Evaluation of a New Combined Antigen and Antibody Human Immunodeficiency Virus Screening Assay, VIDAS HIV DUO Ultra

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0095-1137/02/$04.00⫹0 DOI: 10.1128/JCM.40.4.1420–1426.2002

Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evaluation of a New Combined Antigen and Antibody Human

Immunodeficiency Virus Screening Assay, VIDAS HIV DUO Ultra

Bernard Weber,

1,2

* Annemarie Berger,

2

Holger Rabenau,

2

and Hans Wilhelm Doerr

2

Laboratoires Réunis Kutter-Lieners-Hastert, Junglinster, Luxembourg,1and Institut für Medizinische Virologie,

Universitätskliniken Frankfurt, Frankfurt, Germany2

Received 10 August 2001/Returned for modification 25 September 2001/Accepted 22 January 2002

Combined antigen and antibody screening (fourth-generation) assays reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis by 4 days, on average, in comparison to antibody-only (third generation) enzyme immunoassays (EIAs). The aim of the present study was to assess whether the new VIDAS HIV DUO Ultra (Biomérieux, Marcy-l’Etoile, France) showed an improved sensitivity and specificity in comparison to licensed fourth-generation assays. A total of 16 seroconversion panels, 15 cell culture supernatants infected with different HIV type 1 (HIV-1) subtypes, and 257 potentially cross-reactive serum samples were tested with VIDAS DUO HIV Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen HIV 1/2, version 2 (third-generation EIA), and Genetic Systems HIV-1 Ag EIA (p24 antigen assay). VIDAS HIV DUO Ultra showed a comparable sensitivity to the single p24 antigen assay in seroconversion panels and a dilution series of virus lysates. The diagnostic window was reduced with VIDAS HIV DUO Ultra by 3.82 days, on average, in comparison with the fourth-generation assay with the lowest sensitivity of the antigen detection module. HIV-1 infection was detected 5.88 days earlier than with third-generation EIA. The mean time delay between reverse transcription-PCR and VIDAS HIV DUO Ultra was only 2.31 days. The specificity of fourth-generation assays after retesting ranged between 98.1 and 100%. In conclusion, VIDAS HIV DUO Ultra can replace single-antigen screening for laboratory diagnosis and screening of HIV infection in blood donors. There was no evidence for a second diagnostic window due to impaired sensitivity of the antibody detection module of all the fourth-generation EIAs evaluated in the present study. The specificity after initial and/or repeated testing of VIDAS HIV DUO Ultra was equivalent to that of a third-generation assay.

In order to reduce the diagnostic window period between the time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new screening enzyme-linked immu-nosorbent assays which permit the simultaneous detection of HIV antigen and antibody have been introduced on the inter-national market (4, 8, 9, 11, 13, 16, 17, 21, 23–25, 27). Com-bined antigen and antibody tests show a higher sensitivity for the detection of primary HIV infection than conventional tests in urban centers with high HIV incidences and prevalences (15). Although this new assay generation represents a major improvement in terms of sensitivity in comparison to the former generation through a mean reduction of the diagnostic window by 4 days (4, 8, 9, 11, 13, 16, 23–25, 27), optimization of the performance characteristics is requested for different reasons. Combined assays for antigen and antibody detection cannot substitute single-antigen tests for blood donor screen-ing. The detection limit of fourth-generation assays (20 to

⬎100 pg of p24 antigen [Ag]/ml) is higher than that of antigen

assays (3.5 to 10 pg of p24 Ag/ml). Highly sensitive antigen assays detect primary infection on average 1 to 2 days earlier than fourth-generation enzyme immunoassays (EIAs) (26).

The antigen detection module of fourth-generation assays shows a variable sensitivity for detection of different HIV type 1 (HIV-1) non-B subtypes, including group O, and HIV-2 (1).

Some assays may fail to detect low-level antigens of HIV-1 non-B subtype strains, although monoclonal antibody is di-rected against conserved epitopes of p24 Ag (13, 25). Since the genetic diversity of HIV is rapidly increasing worldwide, in-cluding in industrialized countries, fourth-generation assays need to be optimized in order to accurately detect all HIV-1 subtypes and HIV-2.

