# Presentation: 745857

## Supplemental Testing for HIV-1 and HCV

- Blood Products Advisory Committee Meeting
- September 18, 2003

- Susan L. Stramer, Ph.D.
- National Testing andReference Laboratories
- American Red Cross

- Jaye P. Brodsky
- Quality Analytics

## Background

- HIV-1 and HCV algorithms utilize immunoassays for confirmation
- HIV-1 uses Western Blot (WB) containing electrophoresed whole HIV lysate
- HCV uses Recombinant Immunoblot Assay (RIBA) containing painted recombinant HCV antigens
- Issues with supplemental assays
- Poor performance
- Unreadable or invalid
- False positivity; false negativity
- High rates of indeterminate results (neither pos or neg) in healthy individuals

## Background

- Issues with supplemental assays
- High costs
- Inconsistent availability (HIV)
- Is all the testing necessary, or is redundant testing being performed?
- Can alternatives be validated?

## Alternative Algorithms

- HCV Alternative – 1
- Use of HCV NAT in supplemental algorithms
- Already being performed as a screening test for all donations
- NAT results integrated into donor counseling
- Sensitivity high relative to RIBA even though NAT is performed in pools (16-24)
- Specificity high (false-positive donor rates less than 1:40,000)
- Already recommended for diagnostic use by CDC
- If NAT reactive, RIBA not needed
- HCV Alternative – 2
- Use of S/CO to further reduce the need for RIBA in HCV NAT nonreactive individuals

## Alternative Algorithms

- HIV Alternative – 1
- Use of NAT and/or S/CO as proposed for HCV
- HIV Alternative – 2
- Use of a second EIA where discordant EIA reactives (EIA-1 Rx and EIA-2 nonRx) are not further tested
- Feasibility for HIV versus HCV
- Anti-HCV licensed screening assays use same recombinant antigens, same assay format; overlapping populations of false positives
- Anti-HIV licensed screening assays use different antigens (viral lysate, recombinants and peptides); populations of false positives are unique

## Alternative Algorithms

- Issues for HIV or HCV alternatives
- Potential misclassification of donors
- Current algorithms containing RIBA or WB in combination with NAT (and screening S/CO value) provide specific counseling messages
- Kits will require labeling for intended use (supplemental claims to NAT assays or EIAs)
- 21 CFR 610.40 Subpart E
- “You must further test each donation, including autologous donations, found to be reactive by a screening test ...whenever a supplemental (additional, most specific) test has been approved for such use by FDA....”

## Current: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive

**Perform RIBA 3.0 SIA****N = 34,656**

**Ind**

**Pos**

**Reactive****Individually**

**Neg**

**NonRx****in Pool or****Individually**

**Reactive****Individually**

**NonRx****in Pool or****Individually**

**Reactive****Individually**

**NonRx****in Pool or****Individually**

**HCV****Infected**

**HCV Infected?****Resolved infection (≈20%)****Viral load < NAT cutoff****False Pos RIBA**

**38%**

**13,198**

**HCV Not****Infected**

**HCV Infected?****Early seroconversion****False Rx NAT**

**HCV Infected?**

**17.2%**

**5,967**

**0.15%**

**50**

**34.5%**

**11,963**

**HCV Infected****Early seroconversion**

**0.5%**

**159**

**9.6%**

**3,319**

**HCV NAT**

**HCV NAT**

**HCV NAT**

## Option 1: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive

**Review HCV NAT Results****N = 34,656**

**Pool or Individual Unit****NonRx****(16 dtns ******** HIV-1/HCV TMA,****24 dtns ******** HCV PCR)**

**Individual Unit****NAT Rx****(dHCV TMA or ****HCV PCR)**

**No Further Testing****HCV Infected**

**38.7%**

**13,407**

**98.4% PPV**

**13,198 RIBA Pos**

**61.3%**

**21,249**

**Perform RIBA**

**Ind**

**Pos**

**Neg**

**28.1%**

**5,967**

**15.6%**

**3,319**

**56.3%**

**11,963**

**79.9% Sensitivity**

## Option 2: HCV EIA 2.0 (Abbott) or 3.0 (Ortho) Repeat Reactive

**Review HCV NAT Results****N = 34,656**

**Pool or Individual Unit****NonRx****(16 dtns ******** HIV-1/HCV TMA,****24 dtns ******** HCV PCR)**

**Individual Unit****NAT Rx****(dHCV TMA or ****HCV PCR)**

**No Further Testing****HCV Infected**

**38.7%**

**13,407**

**98.4% PPV**

**13,198 RIBA Pos**

**61.3%**

**21,249**

**82.9%****Sensitivity**

**Perform RIBA**

**Ind**

**Pos**

**Neg**

**31%**

**5,055**

**3.5%**

**566**

**65.5%**

**10,692**

**Review S/CO Values of EIA**

**S/CO ≥ 3.8**

**23.2%**

**4,936**

**No Further Testing****HCV Infected?**

**55.8% PPV**

**2,753 RIBA Pos**

**S/CO < 3.8**

**76.8%**

**16,313**

**99.8% Sensitivity**

**13,177**

**S/CO ≥ 3.8**

## Correlation of NAT Screening withSupplemental HCV Serological Results(36,536 RR donations of which 34,656 (94.9%) had EIA and RIBA data)

