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.
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.
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 3rd
generation immunoassays.
• TP = 92 (3.3%), 90 WBlot +, 1 NAAT +, 1 subsequent
seroconversion
• TN = 2,673
Strategy 3. HIV 1/2 Dual Immunoassay
A1 = HIV1/2 immunoassay (e.g. EIA or CIA)
A1 (+) BR1/2/O = 98
E1/2/O = 98
n = 2765
TP = 92 (3.3%)
A1 (-) Negative for HIV-1 & HIV-2
antibodies
BR1/2/O = 2667
E1/2/O = 2667
A1 (- -)
A2 = HIV 1/2 immunoassay in duplicate
(e.g. EIA or CIA or non-waived rapid)
A2(- -) E1/2/O = 2
BR1/2/O = 2
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%
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
Repeat A1*
(duplicate) ? ?
*This study did not include the
repeat AI pathway.
A1 (++ or +-)
Inconclusive for HIV antibodies;
request plasma redraw for NAAT *.
Requires medical follow-up for further
evaluation and testing.
*If window period infection is suspected, refer to Acute
HIV Infection Testing, Strategy 4.
Table 1
Individual Assay & Strategy Performance
Assay(s)
Clinical Sensitivity
Clinical Specificity
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)
BioRad’s
HIV-1/2 Plus O EIA
* Optional replacement with Advia HIV-1/O/2 w/ same performance.
** Based on Western Blot negative and/or limited seronegative follow up.
Table 2
Discordant Results
Specimen
HIV-1/2 Plus O
S/CO*
Advia HIV-1/O/2
Index*
Western
Blot (HIV-1)
NAAT
(Aptima HIV-1 RNA)
Follow up
1.1
3.25
no bands
negative
seronegative
2
1.1
20.45
no bands
negative
none
3
1.13
<0.05
no bands
negative
none
4
0.10
2.25
not performed
negative
none
5
0.24
1.08
not performed
negative
none
6
8.33
5.40
no bands
negative
seronegative
7
1.43
3.46
p24 only
not performed
seronegative
8
1.62
<0.05
no bands
not performed
seronegative
9
2.82
6.31
no bands
positive
seroconvert
10
7.28
1.56
no bands
negative
seroconvert
11
0.19
0.25
no bands
positive
seroconvert
12
0.39
0.07
no bands
positive
seroconvert
13
0.22
0.31
no bands
positive
seroconvert
14
0.19
0.23
no bands
positive
seroconvert
1
* <1.0 s/co or index values are exact, >1.0 s/co or index values are averages of replicates.
Color representations; false positive screens, false negative blots, confirmed HIV-1 acute infections.
Conclusions
• Increased sensitivity over the reference method.
• Increased specificity over either 3rd 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 nonnegative specimens by 26-43%.
Table 3
Application Comparisons
Strategy
Turn-Around-Time (non-negatives)
Reagent Costs
~3-4 days (laboratory time only)
~$28.80 per non-negative
result
($1.50 x 3 + $24.30)
Dual Immunoassay
Algorithm
(EIA 1st, CIA 2nd)
~1-2 days (laboratory time only)
~$12.30 per non-negative
result
($1.50 + $5.40 x 2)
(based on 5 nonnegatives/run)
Dual Immunoassay
Algorithm
(EIA 1st, CIA 2nd)
~2-3 days (laboratory time only)
~$8.70 per non-negative result
($1.50 + $3.60 x 2)
(based on 20 nonnegatives/run)
Dual Immunoassay
Algorithm
(CIA 1st, EIA 2nd)
~2-3 days (laboratory time only)
~$8.40 per non-negative result
($1.50 + $3.45 x 2)
(based on 20 nonnegatives/run)
3rd gen. EIA / Western
Blot Algorithm
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 3rd 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.