A further potential risk for impaired sensitivity is that a more-limited space of the solid phase can be used for antibody detection since about one-third of the binding sites are oc-cupied by anti-p24 antibody for HIV antigen detection; therefore, the antibody detection module may be less sensitive than single third-generation antibody assays. Antibody detec-tion may be delayed in seroconversion panels without antigen-emia, and a second diagnostic window may be observed in the early seroconversion phase when low antibody titers are present and antigenemia declines (11).

Since fourth-generation EIAs combine two different test principles in one assay, the potential risk for nonspecific reac-tivity may be higher than for second- and third-generation antibody assays. The rate of false-positive results obtained with blood donors and interfering samples varies from 0.3 to 0.8% (versus a maximum of 0.2% for third-generation EIAs), de-pending on the donor background (1).

Fourth-generation assays demand a special algorithm for the analysis of reactive samples. For the anti-HIV part of the assay, confirmation of reactivity should be done first with an assay that lacks the p24 Ag detection module, and when reactivity persists, immunoblotting should be used. For the p24 Ag part,

* Corresponding author. Mailing address: Laboratoires Réunis Kut-ter-Lieners-Hastert, Centre Langwies, L-6131 Junglinster, Luxem-bourg. Phone: (352) 78 02 90 309. Fax: (352) 78 88 94. E-mail: web @labo.lu.

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confirmation of reactivity should be analyzed in an assay that lacks the anti-HIV detection part and when reactivity persists, a nucleic acid-based assay should be used. Confirmation of this part of reactivity is hampered by the fact that actually none of the commercially available nucleic acid-based assays is able to detect HIV-1 group O and the HIV-2 genome.

The new VIDAS HIV DUO Ultra, which has an improved sensitivity of the antigen detection module and has also im-proved antibody detection through a double sandwich antigen EIA test principle, was evaluated in comparison with three licensed fourth-generation, one third-generation, and one p24 Ag EIA.

MATERIALS AND METHODS

VIDAS HIV DUO Ultra.VIDAS HIV DUO Ultra is an enzyme-linked

fluo-rescent assay which permits the simultaneous detection of p24 Ag and antibodies against HIV-1 (including group O) and HIV-2. The upper part of the solid phase receptacle (SPR) coated with three different anti-p24 monoclonal antibodies is used for p24 Ag detection. The lower part of the SPR serves for immunoglobulin G (IgG), IgM, and IgA antibody detection and is sensitized with gp160 of HIV-1 and peptides representing immunodominant epitopes of gp41 from HIV-1 group O and gp36 from HIV-2.

During the first incubation step, the sample and biotinylated rabbit anti-p24 antibody are aspirated into the SPR. p24 Ag is released through virus lysis and binds to the monoclonal antibodies on the SPR and also to the biotinylated anti-p24 antibody. Simultaneously, anti-HIV-1 and anti-HIV-2 antibodies bind to gp160 and/or gp36/gp41 peptides on the lower part of the SPR.

The second incubation with biotinylated antigens (identical to those coated to the solid phase) is only performed on the lower part of the SPR.

The third reaction step is performed with the entire SPR. Alkaline phos-phatase-labeled streptavidin is added and binds to biotinylated anti-p24 antibody on the upper part of the SPR, if present, and to biotinylated antigens in the lower part, if present.

The substrate (4-methyl-umbelliferyl phosphate) is first incubated with the lower part of the SPR, and a first measurement of fluorescence is performed at a wavelength of 450 nm. The intensity of fluorescence is proportional to anti-HIV antibody activity. Afterwards, the substrate is incubated with the entire SPR and the fluorescence is measured a second time. The intensity of the reaction is proportional to the p24 Ag concentration.

At the end of the assay, results are automatically calculated by VIDAS in relation to the standards and printed. Separate test values for p24 Ag and HIV antibody are calculated by dividing the patient references values by the reference value of the respective standard. A test value ofⱖ0.25 is considered to be positive for HIV antibody and/or p24 Ag.

Comparative assays.VIDAS HIV DUO Ultra was compared to combined

HIV antigen and antibody assays, including Enzymun-Test HIV Combi (Roche Diagnostics, Penzberg, Germany), Enzygnost HIV Integral (Dade Behring, Mar-burg, Germany), and Genscreen Plus HIV Ag-Ab (Bio-Rad, Marnes la Co-quette, France), to a third-generation HIV antibody assay (Genscreen HIV 1/2, version 2) and to an HIV antigen assay (Genetic Systems HIV-1 Ag EIA). The characteristics of the different immunoassays are shown in Table 1. INNO-LIA HIV (Innogenetics, Zwijnwarde, Belgium) was used as a confirmatory assay for HIV antibody testing. All the tests were performed and interpreted in accor-dance with the manufacturers’ recommendations.

