Comparison of the HIV LIA vs WB on HIV-Negative Samples
CDC-HIV Diagnostics Meeting
“New Diagnostic Technologies”
Dec 5-7, 2007
Dr. John Kim
National Laboratory for HIV Reference Services
Public Health Agency of Canada
Ottawa, Ontario
Figure 1. HIV Testing (general)
sample
screening
confirmatory
EIA non-reactive
REPORT
Repeat reactive
Final Diagnosis
(a) Western blot (HIV-1)
(b) RIPA (HIV-1, HIV-2)
(C) LIA (HIV-1/HIV-2)
(d) PCR –Roche, Hpol, gp41, HIV-2
RNA – bDNA, Roche, Abbott, HIV-2 Q
Positive Predictive Values
(Importance of Supplementary Tests)Prevalence EIA EIA +
WB
0.02 % 9% 89.3 %*
0.04 % 16.7 % 95.2 %
0.40 % 66.7 % 99.5 %
1.0 % 83.5 % 99.8 %
INNO-LIA HIV(new technology ?)
• New anti-human immunodeficiency virus immunoblot assays resolve non-specific Western blot results. (Zaaijer et al, Transfusion, 37:193, 1997).
• Validation of a new immunoblot assay for confirmation of human immunodeficiency virus infection (Zaaijer et al, Transfusion, 38:776, 1998).
• Ability of the Innogenetics Inno-Lia to distinguish HIV-1, HIV-2 and dually reactive sera. (Granade et al, Abstr. Gen Meet Am Soc Microbiol, 92:416 [T-47[, 1992.
• Confirmation of HIV infection with the line immune assay (INNO-LIA) using whole blood impregnated filter paper discs. (Kestens et al, Abstr. Int Conf Aids 5:309 [TBP 135], 1989.
gp160gp120
p24
p31
p17
gp41
HIV-2
HIV-1
“O” ? A D C E F F 2 2
“O” ? A D C E F F +
Detection of non-Subtype B HIV reference samples on Subtype B HIV-1 (WB & LIA) antigen kits
HIV-2
HIV-1
gp160gp120
p24
p31
p17
gp41
HIV-2 Reference Samples and atypical HIV-1 WB Patterns
1 2 3 4 5 6 7
1 2 3 4 5 6 7
HIV WB Quality ISSUES-Canada
Site Lot # Date Problems
Sask 927304 Sep 04
Negative control had a few faint bands, slightly above where you would expect the gp160 and slightly below p18
Alberta 931422 Apr 05 Weak positive control strips of Biorad Western Blot kit that the 120 band is not visible after the maximum incubation time
Halifax
928718 &
928634
Mar-Apr 05
NFLD 929713 May 05
Quebec 935405 Jan 06
Faint band at p18 on the negative control
Methods
• BIO-RAD Wb vs INNOGenetics HIV 1/2 Score
• > 1000 samples, (2002-2007)
• n=823 (77-pos; 746-neg), final status known/WB-LIA results
• Neg samples (746)
n=472, LIA-neg/Wb-neg
n=232*, LIA-neg/Wb-ind
n=42, LIA-ind/Wb-ind [23],LIA-ind/Wb-neg [15],
LIA-pos/Wb-pos/neg/ind [4]
HIV Wb vs NLHRS
n=823
positive negative
negative
positive 73
4 488
258
NLHRSW
b
TP
TN
FP
FN
sens – 73/(73 +4)= 95%
spec – 488/(488+258)= 65%
PPV – 73/(73 +258)= 22%
NPV – 488/(488+4)= 99%
HIV LIA vs NLHRS
n=823
positive negative
negative
positive 77
0 704
42
NLHRSLIA
TP
TN
FP
FN
sens – 77/(77 +0)= 100%
spec – 704/(704+42)= 94%
PPV – 77/(77 +42)= 65%
NPV – 704/(704+0)= 100%
Sample #
Province
DETECT rLAV RIPA PCR
WB BLOT #
WB gp160
WB gp120
WB p66
WB p55
WB p51
WB gp41
WB gp40
WB p31
WB p24
WB p17
WB Result
INNO Blot #
INNO gp120
INNO gp41
INNO gp31
INNO gp24
INNO gp17
INNO gp105
INNO gp36
INNO Result
