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%)

% Prevalence

% False PositivesOMIT SLIDE ?

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 ???