**RIBA Result**

***Correlation of NAT Screening with******Supplemental HCV Serological Results******(36,536 RR donations of which 34,656 (94.9%) had EIA and RIBA data)***

**NAT****Result**

- Rx
- NonRx
- Total

**Pos**

**Ind**

**Neg**

**13,182****(13,198***)****(80%)**
- 3,319
**16,502****(16,517***)****(48%)**

- 174*(159***)
- 5,967
- 6,141(6,126***)

- 50**
- 11,963
- 12,013

- (1.7%)

**Total**

- 13,407
- 21,249
- 34,656

**9/8/99 to 6/30/03**

- 	*	140/174 (80%) dHCV and PCR Rx (<100-48,000,000 copies/mL)	**	16/50 (32%) dHCV and PCR Rx (<100-24,000,000 copies/mL)	***	15 RIBA Ind due to 1 + hSOD (1:2310 in RR samples)

## Identification of HCV RIBA-Positive, NAT-Nonreactive Samples

**9/8/99 – 12/31/02**

**2,255****HCV RIBA Pos, NAT Neg Neat**

**812****Screened Neat,****Neat Neg (36%)**
**730**

**29****Screened Pools,****Pool Rx, Neat Neg (1%)**
**25**

**1,414****Screened Pools,****Pool Neg (63%)**
**1,397**

- * A minimum of one of two reps positive

**Pos*****15****(2.1%)**

**Neg****715**

**Pos****1****(3.7%)**

**Neg****24**

**Pos*****29****(2.1%)**

**Neg****1,368**

**Neat Retest:****NGI x1, or****Gen-Probe x2**

## Viral Loads of HCV RIBA-Positive Samples Screened NAT Nonreactive in Pools or Individually and that Retested NAT Reactive

| 	              		 | Copies/mL at NGI |  |  |  |
| --- | --- | --- | --- | --- |
| Screened in Pools (N=29) 20+ Quant/24 sent | 100 (11)1*
	380 (2)
	460
	910 | 1,500* 
7,700*
9,400* | 34,000* | 5,200,000* |
| Screened Individually (N=15) 10+ Quant/12 sent | 100 (5)1*
	200 
	430* | 2,000
7,400* | 12,000* |  |

-   * Of 30+ Quant, 10 (30%) tested PCR positive when diluted 1:16; frequency 8 of 2255 (1:282) total with viral loads >1000 copies/mL representing >16 million donations screened

## Relationship between HCV 3.0 EIA and RIBAAll Samples 9/8/99 to 6/30/03

***Relationship between HCV 3.0 EIA and RIBA******All Samples 9/8/99 to 6/30/03***

***N = 34,656***

- S/CO

- HCV SIA (3.0) Results

**2.433**

**1.637**

**1.274**

**3.079**

**1.876**

**>5.000**

**Neg****N = 12,013****34.7%**

**Ind****N = 6,126****17.7%**

**Pos****N = 16,517****47.6%**

- 1.024 - >5.000

- 95% Range

- 1.032 - >5.000

- 3.077 - >5.000

**1.354**

**4.943**

**4.583**

## Relationship between HCV 3.0 EIA and RIBANAT Reactive Samples 9/8/99 to 6/30/03

***N = 13,407***

- S/CO

- HCV SIA (3.0) Results

**4.393**

**1.800**

**1.354**

**>5.000**

**4.763**

**4.221**

**>5.000****4.971****4.627**

**Neg****N = 50****0.4%**

**Ind****N = 159****1.2%**

**Pos****N = 13,198****98.4%**

- 1.026 - >5.000

- 95% Range

- 1.212 - >5.000

- 4.145 - >5.000

## Relationship between HCV 3.0 EIA and RIBANAT Nonreactive Samples 9/8/99 to 6/30/03

***N = 21,249***

- S/CO

- HCV SIA (3.0) Results

**2.428**

**1.636**

**1.274**

**2.955**

**1.840**

**>5.000**

**Neg****N = 11,963****56.3%**

**Ind****N = 5,967****28.1%**

**Pos****N = 3,319****15.6%**

- 1.024 - >5.000

- 95% Range

- 1.031 - >5.000

- 1.388 - >5.000

**1.348**

**4.795**

**4.311**

## ARC HCV Testing 9/8/99 Through 6/30/03NAT Reactive Specimens Only (N = 13,407)

| S/CO | RIBANEG | RIBAIND | RIBAPOS | Total | %RIBAPOS |
| --- | --- | --- | --- | --- | --- |
| 1.0 to 1.999 | 27 | 17 | 8 | 52 | 15.4% |
| 2.0 to 2.999 | 8 | 6 | 5 | 19 | 26.3% |
| 3.0 to 3.799 | 1 | 4 | 8 | 13 | 61.5% |
| 3.8 to 4.999 | 8 | 70 | 6,954 | 7,032 | 98.9% |
| > 5.0 | 6 | 62 | 6,223 | 6,291 | 98.9% |