Specimens.The following specimens were tested to evaluate sensitivity. (i)

Sixteen seroconversion panels, provided by different suppliers, were tested. Pan-els W, Z, AD, AE, AI, AK, AS, AT, AZ, and BA were from Boston Biomedica, Inc. (BBI) (West Bridgewater, Mass.), panels SV-0271-1, SV-0331, and SV-0361 were from North American Biologicals (NABI) (Boca Raton, Fla.), and panels ANT 9017 and BCP 47019 were from BioClinical Partners (Franklin, Mass.). For seroconversion panels BBI W, Z, AD, AE, AI, AK, AS, AT, AZ, and BA and NABI SV-0271, SV-0331, and SV-0361, HIV-1 RNA detection was performed by BBI and NABI by using quantitative PCR (Amplicor HIV-1 Monitor; Roche Diagnostics) or qualitative transcription-mediated amplification (Gen-Probe, San Diego, Calif.). In addition, all of the seroconversion samples were tested for HIV-1 antibodies by Western blotting (Cambridge Biotech, Worcester, Mass., or Dupont, Wilmington, Del.). Western blots were interpreted in accordance with Centers for Disease Control and Prevention criteria (7). (ii) Dilutions of cell culture supernatants infected with different HIV-1 subtypes, including subtypes

TABLE

1.

Characteristics

of

HIV

screening

and

antigen

assays

a

Assay

Support

Solid

phase

Conjugate

p24

Ag

detection

limit

(pg/ml)

Time

(min)

HIV

antibody

detection

HIV

antigen

detection

HIV

antibody

detection

HIV

antigen

detection

VIDAS

HIV

DUO

Ultra

VIDAS

automated

analyzer

gp160,

immunodominant

epitopes

of

gp41

and

gp36

Anti-p24

MAb

gp160,

immunodominant

epitopes

of

gp41

and

gp26

(biotinylated)

Biotinylated

rabbit

polyclonal

anti-p24

3

120

Genscreen

Plus

HIV

Ag-Ab

Microplate

gp160

(recombinant),

arti

ficial

consensus

poly-peptide

of

HIV-1,

peptide

mimicking

gp36

Anti-p24

MAb

Peptides

mimicking

gp41

and

gp36,

arti

ficial

consensus

polypeptide

of

HIV-1

Biotinylated

polyclonal

sheep

anti-p24

44

120

Enzygnost

HIV

Integral

Microplate

gp41

and

gp36

(recombinant

and

synthetic)

Anti-p24

MAb

gp41

and

gp36

(recombinant

and

synthetic),

biotinylated

Anti-p24

MAb

(biotin-ylated)

30

120

Enzymun-Test

HIV

Combi

Enzymun-Test

gp41

and

gp36

(synthetic),

reverse

transcrip-tase

(recombinant)

Anti-p24

MAb

gp41

and

gp36

(synthetic),

reverse

transcrip-tase

(recombinant)

Anti-p24

MAb

110

240

Genscreen

HIV

1/2,

version

2

Microplate

gp160

and

p24

(recombinant),

peptide

mim-icking

HIV-2

envelope

protein

Peptide

mimicking

HIV-1

and

-2

envelope

protein,

p24

(recombinant)-peroxidase

conjugate

90

90

Genetic

Systems

HIV-1

Ag

EIA

Microplate

Anti-p24

MAb

Biotinylated

polyclonal

sheep

anti-p24

3

150

a

MAb,

monoclonal

antibody.