04-0919 AlbNR NR NR N/A 01-05 0 0 0 0 0 0 0 1 0 0 Ind 45-04 0 0 0 0 0 0 0 NR04-0038 SaskNR NR NR N/A 03-04 0 0 1 0 0 0 0 0 0 0 Ind 03-04 0 0 0 0 0 0 0 NR04-0046 ManNR NR NR N/A 04-04 0 0 0 1 0 0 1 0 1 1 Ind 04-04 0 0 0 0 0 0 0 NR04-0047 ManNR NR NR N/A 04-04 0 0 0 1 0 0 1 0 1 1 Ind 04-04 0 0 0 0 0 0 0 NR04-0106 OntNR NR NR N/A 06-04 1 atyp 0 0 0 atyp 0 0 0 0 Ind 04-04 0 0 0 0 0 0 0 NR04-0115 ManNR NR NR N/A 07-04 0 0 0 0 0 0 0 1 0 0 Ind 05-04 0 0 0 0 0 0 0 NR04-0116 ManNR NR NR N/A 07-04 0 0 0 0 0 1 0 0 1 0 Ind 05-04 0 0 0 0 0 0 0 NR04-0121 SaskNR NR NR N/A 07-04 0 0 0 0 0 0 0 0 1 0 Ind 05-04 0 0 0 0 0 0 0 NR04-0122 SaskNR NR NR N/A 07-04 0 0 0 1 0 0 0 0 0 0 Ind 05-04 0 0 0 0 0 0 0 NR04-0137 ManNR R NR N/A 08-04 0 0 0 1 0 0 0 0 0 0 Ind 06-04 0 0 0 0 0 0 0 NR04-0139 ManNR NR NR N/A 08-04 0 0 1 0 1 0 0 0 0 0 Ind 06-04 0 0 0 0 0 0 0 NR04-0141 ManNR NR NR N/A 08-04 0 0 0 1 0 0 0 0 0 0 Ind 06-04 0 0 0 0 0 0 0 NR04-0143 AlbR NR - 08-04 1 0 1 1 0 0 0 0 0 0 Ind 07-04 0 0 0 0 0 0 0 NR04-0160 SaskNR NR NR N/A 08-04 0 0 0 1 0 0 0 0 0 0 Ind 08-04 0 0 0 0 0 0 0 NR04-0161 AlbNR NR NR N/A 08-04 0 0 0 1 0 0 0 0 0 0 Ind 08-04 0 0 0 0 0 0 0 NR04-0162 AlbNR NR NR N/A 08-04 0 0 0 1 0 0 0 0 1 0 Ind 08-04 0 0 0 0 0 0 0 NR04-0186 ManNR R NR N/A 09-04 0 0 0 1 0 0 0 0 0 0 Ind 08-04 0 0 0 0 0 0 0 NR04-0201 AlbNR NR NR N/A 10-04 0 0 0 1 0 0 0 0 0 1 Ind 10-04 0 0 0 1 0 0 0 NR04-0202 ManNR NR Ind N/A 10-04 0 0 0 1 0 0 0 0 0 1 Ind 10-04 0 0 0 0 0 0 0 NR04-0204 ManNR NR NR N/A 10-04 0 0 0 1 0 0 0 1 1 0 Ind 10-04 0 0 0 0 0 0 0 NR04-0220 ManNR NR NR N/A 10-04 0 0 0 0 0 0 0 0 1 0 Ind 10-04 1 0 0 0 0 0 0 NR04-0222 AlbNR NR NR N/A 10-04 0 0 0 1 0 0 0 0 0 0 Ind 10-04 0 0 0 0 0 0 0 NR04-0223 AlbNR NR Ind 10-04 1 0 0 0 0 atyp 0 0 0 0 Ind 10-04 0 0 0 0 0 0 0 NR04-0249 SaskNR NR NR N/A 12-04 0 0 0 1 0 0 0 0 0 0 Ind 12-04 0 0 0 0 0 0 0 NR04-0260 ManNR NR NR N/A 12-04 0 0 0 1 0 0 0 1 0 0 Ind 12-04 0 0 0 0 0 0 0 NR04-0263 BCNR NR Ind 12-04 1 0 0 0 0 0 0 0 0 0 Ind 12-04 0 0 0 0 0 0 0 NR04-0329 ManNR R Ind N/A 13-04 0 0 0 1 0 0 0 0 0 0 Ind 14-04 0 0 0 1 0 0 0 NR04-0347 AlbNR NR NR N/A 14-04 1 0 0 0 0 0 0 0 0 0 Ind 15-04 0 0 0 0 0 0 0 NR04-0367 SaskNR NR NR N/A 14-04 0 0 1 1 0 0 0 1 1 0 Ind 16-04 0 0 0 0 0 0 0 NR04-0463 ManNR NR NR N/A 15-04 0 0 0 1 0 0 0 0 0 0 Ind 17-04 0 0 0 0 0 0 0 NR04-0469 SaskNR NR Ind N/A 15-04 0 0 0 0 0 0 0 1 0 0 Ind 22-04 0 0 0 0 0 0 0 NR02-0379 ManNR NR NR N/A 05-02 0 0 0 0 0 0 N/A 0 1 0 Ind 05-02 0 0 0 0 0 0 0 NR02-0497 SaskNR NR NR N/A 07-02 0 0 0 1 0 0 N/A 0 0 0 Ind 07-02 0 0 0 0 0 0 0 NR02-0508 ManNR R NR N/A 07-02 1 0 0 0 0 0 N/A 0 0 0 Ind 07-02 0 0 0 0 0 0 0 NR02-0554 NSNR NR NR NR 07-02 0 0 0 0 0 0 N/A 0 1 0 Ind 09-02 0 0 0 0 0 0 0 NR02-0556 SaskNR NR NR N/A 07-02 0 0 0 0 0 0 N/A 0 1 0 Ind 09-02 0 0 0 0 0 0 0 NR02-0558 Alb? ? ? NR 09-02 0 0 0 1 0 0 N/A 0 0 0 Ind 09-02 0 0 0 0 0 0 0 NR02-0605 ManNR NR NR N/A 09-02 0 0 0 0 1 0 N/A 0 0 0 Ind 09-02 0 0 0 0 0 0 0 NR02-0750 ManNR NR NR N/A 10-02 1 0 0 0 0 0 N/A 0 0 0 Ind 09-02 0 0 0 0 0 0 0 NR02-0795 SaskNR NR NR N/A 11-02 0 0 0 0 0 0 N/A 0 1 0 Ind 11-02 0 0 0 0 0 0 0 NR02-0799 SaskNR NR NR N/A 11-02 0 0 1 0 0 0 N/A 0 0 0 Ind 10-02 0 0 0 0 0 0 0 NR02-0800 SaskNR NR NR N/A 11-02 0 0 1 0 0 0 N/A 0 0 0 Ind 10-02 0 0 0 0 0 0 0 NR02-0845 AlbNR NR NR N/A 11-02 0 0 0 0 1 0 N/A 0 0 0 Ind 11-02 0 0 0 0 0 0 0 NR