- Note:  13,323 of 13,407 (99.4%) NAT POS specimens had an S/CO ≥ 3.8;13,177/13,323 (98.9%) specimens with S/CO ≥ 3.8 confirmed POS;
- 	 95% CI = 98.7 - 99.1 by binomial distribution
- Chi-square tests for independence were significant (p<0.0001)

## ARC HCV Testing 9/8/99 Through 6/30/03NAT Nonreactive Specimens Only (N = 21,249)

| S/CO | RIBANEG | RIBAIND | RIBAPOS | Total | %RIBAPOS |
| --- | --- | --- | --- | --- | --- |
| 1.0 to 1.999 | 7,731 | 3301 | 198 | 11,230 | 1.8% |
| 2.0 to 2.999 | 2,191 | 1,209 | 181 | 3,581 | 5.1% |
| 3.0 to 3.799 | 770 | 545 | 187 | 1,502 | 12.5% |
| 3.8 to 4.999 | 953 | 678 | 1,522 | 3,153 | 48.3% |
| > 5.0 | 318 | 234 | 1,231 | 1,783 | 69.0% |

- Note:  4,936 of 21,249 (23.2%) NAT NEG specimens had an S/CO ≥ 3.8;2,753/4,936 (55.8%) specimens with S/CO ≥ 3.8 confirmed POS;
- 	95% CI = 54.4 – 57.2 by binomial distribution
- Chi-square tests for independence were significant (p<0.0001)

***ARC HCV Testing 9/8/99 Through 6/30/03******NAT Nonreactive Specimens Only (N = 21,249)***

## PPV and Sensitivity of EIA and NAT vs RIBA 

|  | RIBA |  |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, NAT Rx | 13,198 | 209 | 13,407 |
| EIA RR, NAT NR | 3,319 | 17,930 | 21,249 |
| TOTAL | 16,517 | 18,139 | 34,656 |

- PPV = 13,198/13,407 = 98.4%
- Sens = 13,198/16,517 = 79.9%

## PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA 

|  |  | RIBA |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 3.8  | 15,930 | 2,329 | 18,259 |
| EIA RR, S/CO < 3.8  | 587 | 15,810 | 16,397 |
| TOTAL | 16,517 | 18,139 | 34,656 |

- PPV = 15,930/18,259 = 87.2%
- Sens = 15,930/16,517 = 96.4%
- Note:	Increasing the S/CO limit to ≥ 5.0 increases the PPV to 92.3% and decreases the sensitivity to 45.1%

## PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA Where NAT is Reactive  

|  |  | RIBA |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 3.8  | 13,177 | 146 | 13,323 |
| EIA RR, S/CO < 3.8  | 21 | 63 | 84 |
| TOTAL | 13,198 | 209 | 13,407 |

- PPV = 13,177/13,323 = 98.9%
- Sens = 13,177/13,198 = 99.8%

## PPV and Sensitivity of High S/CO (≥ 3.8) vs RIBA Where NAT is Nonreactive  

|  |  | RIBA |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 3.8  | 2,753 | 2,183 | 4,936 |
| EIA RR, S/CO < 3.8  | 566 | 15,747 | 16,313 |
| TOTAL | 3,319 | 17,930 | 21,249 |

- PPV = 2,753/4,936 = 55.8%
- Sens = 2,753/3,319 = 82.9%

## HCV Alternate Algorithm Summary

- Use of NAT as the first step of the supplemental test algorithm will reduce the amount of RIBA performed by approximately 40%
- HCV NAT-reactive samples (even using pooled NAT for screening):
- Sensitivity of 79.9% (detection of RIBA confirmed positives)
- Those not detected by NAT will be tested by RIBA (as true today)
- 98.4% are RIBA pos (PPV)
-  Small percent of NAT-reactive samples likely representing early seroconversion are RIBA ind or neg

## HCV Alternate Algorithm Summary

- Use of S/CO of _>_ 3.8 can be applied following separation of RR population into NAT Rx and nonRx
- Since NAT results are available for all donations, and is a specific test for HCV, S/CO introduced for NAT nonRx to further reduce the dependence on RIBA
- S/CO _>_ 3.8 does have high sensitivity and PPV relative to RIBA in populations where NAT is not performed
- Sensitivity of 96.4%
- PPV of 87.2%
- However, use of a high S/CO for NAT nonRxs would only eliminate 23% of RIBAs performed with poor performance
- Sensitivity of 82.9%
- PPV of 55.8%

## Cambridge BiotechHuman Immunodeficiency Virus Type 1 (HIV -1)Western Blot Kit

***March 16, 2000 BPAC***
- Any bands present but pattern does not meet criteria for POSITIVE = INDETERMINATE
- Non-viral bands have been observed with certain specimens. These bands are not usually accompanied by any of the major viral bands of diagnostic significance (p24, gp41/120/160). The non-viral bands appear to be cell related with the most common in the molecular weight range of 70K, 51-55K (possible HLA-DR) and 43K (possible HLA-ABC).