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B, E (CRF01__AE), F, G, and H and group O, were tested in order to investigate the influence of the genetic variability of HIV on antigen detection. Virus isolates had been genotyped by the sequencing of PCR-amplified fragments of the V3 genome region (P. Kasper, A. N. Smith, G. Duraisamy, B. Ofenlock, and E. Faatz, Infektionsepidemiologische Forschung, abstr. V089, p. P32, 1996). All of the supernatants were diluted in HIV-negative serum. All of the viral stocks were in the same concentration range in terms of picograms of p24 Ag per milliliter so that the highest dilution available for testing was in the range between 10 and 15 pg of p24 Ag/ml in order to give a relatively strong signal in p24 Ag assays. (iii) Two hundred fifty-seven potentially cross-reacting samples were tested to challenge the specificity of the assays. These included samples from pregnant women and patients suffering from autoimmune diseases; samples which were positive for IgM antibodies against cytomegalovirus, herpes simplex virus, rubella virus, orToxoplasma gondii; samples positive for rheumatoid factor, hepatitis C virus antibody, or Epstein-Barr virus capsid antigen antibody; and sera that were HIV EIA reactive on tests different from those evaluated in the present study but Western blot negative, i.e., unconfirmed.

Statistical analysis.The performance of VIDAS HIV DUO Ultra was

com-pared with alternative screening assays, HIV antigen, and HIV-1 RNA detection for the seroconversion panels. The mean number of days by which the diagnostic window period was reduced with VIDAS HIV DUO Ultra in comparison with the third-generation assay (Genscreen HIV 1/2, version 2) and fourth-generation EIAs was determined. The statistical significance of the reduction for each test was determined by using the Wilcoxon test for matched pairs (5).

The time delay between blood sampling points in commercially available seroconversion panels used for the present study is on average relatively short (2 to 7 days) but may last up to 37 days, for example, for panel W. The calculation model for time delays between assays established by the Paul Ehrlich Institute (8) was used. This method considers that seroconversion is theoretically possible the following day after the last negative follow-up sample. The total and average number of days of time delay for the 16 panels were calculated in comparison with the most sensitive assay

For the calculation of sensitivity and specificity, samples were considered to be HIV-1 positive if any of the following tests were positive: Western blotting interpreted according to Centers for Disease Control and Prevention criteria (5), HIV-1 p24 Ag assay, and HIV-1 RNA assay. Patients were considered HIV negative if all of the screening assays were negative or, in the case of the EIA-reactive samples, if they were negative or indeterminate on a Western blot.

RESULTS

The results obtained for the 16 seroconversion panels are summarized in Table 2. Eight of the 16 primary HIV infections were detected at least one blood sampling point earlier with all fourth-generation assays than with Genscreen HIV 1/2, version 2. Conversely, HIV antibody detection was detected in panel ANT 9017 one blood sampling point earlier with Genscreen HIV 1/2, version 2, than with the most sensitive fourth-gener-ation EIA. ANT 9017 is an atypical panel since an antibody response is present before the appearance of p24 Ag.

VIDAS HIV DUO Ultra showed the best performance of the combined antigen and antibody assays (Table 2). Com-pared with Genscreen Plus HIV Ag-Ab, it detected primary infections at an earlier stage in 8 of 16 panels. Genscreen Plus HIV Ag-Ab detected HIV infection one blood sampling point earlier than VIDAS DUO Ultra in panel ANT 9017. HIV infections were detected at least one blood sampling point earlier with VIDAS HIV DUO Ultra in comparison to Enzyg-nost Integral in 13 of 16 panels and Enzymun-Test HIV Combi in 11 of 16 seroconversions. It showed a comparable perfor-mance to Genetic Systems HIV-1 Ag. Fourteen panels were detected at the same time point, one was detected earlier with VIDAS HIV DUO Ultra (ANT 9017), and one was detected earlier with Genetic Systems HIV-1 Ag (BBI AT). HIV p24 Ag was detected 2 days earlier with Genetic Systems HIV-Ag EIA in comparison with VIDAS HIV DUO Ultra for panel AT. Conversely, primary infection was detected one blood

sam-TABLE 2. Comparison of the performances of VIDAS HIV DUO Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen H IV 1/2, version 2, Genetic Systems HIV-1 Ag EIA, and HIV-1 RNA RT-PCR in seroconversion panels Assay Blood sampling day with first positive result in seroconversion panel: No. of posi-tive samples/ total no. of samples (%) No. of posi-tive samples/ no. of positive

RT-PCR samples (%)