Comparison of WB vs LIA Patterns on HIV-Negative Samples (NLHRS)
gp 160gp 120
p66
p55p51
gp 41
p31p24p17
gp 120
gp 41
p31p24
p17
gp 105
gp 36
HIV-1
HIV-2
HIV-1
INNO-LIA – HIV 1&2
HIV-1 WB (BIO-RAD)
gp 160gp 120
p66
p55p51
gp 41
p31p24p17
gp 120
gp 41
p31p24
p17
gp 105
gp 36
HIV-1
HIV-2
HIV-1
INNO-LIA – HIV 1&2
HIV-1 WB (BIO-RAD)
WB Cross reactivity
gp 160 15 gag p55 66
p24 117
gp 120 1 p17 24
207
p66 27
p66 27
p55 66 pol p51 30
p31 29
p51 30 86
gp41 4 gp160 15
env gp120 1
p31 29 gp41 4
20
p24 117
p17 24
a) I ndividual Antigen b) By class
n=232 (2002- 2007 [May])
gp 160gp 120
p66
p55p51
gp 41
p31p24p17
gp 120
gp 41
p31p24
p17
gp 105
gp 36
HIV-1
HIV-2
HIV-1
INNO-LIA – HIV 1&2
HIV-1 WB (BIO-RAD)
LI A Cross reactivity
gp 160 N/A p55 N/A
gag p24 12
gp 120 1 p17 0
12
p66 N/A
p66 N/A
p55 N/A pol p51 N/A
p31 1
p51 N/A 1
gp41 3 gp160 N/A
env gp120 1
p31 1 gp41 3
4
p24 12
p17 0
a) I ndividual Antigen b) By class
Kit Name
(Manufacturer)
Source of
antigen Purified
Mode of
I nactivation Membrane
Conjugate/
Substrate
HI V- 2
criteria
Cambridge Biotech
HI V- 1
Western Blot Kit
(Calypte) H9/HTLV- I I I B T cell linepartial psoralen/UV nitrocell
I gG HRP/
chloro- napthol NO
Bio- Rad Novapath
HI V- 1 I munoblot
(Sanofi) HUT- 78 T cell line partial /SDS nitrocell
I gG AP/
BCI P- NBT NO
HI V- 1 Western
Blot Kit
(Organon Teknika) H9/HTLV- I I I B T cell line
ultra-
centrifuge
detergent/
heat nitrocell
I gG HRP/
proprietary NO
I NNO- LI A HI V
Confirmation
(I nnogenetics)
recombinant protein/
synthetic peptide N/A N/A nylon
I gG AP/
BCI P- NBT YES
Table 1. Characteristics of HI V Confirmatory Tests Used
Living with the Wb-Improvements
• Wb still most widely used (licensed) confirm tool
• Known for years (89.3% [266/298]; BIORad; p20)
• Review/Change interpretative criteria…Harmonize international criterias
• Combine wb interpretative criteria
eg: CDC/ASTPHLD + WHO [Tebourski et al, 2004]
• License new tests (eg. LIA) – patent issues ?
• LIA – test of choice in NLHRS
sens (100% vs 95%), spec (94% vs 65%)
PPV (65% vs 22%), NPV (100% vs 99%)
Predictive Values
Positive predictive value (PPV)
PPV = TP/TP +FP (x 100)
Negative predictive value (NPV)
NPV = TN/TN+FN (x100)
How accurately a test predicts the presence or absence of analyte (disease) !
(i) sensitivity - incidence of true positives in those with disease
(ii) specificity - incidence of true of negatives in those without disease
(iii) prevalence - number of actual cases (of disease) in the population.
Galen RS and Gambino SR. Beyond Normality: The Predictive Value and Efficiency of Medical Diagnosis. New York: Wiley, 1975.
OMIT SLIDE ???