## HIV-1 Blood Donor Screening/SupplementalTest Results (ARC); March 16, 2000 BPAC

**12.4 Million Donations Screened**

**Positive**
**791 (7.1%)**

**Negative**
**5,128 (46.3%)**

**Indeterminate**
**5,161 (46.6%)**

**1998-1999**

**11,080 Repeat Reactive (0.09%)****HIV-1 WB (Cambridge Biotech)**

## HIV-1 Blood Donor Screening/SupplementalTest Results (ARC) (Cont.); March 16, 2000 BPAC

**5,161 Indeterminate (46.6%)**

**One**
**Viral**
**Band**

**2,752 (53.3%)**

**Multiple**
**Viral**
**Bands**

**589 (11.4%)**

**Background****Only**
**(obscures**
**reading)**

**1,050 (20.3%)**

**Non-**
**Viral****Bands**

**724 (14%)**

**16 (35%)****“ENV” only**

**1,925 (70%)****p24 “GAG” only**

**464 (79%) ****“GAG” reactivity with or without ****other banding**

**569 (79%)****“p70”**

**65.7% Viral Banding**

**1998-1999**

## Calypte Biotech HIV-1 Western Blot

- gp160

- gp120

- p66

- p55/51

- gp41

- p31

- p24

- p17

## Calypte HIV-1 Western Blot

- gp160

- gp120

- p66

- p55/51

- gp41

- p31

- p24

- p17

## HIV-1 RNA Concentrations During SC Based on NGI’s PCR of 28 Plasma Donor Panels (N=221) 

- Categories

- E001372A

## Comparison of HIV-1 p24 Ag PositiveWindow-Case U.S. Blood Donors

***Comparison of HIV-1 p24 Ag Positive******Window-Case U.S. Blood Donors***

- 1	Index	**6 x 10****5*******	**14.0***	95	0.1		12	**3 x 10****4**	**1.2**	95	**2.3**	POS – minus p31	22	**1 x 10****5**	0.3	 	**5.6**	POS – all bands
- 2	Index	**2 x 10****6*******	**28.5***	94	0.8		20	**1 x 10****5**	0.7	 	**1.6**	POS – minus p31	43	**1 x 10****4**	0.1	 	**5.3**	POS – all bands
- 3	Index	**1 x 10****5**	**8.1**	95	0.1		13	**5 x 10****5*******	**26.5***	90	**8.8**	POS – minus p31
- 4	Index	**1 x 10****5**	**2.4**	95	0.0		13	**1 x 10****6*******	**17.5***	89	**2.7**	IND – p66	75	**5 x 10****4**	0.1	 	**17.4**	POS – all bands
- 5	Index	**3 x 10****5**	**18.6***	99	0.3		10	**4 x 10****5*******	0.2		**4.3**	POS – minus p31	17	**1 x 10****5**	0.9	 	**12.5**	POS – minus p31	24	**3 x 10****4**	0.7	 	**8.5**	POS – minus p31	31	**2 x 10****4**	0.1	 	**13.1**	POS – minus p31