BBI W BBI Z BBI AD BBI AE BBI AK BBI AI BBI AS BBI AT BBI AZ BBI BA ANT 9017 BCP 47019 NABI SV-0271-1 NABI SV-0331 NABI SV-0351 NABI SV-0361 VIDAS HIV DUO Ultra 37 7 14 0 12 0 7 2 18 8 21 17 8 13 8 9 64/104 (61.5) 53/75 (70.7) a Genscreen Plus HIV Ag-Ab 47 7 18 7 12 0 12 7 21 11 15 20 8 13 8 9 60/104 (57.7) 48/75 (64.0) a Enzygnost HIV Integral 47 9 18 0 12 0 12 14 28 15 25 20 15 20 11 11 48/104 (46.2) 41/75 (54.7) a Enzymun-Test HIV Combi 47 9 18 7 12 0 12 9 28 11 19 20 13 15 8 9 52/104 (50.0) 42/75 (56.0) a Genscreen HIV 1/2, version 2 84 27 25 7 19 0 19 9 28 19 12 47 15 20 11 16 41/104 (39.4) 31/75 (41.3) a Genetic Systems HIV-1 Ag EIA 37 7 14 0 12 0 7 0 18 8 25 17 8 13 8 9 58/104 (55.8) 53/75 (70.7) a RT-PCR b 35 2 14 0 5 0 5 0 18 8 0 ND c ND c 6 1 1 75/90 (83.3) aThe dif ferences in performance between RT-PCR and VIDAS HIV DUO Ultra ( P ⫽ 0.027), Genetic Systems HIV-1 Ag EIA ( P ⫽ 0.016), Enzygnost HIV Integral ( P ⫽ 0.001), Enzymun-Test HIV Combi ( P ⫽ 0.001), and Genscreen HIV 1/2, version 2 ( P ⫽ 0.005) were statistically signi ficant. bHIV-1 RNA RT-PCR data are from BBI and NABI. cND, no data. No data on HIV-1 viral load were available for seroconversion panels BCP 47019 and NABI SV-0271-1.

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pling point (4 days) earlier with VIDAS HIV DUO Ultra than with Genetic Systems HIV-1 Ag in panel ANT9017. HIV-1 RNA reverse transcription-PCR (RT-PCR) detected primary infection one blood sampling point earlier than VIDAS HIV DUO Ultra in 9 of 13 panels tested with both methods. HIV primary infection was detected eight blood sampling points later with VIDAS HIV DUO Ultra than with PCR in serocon-version panel ANT 9017.

The time delay in days for detection of primary HIV infec-tion in comparison with the most sensitive assay for each panel (last negative sample plus 1 day) ranged from 0 (RT-PCR) to 8.19 (Genscreen HIV 1/2, version 2) days. The mean time delay between RT-PCR and VIDAS DUO HIV Ultra was only 2.31 days (Table 3). If panel ANT 9017 was excluded from analysis, the mean time delay between HIV-1 RNA detection and the first positive VIDAS HIV Duo Ultra result was only 0.85 days. The highest number of seroconversion panel sam-ples (83.3%) was detected by RT-PCR (Table 2). Among RT-PCR positive seroconversion panel members (Table 2), VIDAS DUO HIV Ultra detected the highest number (70.7%). The lowest performance was achieved with a third-generation EIA (41.3%).

The performance of combined antigen and antibody assays was significantly better (Table 3) than that of the third-gener-ation screening EIAs. There was no statistical difference in sensitivity between VIDAS HIV DUO Ultra and Genscreen Plus HIV Ag-Ab (Table 3). In contrast, the difference in per-formance between VIDAS HIV DUO Ultra, Enzygnost HIV Integral, and Enzymun-Test HIV Combi was statistically sig-nificant (Table 3).

VIDAS HIV DUO Ultra showed the highest sensitivity for the detection of HIV antigen in a dilution series of cell culture supernatants infected with different HIV-1 subtypes (Table 4). It showed an equivalent sensitivity to the single-antigen assay (Genetic Systems HIV-1 Ag).

A variable number of false-positive results were obtained with the different assays among potentially interfering serum samples (Table 5). Enzygnost HIV Integral and Enzymun-Test HIV Combi obtained the best performances. Only 4 and 2 samples were initially and repeatedly reactive, respectively (Table 5). Genetic Systems HIV-1 Ag EIA (6.3%) and Gen-screen Plus HIV Ag-Ab (6.4%) showed a high percentage of initial false-positive results. After retesting, Genscreen Plus HIV Ag-Ab showed 100% specificity. The differences in spec-ificity between the HIV screening assays were not statistically significant.

