
1:24,000
1:1,500
302-28
HIV-1 Clade F
1:12,800
1:1,600
302-17
HIV-1 Clade D
1:4,000
1:250
( 1 ) The licensed Vironostika HIV-1 Microelisa System
( 2 ) Non-reactive
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Detection of HIV-1 Group O Specimens
Specimen ID
Sample
Dilution
1
1:100
2
1:50
3
1:50
4
1:50
5
1:50
6
1:50
7
1:50
8
1:50
9
1:50
10
1:50
11
1:50
Total Repeatedly Reactive
1
2
Vironostika HIV-1 Plus O
System
2
Mean SCR
Comparative Test
Mean SCR
6.1
4.0
7.4
5.6
7.7
7.7
5.3
7.6
5.9
3.9
7.6
11/11
2.0
0.4
1.6
1.1
2.0
7.2
0.5
2.2
0.4
0.4
4.5
7/11
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The licensed Vironostika HIV-1 Microelisa System
SCR = Signal to Cutoff Ratio
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Detection of HIV-2 Positive Specimens
Specimen ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
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Sample Dilution
1:10
1:10
1:500
1:10
1:50
1:10
1:10
1:10
1:100
1:1,000
1:200
1:200
1:10
1:50
1:1,000
1:1,000
1:50
1:1,000
1:10
1:10
Mean SCR*
3.3
5.8
1.5
4.3
1.7
5.5
2.5
5.0
3.7
1.8
1.3
1.4
1.9
1.3
4.9
3.6
1.7
2.7
2.0
1.8
*Specimens with SCR equal to or greater than 1.0 are considered reactive with the test.
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Conclusions
•
Vironostika® HIV-1 Plus O is intended for use as an aid in diagnosis of
infection with HIV-1 and not for use in screening blood donors.
•
This assay uses three solid phase antigens; inactivated, purified HIV-1
viral lysate proteins, a purified viral envelope protein (native gp160), and
a synthetic group O peptide.
•
Assay procedure similar to the current licensed Vironostika® HIV with
added features to improve ease of use.
•
The assay design sought to improve overall sensitivity to Group M and
Group O while maintaining the specificity compared to the current
licensed Vironostika® HIV-1.
•
Detection of HIV-1 Group M antibodies was demonstrated with subtypes
A, B, B/D, C, C/E, D, E, E/A, E/C, E/F, F, G and H.
•
The assay exhibited higher analytical sensitivity with dilutional panels
from clinical specimens for both HIV-1 Group M and HIV-1 Group O
specimens compared to the current licensed Vironostika® HIV-1.
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