**SC**

**Sample ****Collection****(days)**

**WB**

**RNA ****Copies/mL**

**p24 Ag ****S/CO**

**Percent****Neut.**

**HIV-1/****HIV-2 Ab ****S/CO**

## Current: HIV-1/HIV-2 EIA(Abbott rDNA or GSC pEIA)

**Perform WB****N = 17,090**

**Ind**

**Pos**

**Reactive****Individually**

**Neg**

**NonRx****in Pool or****Individually**

**Reactive****Individually**

**NonRx****in Pool or****Individually**

**Reactive****Individually**

**NonRx****in Pool or****Individually**

**HIV****Infected**

**HIV Infected?****Viral load < NAT cutoff****False Pos WB**

**4.4%**

**759**

**HIV Not****Infected**

**HIV Infected?****Early seroconversion****False Rx NAT**

**HIV Not****Infected?**

**51%**

**8,654**

**0.2%**

**37 (0)**

**44%**

**7,525**

**HIV Infected****Early seroconversion**

**0.3%**

**56 (6)**

**0.3%**

**59**

**HIV NAT**

**HIV NAT**

**HIV NAT**

## Option 1: HIV-1/HIV-2 EIA(Abbott rDNA or GSC pEIA)

**Review HIV-1 NAT Results****N = 17,090**

**Pool or Individual Unit****NonRx****(16 dtns ******** HIV-1/HCV TMA,****24 dtns ******** HIV PCR)**

**Individual Unit****NAT Rx****(dHIV TMA or ****HIV PCR)**

**No Further Testing****HIV Infected**

**5%**

**852 (765)**

**89.1% PPV****(99.2%)**

**759 WB Pos**

**95%**

**16,238**

**Perform WB**

**Ind**

**Pos**

**Neg**

**53.3%**

**8,654**

**0.4%**

**59**

**46.3%**

**7,525**

**92.8% Sensitivity**

## Option 2: HIV-1/HIV-2 EIA(Abbott rDNA or GSC pEIA)

**Review HIV-1 NAT Results****N = 17,090**

**Pool or Individual Unit****NonRx****(16 dtns ******** HIV-1/HCV TMA,****24 dtns ******** HIV PCR)**

**Individual Unit****NAT Rx****(dHIV TMA or ****HIV PCR)**

**No Further Testing****HCV Infected**

**5%**

**852 (765)**

**89.1% PPV****(99.2%)**

**759 WB Pos**

**95%**

**16,238**

**33.9%****Sensitivity**

**Perform WB**

**Ind**

**Pos**

**Neg**

**53.3%**

**8,510**

**0.2%**

**39**

**46.5%**

**7,428**

**Review S/CO Values of EIA**

**S/CO ≥ 15**

**1.6%**

**261**

**No Further Testing****HIV Infected?**

**7.7% PPV**

**20 WB Pos**

**S/CO < 15**

**98.4%**

**15,977**

**99.6% Sensitivity**

**694**

**S/CO**_** >**_** 15**

## Correlation of NAT withSupplemental HIV Serological Data(17,791 RR donations of which 17,090 (96.1%) had EIA and WB data)

**Western Blot Result**

***Correlation of NAT with******Supplemental HIV Serological Data******(17,791 RR donations of which 17,090 (96.1%) had EIA and WB data)***

**NAT****Result**

- Rx
- NonRx
- Total

**Pos**

**Ind**

**Neg**

**759****(89.1%)**
- 59(0.4%)
**818****(4.8%)**

- 56*
- 8,654
- 8,710

- 37**
- 7,525
- 7,562

-  (10.9%)

**Total**

- 852
- 16,238
- 17,090

**9/8/99 to 6/30/03**

- 	*	6/56 (11%) dHIV and PCR Rx (9,500-800,000 copies/mL)	**	0/37 (0%) dHIV and PCR Rx

## Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples

***Characteristics of HIV-1 WB ******Pos/TMA Nonreactive Samples***

- All samples p24 Ag negative

**	****Sample****	****Pool****	****Neat****	****HIV-1/HIV-2 S/CO****	****WB****	****HIV PCR**
**	**1		NR	1.43	41, 120, 160	Neg
- 	2	NR		1.03	41, 160	Neg
- 	3		NR	1.62	41, 160	Neg
- 	4		NR	1.11	41, 55, 160	Neg
- 	5		NR	2.50	24, 41, 51, 61, 160	Neg
- 	6**	****	****NR****	****20.18****	****all bands****	****Neg**
- 	7		NR	1.22	24, 41, 160	Neg
- 	8	NR		1.11	17, 41, 120, 160	Neg
- 	9	NR		1.84	41, 160	Neg
- 	10		NR	1.40	24, 41, 160	Neg
- 	11		NR	1.70	17, 24, 41, 51, 160	Neg
**	**12**	****NR****	****	****20.00****	****all bands****	****Pos (200 copies/mL)**
**	**13**	****	****NR****	****17.84****	****all bands****	****Neg**

**9/8/99 – 8/31/00**

## Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples

***Characteristics of HIV-1 WB ******Pos/TMA Nonreactive Samples***

- All samples p24 Ag negative

**	****Sample****	****Pool****	****Neat****	****HIV-1/HIV-2 S/CO****	****WB****	****HIV PCR**
**	**14		NR	8.73	17, 24, 160	Neg
- 	15	NR		10.56	24, 55, 120, 160	Neg
**	****16****	****NR****	****	****19.30****	****all bands****	****Neg**
**	****17****	****NR****	****	****19.47****	****all bands****	**** Pos (200 copies/mL)**
- 	18	NR		1.04	41, 66, 120, 160	Neg
- 	19	NR		4.72	41, 160	Neg
- 	20	NR		1.50	24, 41, 160	Neg
- 	21	NR		1.53	24, 41, 160	Neg
- 	22	NR		1.60	24, 160	Neg
**	****23****	****NR****	****	****17.60****	****all bands****	****Neg**
**	****24****	****	****NR****	****19.30****	****all bands****	****Neg**