DISCUSSION

Although the residual risk of HIV transmission by blood and blood products is very small (1, 2, 3, 6, 10), the safety of donated blood could be improved by the combined use of antibody and p24 Ag detection (9). Fourth-generation assays permit an earlier diagnosis of HIV infection than third-gener-ation double-antigen sandwich assays by detecting p24 Ag, which may be present in recent HIV infection prior to sero-conversion. However, until now, these assays could not substi-tute for single-antigen testing since their detection threshold is equal to or higher than 20 pg of p24 Ag/ml (8). With Genetic Systems HIV-1 Ag, the diagnostic window in comparison to

TABLE 3. Time delay for detection of primary HIV infection in comparison with the most sensitive assay for each panel (last negative sample plus 1 day) Assay Blood sampling day with first positive result in seroconversion panel: Total no. of days Mean no. of days BBI W BBI Z BBI AD BBI AE BBI AK BBI AI BBI AS BBI AT BBI AZ BBI BA ANT 9017 BCP 47019 NABI SV-0271-1 NABI SV-0331 NABI SV-0351 NABI SV-0361 VIDAS HIV DUO Ultra 1 3 0 0 3 0 1 1 0 0 20 0 0 3 2 3 37 2.31 a , b Genscreen Plus HIV Ag-Ab 3 3 1 4 3 0 3 3 1 1 13 1 0 3 2 3 44 2.75 a Enzygnost HIV Integral 3 6 1 0 3 0 3 10 4 4 22 1 6 8 8 9 98 6.13 b Enzymun-Test HIV Combi 3 6 1 4 3 0 3 8 4 1 16 1 1 4 2 3 60 3.75 b Genscreen HIV 1/2, version 2 13 8 8 4 10 0 10 8 4 8 9 14 6 10 8 11 13 8.19 b Genetic Systems HIV-1 Ag EIA 1 3 0 0 3 0 1 0 0 0 22 0 0 3 2 3 38 2.38 a RT-PCR d 00 0 0 0 0 0 0 0 0 0 N D c ND c 00 0 0 0 a The performances of VIDAS HIV DUO Ultra and Genscreen Plus HIV Ag-Ab ( P ⫽ 0.129) and Genetic Systems HIV-1 Ag ( P ⫽ 1) were not statistically dif ferent (Wilcoxon matched pairs test). b The performances of VIDAS HIV DUO Ultra and Enzygnost Integral ( P ⫽ 0.000244), Enzymun-Test HIV Combi ( P ⫽ 0.021), and Genscreen HIV 1/2, version 2 ( P ⫽ 0.0034), were statistically dif ferent (Wilcoxon matched pairs test). c ND, no data. d Values for RT-PCR were always 0 (days), since it was the most sensitive assay.

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RT-PCR is reduced between 1.37 and 3.75 days (Table 3) in comparison to fourth-generation EIAs with a relatively low sensitivity of the antigen detection module (Enzygnost HIV Integral and Enzymun-Test HIV Combi). In contrast, with the more-sensitive VIDAS HIV DUO Ultra (2.31 days) and Gen-screen Plus HIV Ag-Ab (2.75 days), the performance was comparable to that of Genetic Systems HIV-1 Ag EIA (2.38 days).

In comparison to Genscreen HIV 1/2, version 2, which is one of the most sensitive commercially available third-generation screening EIAs, the diagnostic window was reduced by 5.88 days with VIDAS HIV DUO Ultra (Table 3). In a former study, combined antigen and antibody assays detected HIV-1 infection an average of 4.8 and 4.4 days earlier than a third-generation EIA (24).

In 9 of 14 seroconversion panels tested in parallel with RT-PCR, the diagnostic window period would be reduced by 1 to 20 days (mean time interval, 2.31 days) by amplification of HIV-1 RNA from plasma or serum. If the atypical panel ANT 9017 is not considered, the time delay ranges between 1 and 3 days for 8 of 13 seroconversions (mean time interval, 0.85 days). Cost-benefit analysis of expanded HIV-testing protocols for donated blood has shown that RNA PCR testing would prevent more additional cases of transfusion-associated HIV infection annually than combined p24 Ag and antibody detec-tion; however, the costs would be considerably higher (2). Current commercially available PCR protocols are not adapt-ed to large-scale screening of single blood donations, and false-negative reactions have been reported in patients with low HIV-1 RNA or cDNA copy number, irrespective of the HIV-1 subtype (18).

