1. Comparative Performance of Dual 3rd Generation Immunoassays as a Potential Laboratory-Based HIV-1/2 Testing Strategy. B. Bennett, S. Fordan, O. David, M. Salfinger, M. Chan, D. Willis, S. Crowe, Florida Department of Health, Bureau of Laboratories-Jacksonville, FL., USA.

2. Objectives Primary: To assess clinical performance of each immunoassay for possible ITN purposes within our current algorithm, not specifically for a dual immunoassay algorithm design. Strategy-specific: Increase algorithm sensitivity w/o compromising specificity as compared to a traditional algorithm. Demonstrate potential reagent cost-effectiveness on seropositive results as compared to a traditional algorithm. Demonstrate improve testing TAT on non-seronegative reporting. Reduce the number of inconclusive reports.

3. Methods Assay selection; BioRad’s HIV-1/2 Plus O EIA Siemens Advia Enhanced HIV-1/O/2 n = 2,765 fresh serum samples tested by both 3 rd generation immunoassays. TP = 92 (3.3%), 90 WBlot +, 1 NAAT +, 1 subsequent seroconversion TN = 2,673

4. A1 = HIV1/2 immunoassay (e.g. EIA or CIA) n = 2765 TP = 92 (3.3%) A1 (+) BR1/2/O = 98 E1/2/O = 98 A1 (-) Negative for HIV-1 & HIV-2 antibodies BR1/2/O = 2667 E1/2/O = 2667 A2 = HIV 1/2 immunoassay in duplicate (e.g. EIA or CIA or non-waived rapid) A2 (++) (+-) Presumptive positive for HIV-1 or HIV-2 antibodies; requires medical follow-up for further evaluation and testing E1/2/O = 96 BR1/2/0 = 96 A1/A2 sensitivity = 100% PPV = 95.8% A1/A2 specificity = 99.85%NPV = 100% Inconclusive for HIV antibodies; request plasma redraw for NAAT *. Requires medical follow-up for further evaluation and testing. A2(- -) E1/2/O = 2 BR1/2/O = 2 A1 (- -) Repeat A1* *This study did not include the (duplicate) ? ? repeat AI pathway. A1 (++ or +-) HIV-2 testing; Strategy 5, if 1 or more of the following apply: 1) Indicated by clinical presentation 2) Indicated by local HIV-2 prevalence 3) Indicated by client/patient travel or risk history * If window period infection is suspected, refer to Acute HIV Infection Testing, Strategy 4. Strategy 3. HIV 1/2 Dual Immunoassay

5. Table 1 Individual Assay & Strategy Performance Assay(s) Clinical SensitivityClinical Specificity BioRad’s HIV-1/2 Plus O EIA 100% (92/92) 99.78%** (2667/2673) Siemens Advia HIV-1/O/2 CIA 100% (92/92) 99.78%** (2667/2673) BioRad/Siemens Dual Algorithm 100% (92/92) 99.85%** (2669/2673) Siemens/BioRad Dual Algorithm 100% (92/92) 99.85%** (2669/2673) HIV-1/2 Plus O EIA*/ HIV-1 Wblot Algorithm 97.8% (90/92) 100% (2673/2673) * Optional replacement with Advia HIV-1/O/2 w/ same performance. ** Based on Western Blot negative and/or limited seronegative follow up.

6. Table 2 Discordant Results Specimen HIV-1/2 Plus O Advia HIV-1/O/2 Western NAAT Follow up S/CO* Index* Blot (HIV-1) (Aptima HIV-1 RNA) 1 1.13.25no bandsnegativeseronegative 21.120.45no bandsnegativenone 31.13<0.05no bandsnegativenone 40.102.25not performednegativenone 50.241.08not performednegativenone 68.335.40no bandsnegativeseronegative 71.433.46p24 onlynot performedseronegative 81.62<0.05no bandsnot performedseronegative 92.826.31no bandspositiveseroconvert 107.281.56no bandsnegativeseroconvert 110.190.25no bandspositiveseroconvert 120.390.07no bandspositiveseroconvert 130.220.31no bandspositiveseroconvert 140.190.23no bandspositiveseroconvert * 1.0 s/co or index values are averages of replicates. Color representations; false positive screens, false negative blots, confirmed HIV-1 acute infections.

7. Conclusions Increased sensitivity over the reference method. Increased specificity over either 3 rd generation immunoassay in stand-alone use. Comparable specificity to a licensed HIV-1 WBlot, the licensed HIV-1 NAAT assay and several POC HIV-1/2 rapid devices. Did not demonstrate the ability to detect a very limited number of confirmed HIV-1 acute infections. Laboratory-TAT of seropositive results potentially could be reduced by 1-3 days. Diagnostic reagent costs could be reduced for non- negative specimens by 26-43%.

8. Table 3 Application Comparisons Strategy Turn-Around-Time (non-negatives)Reagent Costs 3 rd gen. EIA / Western Blot Algorithm ~3-4 days (laboratory time only)~$28.80 per non-negative result ($1.50 x 3 + $24.30) Dual Immunoassay Algorithm (EIA 1 st, CIA 2 nd ) ~1-2 days (laboratory time only)~$12.30 per non-negative result ($1.50 + $5.40 x 2) (based on 5 non- negatives/run) Dual Immunoassay Algorithm (EIA 1 st, CIA 2 nd ) ~2-3 days (laboratory time only)~$8.70 per non-negative result ($1.50 + $3.60 x 2) (based on 20 non- negatives/run) Dual Immunoassay Algorithm (CIA 1 st, EIA 2 nd ) ~2-3 days (laboratory time only)~$8.40 per non-negative result ($1.50 + $3.45 x 2) (based on 20 non- negatives/run)

9. Study Notes & Needs Need to expand study size & include more acute and early infections (focus on increasing specificity w/o compromising the improved sensitivity); - Better assay(s) selections? - The addition of a 3 rd assay? - The use of a S/CO threshold value to assist in further testing, referrals and/or reporting needs? Need for reproducibility data over more than one lot# per assay. Need for seroconversion panels (budgetary restraints in our case) Note: Based on this study’s limited number of AIs (4) and if AIT is desired, perhaps consider the Strategy 5 add-on. Note: Large automated analysis platforms are often required to accompany these newer diagnostic assays and possibly could impact the cost-effectiveness of a dual immunoassay algorithm. Use of other diagnostic applications (HBV, HCV, etc.) on the secondary platform may counter some of the additional costs. Note: The “Random access” feature of at least one immunoassay has the potential to decrease TAT in a dual immunoassay algorithm but the “control bracketing” requirement may lessen the appeal when testing small numbers of initial reactives.