**9/1/00 – 12/29/01**

## Characteristics of HIV-1 WB Pos/TMA Nonreactive Samples

***Characteristics of HIV-1 WB ******Pos/TMA Nonreactive Samples***

- All samples p24 Ag negative

**	****Sample****	****Pool****	****Neat****	****HIV-1/HIV-2 S/CO****	****WB****	****HIV PCR**
- 	25	NR		1.00	24, 120, 160	Neg
- 	26	NR		1.19	41, 120, 160	Neg
**	****27****	****NR****	****	****17.43****	****all bands****	****Neg**
- 	28	NR		1.37	41, 160	Neg
- 	29	NR		1.40	24, 41, 120, 160	Neg
- 	30	NR		1.43	24, 160	Neg
**	****31****	****NR****	****	****15.20****	****17, 24, 120, 160****	****Neg**
**	****32****	****	****NR****	****18.80****	****all bands****	****Neg**
**	****33****	****NR****	****	****15.93****	****all bands****	****Neg**
- 	34	NR		1.34	41, 160	Neg

**9/1/00 – 12/29/01**

## Relationship between HIV-1/HIV-2 EIA andWestern Blot-All Samples 9/8/99 to 6/30/03

***Relationship between HIV-1/HIV-2 EIA and******Western Blot-All Samples 9/8/99 to 6/30/03***

- EIAS/CO

- Western Blot Results

**2.600**

**1.602**

**1.225**

**2.672**

**1.598**

**19.178**

**Neg****N = 7,562****44.2%**

**Ind****N = 8,710****51.0%**

**Pos****N = 818****4.8%**

- 1.018 – 11.370

- 95% Range

- 1.018 – 12.329

- 1.513 – 20.952

**1.231**

**18.030**

**16.008**

## Relationship between HIV-1/HIV-2 EIA and Western BlotNAT Nonreactive Samples 9/8/99 to 6/30/03

***Relationship between HIV-1/HIV-2 EIA and Western Blot******NAT Nonreactive Samples 9/8/99 to 6/30/03***

***N = 16,238******(95% of total)***

- Western Blot Results

**2.602**

**1.602**

**1.225**

**2.672**

**1.599**

**16.268**

**Neg****N = 7,525****46.3%**

**Ind****N = 8,654****53.5%**

**Pos****N = 59****0.4%**

- 1.018 – 11.368

- 95% Range

- 1.018 – 12.258

- 1.013 – 20.092

**1.230**

**2.390**

**1.400**

- EIAS/CO

## Relationship between HIV-1/HIV-2 EIA and Western BlotNAT Reactive Samples 9/8/99 to 6/30/03

***Relationship between HIV-1/HIV-2 EIA and Western Blot******NAT Reactive Samples 9/8/99 to 6/30/03***

- Western Blot Results

**2.545**

**1.578**

**1.314**

**2.560**

**1.581**

**19.298**

**Neg****N = 37****4.3%**

**Ind****N = 56****6.6%**

**Pos****N = 759****89.1%**

- 1.076 – 12.398

- 95% Range

- 1.012 – 18.385

- 8.130 – 20.952

**1.299**

**18.182**

**16.296**

***N = 852******(5% of total)***

- EIAS/CO

## ARC HIV Testing 9/8/99 Through 6/30/03NAT Reactive Specimens Only (N = 852)

| S/CO | WBNEG | WBIND | WBPOS | Total | %WBPOS |
| --- | --- | --- | --- | --- | --- |
| 1.0 to 4.999 | 35 | 48 | 5 | 88 | 5.7% |
| 5.0 to 9.999 | 1 | 2 | 23 | 26 | 88.5% |
| 10.0 to 14.999 | 1 | 3 | 37 | 41 | 90.2% |
| 15.0 to 19.999 | 0 | 3 | 591 | 594 | 99.5% |
| > 20.0 | 0 | 0 | 103 | 103 | 100.0% |

- Note:  697 of 852 (81.8%) NAT POS specimens had an S/CO ≥ 15.0;694/697 (99.6%) specimens with S/CO ≥ 15.0 confirmed POS;
- 	95% CI = 98.8 - 99.9 by binomial distribution
- Chi-square tests for independence were significant (p<0.0001)

***ARC HIV Testing 9/8/99 Through 6/30/03******NAT Reactive Specimens Only (N = 852)***

## ARC HIV Testing 9/8/99 Through 6/30/03NAT Nonreactive Specimens Only (N = 16,238)

| S/CO | WBNEG | WBIND | WBPOS | Total | %WBPOS |
| --- | --- | --- | --- | --- | --- |
| 1.0 to 4.999 | 6,769 | 7,749 | 35 | 14,553 | 0.2% |
| 5.0 to 9.999 | 515 | 609 | 3 | 1,127 | 0.3% |
| 10.0 to 14.999 | 144 | 152 | 1 | 297 | 0.3% |
| 15.0 to 19.999 | 86 | 133 | 18 | 237 | 7.6% |
| > 20.0 | 11 | 11 | 2 | 24 | 8.3% |

- Note:  261 of 16,238 (1.6%) NAT NEG specimens had an S/CO ≥ 15.0;20/261 (7.7%) specimens with s/co ≥ 15.0 confirmed POS;
- 	95% CI = 4.7 – 11.6 by binomial distribution
- Chi-square tests for independence were significant (p<0.0001)