However, the most efficient way to reduce the diagnostic window in the future will be the screening of blood donations with nucleic acid amplification testing (NAT) techniques, since HIV RNA is detected about 11 days before antibody to HIV is detected (25), and in the chimpanzee model, there is no de-monstrable infectivity in either plasma or peripheral blood

mononuclear cells obtained before molecular markers are de-tectable (19). Roth et al. (20) demonstrated the feasibility and efficacy of routine PCR screening of pooled blood donations for HIV-1 in a blood bank setting. For efficient NAT under routine conditions, assays that are not affected by the genetic variability of HIV, including rapid automated nucleic acid ex-traction procedures, need to be developed. Moreover, the yield of NAT is close to that of combined antigen and antibody tests for high values of the detection threshold and the pool size (14).

Genetic variability does not represent a major challenge for VIDAS HIV DUO Ultra in comparison to combined p24 Ag and antibody assays with a lower sensitivity of the antigen detection module. With Genscreen Plus HIV Ag-Ab, one of three HIV-1 group O strains was not detected (Table 4). Ters-mette et al. (22) reported failure of monoclonal antibody to detect p24 Ag from certain strains of HIV. HIV-1 group O (12), which is highly divergent from other HIV-1 subtypes known so far, may not be detected by assays with monoclonal antibodies for the capture of p24 Ag. An impaired sensitivity for HIV-1 subtype C antigen detection with VIDAS HIV DUO Ultra was not observed in our evaluation in contrast to the findings of Ly et al. (17). The results of dilutions of HIV-1

TABLE 4. End point titration of HIV-infected cell culture supernatantsa

Culture supernatant HIV-1 subtype or group

Highest reciprocal dilution with a positive result with assay: VIDAS HIV

DUO Ultra Genscreen PlusHIV Ag-Ab Enzygnost HIVIntegral Enzymun-TestHIV Combi Genetic SystemsHIV-1 Ag EIA Subtypes (strain)

B (ASM42) 16,000 1,000 500 500 8,000

B (MBAT) 8,000 250 250 125 4,000

C 40,960 10,240 ND 640 20,480

C (MP 37) 32,000 4,000 125 500 64,000

E/CRF01_AE 32,000 5,120 2,560 1,280 20,480

E/CRF01_AE (MP 121) 32,000 2,000 2,000 1,000 32,000

F 10,240 10,240 640 640 20,480

F (J7) 4,000 500 125 250 4,000

G ND 10,240 5,120 2,560 40,960

G (VI 1197) 160 20 20 10 160

H 5,120 1,280 40 640 5,120

H (VI 991) 160 20 5 5 160

Groups

O (FAN) 8,000 ⬍500 1,000 1,000 1,000

O (LOB) 16,000 1,000 500 1,000 16,000

O (MP 331) 5,120 40 160 640 160

aThe sensitivities of VIDAS HIV DUO Ultra and Genetic Systems HIV-1 Ag were not statistically significantly different (P0.547). The sensitivities of VIDAS

HIV DUO Ultra and Genscreen Plus HIV Ag-Ab (P⫽0.000244), Enzygnost HIV integral (P⫽0.000244), and Enzymun-Test HIV Integral were significantly different (Wilcoxon matched pairs test). ND, not determined.

TABLE 5. Specificities of HIV screening assays

Assay

No. of false-positive

samples Specificity (%) Initially

reactive Repeatedlyreactive reactiveInitially Repeatedlyreactive

VIDAS HIV DUO Ultra 6 5 97.7 98.1

Genscreen Plus HIV Ag-Ab 17 0 93.4 100

Enzygnost HIV Integral 4 2 98.4 99.2

Enzymun-Test HIV Combi 4 2 98.4 99.2

Genscreen HIV 1/2, version 2 7 2 97.3 99.2 Genetic Systems HIV-1 Ag 16 8 93.7 96.9

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subtype virus lysates are dependent on the strain used and cannot be generalized for all HIV-1 subtype C isolates.

In contrast to a previous study from Gürtler et al. (11), there was no second diagnostic window in the early seroconversion phase, when low antibody titers are present and antigenemia declines. However, in the atypical seroconversion panel ANT 9017 with a delayed weak antigenemia (and low viral load) after antibody response, third-generation EIA was the first to detect primary infection, before antigen assay and combined p24 Ag and antibody assays. According to the dynamics of HIV replication and immune response during primary infection, it is expected that these cases are very rare. With the exception of this particular panel, antibody detection seems not to be im-paired in combined assays, since there was no significant drop in the measured index values or negative result in the transi-tion period between the decline of antigenemia and the begin-ning of seroconversion (data not shown).

The VIDAS HIV DUO Ultra offers, in comparison to the alternative assays, the advantage that separate test values for p24 Ag and HIV antibody are calculated. Usually, fourth-generation assays demand a special algorithm for the analysis of reactive samples. For the anti-HIV part of the assay, con-firmation of reactivity should be done with an assay that lacks the p24 Ag detection module when reactivity persists subse-quently by immunoblotting. For the p24 Ag part, confirmation of reactivity should be analyzed in an assay that lacks the anti-HIV detection part. With VIDAS HIV DUO Ultra, only one of the parts (HIV antigen or antibody) needs to be verified depending on which of the two is repeatedly reactive. All of the 6 samples that gave false-positive results with VIDAS HIV DUO Ultra (Table 5) showed a positive test value for HIV antibody.

The specificity of fourth-generation assays in 257 potentially interfering serum samples ranged between 98.1 and 100% after retesting. VIDAS HIV DUO Ultra obtained a specificity of 98.1% (Table 4). The specificity of VIDAS DUO Ultra was 99.5% in samples from 2,660 low- and high-risk individuals when compared to Food and Drug Administration-licensed tests (21).

In a previous study, the specificity of combined antigen and antibody assays ranged between 97.6 and 98.4% for a collec-tion of 255 potentially interfering serum samples. Values for specificity of fourth-generation EIAs obtained from studies performed in unselected donors ranged between 99.6 and 99.8% (4, 11). Ly et al. observed a specificity of 99.51% for 29,657 samples tested with VIDAS HIV DUO in a survey performed with unselected samples in French public and pri-vate laboratories (15). Third-generation assays showed a spec-ificity of 99.86%. The cost effectiveness of a more-sensitive but less-specific fourth-generation assay may not be warranted in low incidence populations (higher workload by confirmatory testing of reactive samples and discarding of reactive blood donations).

In conclusion, the new VIDAS DUO HIV Ultra showed a statistically significantly better performance for early diagnosis of HIV infection than alternative fourth-generation assays, with the exception of Genscreen HIV Ag-Ab Plus, and was also more-sensitive than all of the alternative combined anti-gen and antibody assays for the detection of HIV virus lysates of different genotypes. The new VIDAS HIV DUO Ultra

could be an alternative to single-antigen screening and permit a more cost-effective laboratory diagnosis and screening of HIV infection in blood donors than stand-alone HIV antigen and antibody assays.

ACKNOWLEDGMENTS

We thank Emilio Brignoli (Biomérieux, Marcy l’Etoile, France), Dany De Becker (Bio-Rad), Martin Bä␤ler (Bio-Rad), Rita Mertens (Dade-Behring), and Walter Melchior (Roche Diagnostics) for pro-viding test kits and financial support for purchasing commercially avail-able seroconversion panels.

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Figure

TABLE 1. Characteristics of HIV screening and antigen assaysa

TABLE 1.

Characteristics of HIV screening and antigen assaysa p.2
TABLE 2. Comparison of the performances of VIDAS HIV DUO Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen HIV 1/2,version 2, Genetic Systems HIV-1 Ag EIA, and HIV-1 RNA RT-PCR in seroconversion panels

TABLE 2.

Comparison of the performances of VIDAS HIV DUO Ultra, Genscreen Plus HIV Ag-Ab, Enzygnost HIV Integral, Enzymun-Test HIV Combi, Genscreen HIV 1/2,version 2, Genetic Systems HIV-1 Ag EIA, and HIV-1 RNA RT-PCR in seroconversion panels p.3
TABLE 3. Time delay for detection of primary HIV infection in comparison with the most sensitive assay for each panel (last negative sample plus 1 day)

TABLE 3.

Time delay for detection of primary HIV infection in comparison with the most sensitive assay for each panel (last negative sample plus 1 day) p.4
TABLE 4. End point titration of HIV-infected cell culture supernatantsa

TABLE 4.

End point titration of HIV-infected cell culture supernatantsa p.5
TABLE 5. Specificities of HIV screening assays

TABLE 5.

Specificities of HIV screening assays p.5