***ARC HIV Testing 9/8/99 Through 6/30/03******NAT Nonreactive Specimens Only (N = 16,238)***

## PPV and Sensitivity of HIV-1/HIV-2 EIA and NAT vs Western Blot

|  |  | Western Blot |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, NAT Rx  | 759 | 93 (6) | 852 (765) |
| EIA RR, NAT NR  | 59 | 16,179 | 16,238 |
| TOTAL | 818 | 16,272 | 17,090 |

- PPV = 759/852 = 89.1%   (759/765 = 99.2%)
- Sens = 759/818 = 92.8%

***PPV and Sensitivity of HIV-1/HIV-2 EIA and NAT vs Western Blot***

## PPV and Sensitivity of High S/CO (≥ 15.0) vs Western Blot

|  |  | Western Blot |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 15.0  | 714 | 244 | 958 |
| EIA RR, S/CO < 15.0  | 104 | 16,028 | 16,132 |
| TOTAL | 818 | 16,272 | 17,090 |

- PPV = 714/958 = 74.5%
- Sens = 714/818 = 87.3%

***PPV and Sensitivity of High S/CO ******(≥ 15.0) vs Western Blot***

## PPV and Sensitivity of High S/CO (≥ 15.0) vs WB Where NAT is Reactive

|  |  | Western Blot |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 15.0  | 694 | 3 | 697 |
| EIA RR, S/CO < 15.0  | 65 | 90 | 155 |
| TOTAL | 759 | 93 (6) | 852 (765) |

- PPV = 694/697 = 99.6%
- Sens = 694/759 = 91.4%

***PPV and Sensitivity of High S/CO ******(≥ 15.0) vs WB Where NAT is Reactive***

## PPV and Sensitivity of High S/CO (≥ 15.0) vs WB Where NAT is Nonreactive

|  |  | Western Blot |  |
| --- | --- | --- | --- |
|  | POS | NEG or IND | TOTAL |
| EIA RR, S/CO ≥ 15.0  | 20 | 241 | 261 |
| EIA RR, S/CO < 15.0  | 39 | 15,938 | 15,977 |
| TOTAL | 59 | 16,179 | 16,238 |

- PPV = 20/261 = 7.7%
- Sens = 20/59 = 33.9%

***PPV and Sensitivity of High S/CO ******(≥ 15.0) vs WB Where NAT is Nonreactive***

## HIV Alternate Algorithm Summary

- Use of NAT as the first step of the supplemental test algorithm will reduce the amount of HIV WB performed by only 5%
- HIV NAT-reactive samples (even using pooled NAT for screening):
- Sensitivity of 92.8% (detection of WB confirmed positives)
- Those not detected will be tested by WB; only 0.4% of positives remain and majority are false positive on WB (as indicated by repeat NAT nonRx, low EIA S/CO and weak WB banding patterns)

## HIV Alternate Algorithm Summary

- Use of S/CO _>_ 15 following separation of RR population into NAT Rx and nonRx is not practical
- Vast majority of HIV RR samples are not from individuals with HIV infection and consequently are NAT nonRx and have low S/CO values
- Of those with an S/CO _>_ 15, the PPV is 7.7% (20/261)
- Of those with an S/CO < 15, it is likely that all WB pos are false pos
- Other options
- Dual EIA Algorithm

## Dual EIA Algorithm

- Feasibility based on the concept:
- If two assays with comparable sensitivity are composed of differing rare reagents and have a different format, the false positive populations should have limited cross over; the more unique the tests, the greater the separation of false positive populations
- Used successfully for HTLV and HBsAg to eliminate >60% of repeat reactives requiring further testing by expensive, complicated, error prone and unavailable/unlicensed tests (e.g., HTLV)

## ARC HTLV Supplemental Results 

- ARC HTLV Supplemental Results

- *

- * State of California Testing Initiated

**Month (2000-2002)**

**Number**

## HIV Dual EIA Algorithm Qualification

- Qualified in both directions based on the two FDA licensed HIV-1/HIV-2 EIAs:
- Abbott (EIA-1)  Genetic Systems (EIA-2)  (ARC)
- Genetic Systems (EIA-1)  Abbott (EIA-2)  (BSL)
- All HIV EIA repeat reactive samples from 1/1/00-3/31/02 having adequate volume for additional testing were evaluated
- Western Blot and NAT (TMA pools of 16) test-of-record data were used for analysis
- All 2nd EIA testing was performed centrally at BSL

## HIV Dual EIA AlgorithmGenetic Systems pEIA (EIA-1) at BSL

**N = 1,657 RR Samples (1/1/00 – 3/31/02)**

**Ind****544 (32.8%)**

**Pos****80 (4.8%)**

**RR**
**80**

**Neg****1,033 (62.4%)**

**NR**
**0**

**RR**
**31**

**NR**
**513**

**RR**
**49**

**NR**
**984**

**Tested by Western Blot (Bio-Rad)**

**Abbott**

**EIA-2**

**Abbott**

**EIA-2**

**Abbott**

**EIA-2**

**+**
**79**

**–**
**1**

**+**
**0**

**–**
**31**

**+**
**0**

**–**
**513**

**+**
**0**

**–**
**49**

**+**
**0**

**–**
**984**

**NAT**

**NAT**

**NAT**

**(all WB bands;****high S/CO****both EIAs)**

## HIV Dual EIA AlgorithmAbbott rDNA EIA (EIA-1) at ARC

**N = 6,227 RR Samples (1/1/00 – 3/31/02)**

**Ind****2,890 (46.4%)**

**Pos****266 (4.3%)**

**RR**
**250**

**Neg****3,071 (49.3%)**

**NR**
**16**

**RR**
**16**

**NR**
**2,874**

**RR**
**13**

**NR**
**3,058**

**Tested by Western Blot (Calypte)**

**GSC**

**EIA-2**

**GSC**

**EIA-2**

**GSC**

**EIA-2**

**+**
**237**

**–**
**13**

**+**
**1**

**–**
**15**

**+**
**0**

**–**
**2,874**

**+**
**0**

**–**
**13**

**+**
**0**

**–**
**3,058**

**NAT**

**NAT**

**NAT**

**+**
**0**

**–**
**16**

**(+/- 24, 160)**

## N = 13, Abbott RR, WB Pos, GSC RR, NAT NR

|  | WB Banding Pattern | Abbott S/CO | GSC S/CO |
| --- | --- | --- | --- |
| 1 | 24, 31, 41, 51, 66, 120, 160 | 17.43 | 5.80 |
| 2 | 17, 24, 120, 160 | 15.52 | 2.91 |
| 3 | all bands | 15.93 | 9.30 |
| 4 | all bands | 17.19 | 8.62 |
| 5 | all bands | 19.30 | 8.87 |
| 6 | 17, 24, 61, 160 | 2.40 | 7.06 |
| 7 | 24, 31, 41, 51, 66, 120, 160 | 20.95 | 4.39 |
| 8 | 24, 41, 120, 160 | 1.40 | 1.33 |
| 9 | all bands | 19.30 | 8.07 |
| 10 | 41, 66, 120, 160 | 1.04 | 9.38 |
| 11 | all bands | 17.60 | 8.59 |
| 12 | all bands | 17.84 | 8.93 |
| 13 | all bands | 18.80 | 11.12 |

## N = 16, Abbott RR, WB Pos, GSC NR, NAT NR

|  | WB Banding Pattern | Abbott S/CO | GSC S/CO |
| --- | --- | --- | --- |
| 1 | 17, 41, 160 | 1.53 | 0.17 |
| 2 | 17, 41, 160 | 1.50 | 0.20 |
| 3 | 41, 160 | 1.34 | 0.30 |
| 4 | 41, 120, 160 | 4.90 | 0.25 |
| 5 | 41, 160 | 4.72 | 0.31 |
| 6 | 24, 120 | 1.52 | 0.29 |
| 7 | 41, 120, 160 | 4.92 | 0.25 |
| 8 | 24, 41, 160 | 1.00 | 0.38 |
| 9 | 41, 120, 160 | 1.19 | 0.29 |
| 10 | 24, 160 | 1.43 | 0.22 |
| 11 | 24, 160 | 1.60 | 0.32 |
| 12 | 17, 24, 41, 120, 160 | 2.36 | 0.33 |
| 13 | 17, 41, 120, 160 | 1.11 | 0.17 |
| 14 | 24, 55, 120, 160 | 10.56 |   0.57* |
| 15 | 17, 24, 160 | 8.73 |   0.62* |
| 16 | 41, 160 | 1.37 | 0.28 |

*** Both samples PCR and repeat TMA (undilute) NR; one of two reported participation in an HIV vaccine trial**

## HIV Dual EIA Summary

| GSC – BSL | N = 1,657 | No. Abbott RR | No. NAT Rx |
| --- | --- | --- | --- |
| No. WB: | Pos = 80 (5%) | 80 (100%) | 79 |
| (Bio-Rad) | Ind = 544 (33%) | 31 (6%) | 0 |
|  | Neg = 1,033 (62%) | 49 (5%) | 0 |

***HIV Dual EIA Summary***

| Abbott – ARC | N = 6,227 | No. GSC RR | No. NAT Rx |
| --- | --- | --- | --- |
| No. WB: | Pos = 266 (4%) | 250 (94%) | 237 |
| (Calypte) | Ind = 2,890 (46%) | 16 (0.6%) | 1 |
|  | Neg = 3,071 (49%) | 13 (0.4%) | 0 |

## HIV Dual EIA Algorithm Findings

- Using NAT as the “gold standard”
- Sensitivity = 100% (317/317)
- 95% CI 98.84-100%
- Specificity = 98.4% (7,447/7,567)
- 95% CI 98.11-98.68%
- No. WBs eliminated = 98.4% (7,764/7,884)
- No. indeterminate interpretations eliminated = 98.6% (3,387/3,434)