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Delayed HIV primary infection diagnosis with a fourth-generation HIV assay

Delforge, Marie-Lucea; Dollé, Nicoleb; Hermans, Philippec; Liesnard, Corinnea

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Fourth-generation HIV assays are now available in Europe. These screening assays allow the combined detection of HIV antigen and antibody (Ag/Ab) in order to reduce the diagnostic window between infection and antibody detection in primary HIV infection (PHI). We report on a case of PHI that was not detected with one of these available fourth-generation HIV assay in spite of the presence of a high p24 antigen level.

A 35-year-old man presented to the emergency room with fever, myalgia and skin rash. Serological tests were performed, including a fourth-generation HIV screening test (Vironostika HIV Uni-Form II Ag/Ab assay, Organon Teknika, Boxtel, the Netherlands) and all the results were negative. Seven days later, a second blood sample was performed and tested for HIV antibodies in another laboratory. The HIV antibody test was positive (Ortho HIV1/2 Ab Capture ELISA, Ortho Diagnostic System, NJ, USA). The Western blot (HIV blot 2.2, Genelabs Diagnostics, Singapore) showed antibodies weakly reactive with p24 and gp160 proteins. p24 antigen (Innotest HIV Antigen mAb, Innogenetics, Ghent, Belgium) was positive at a level of 1300 pg/ml. The plasma HIV-1-RNA load (HIV-1 Amplicor Monitor 1.5, Roche Diagnostics, Branchburg, NJ, USA) was greater than 750 000 copies/ml. All these results together supported a diagnosis of PHI. The patient reported being homosexual and had had a risk contact for HIV 2 weeks before the onset of symptoms.

The first sample was then re-analysed using different screening and confirmatory tests. Three antibody screening tests were used: the Murex HIV-1.2.O (Murex Biotech, Dartford, UK), the Ortho HIV1/HIV2 Ab Capture ELISA and the Vitros Immunodiagnostic anti-HIV-1+2 (Ortho Clinical Diagnostic, Amersham, UK). Three Ag/Ab combined assays were tested: the Vironostika HIV Uni-Form II Ag/Ab, which was initially non-reactive, the Vidas Duo (BioMérieux, Marcy l'Etoile, France) and the Murex HIV Ag/Ab combination (Murex Biotech). Quantification of p24 antigen was performed using the Innotest HIV Antigen mAb. Western blot was performed using the HIV Blot 2.2. All the tests were performed and interpreted according to the manufacturers’ instructions.

The initially negative result obtained with the Vironostika HIV Uni-Form II Ag/Ab was confirmed. The sample, tested undiluted and at five- and 10-fold dilutions remained negative, excluding the possibility of a prozone effect. On the contrary, the quantitative p24 antigen detection assay showed a high level of p24 antigen. Two out of the three antibody screening tests were positive. The complete results are shown in Table 1.

Table 1
Table 1:
Results obtained with the different tests on the two consecutive samples.

According to the results of Kabamba et al. (see previous letter), the Vironostika Ag/Ab test seems to be the least sensitive of the three Ag/Ab tests they investigated. In our hands, it was even less sensitive than two classic third-generation antibody tests. The reason for this lack of sensitivity is not clear. The level of p24 antigen was high in the first serum sample and should have been detected by the Vironostika Ag/Ab test, which claims to have a detection limit of 30 pg/ml [1]. A prozone effect was excluded by the negative results obtained after serum dilution. Neither can the failure to detect p24 antigen be explained by the genetic variability of the HIV-1 patient strain, which is a B subtype. It is of note that Laperche et al. [2] also observed a negative Vironostika Ag/Ab test result in a seroconversion serum containing 34000 pg/ml p24 antigen that was detected by the other Ag/Ab tests.

In our case, as in the case of Kabamba et al. (see previous letter), the positivity of the Vironostika Ag/Ab test corresponded to the appearance of envelope antibodies in the Western blot assay. Glycoprotein 160 antigen is actually the HIV-1 antigen that is used for the detection of HIV-1 antibodies in the Virostika Ag/Ab test. On the contrary, in our case, the positivity of the Vidas Ag/Ab test correlated with the presence of p24 antigen. It thus seems that the failure to detect early primary infection by the Vironostika Ag/Ab test is caused by its low sensitivity in detecting p24 antigen. Moreover Ag/Ab tests have no gag antigen included among the antigens intended to detect HIV antibodies. In the case of the Vironostika Ag/Ab test, this also could explain the delayed diagnosis compared with classic HIV antibody tests such as the Ortho HIV1/2 Ab Capture ELISA, which includes a gag antigen in their design. Consecutively to the incorporation of HIV antigen detection in HIV antibody tests, the antibody detection part of the fourth-generation assays may be less sensitive than that of third-generation assays. Although the concept of fourth-generation HIV tests presents an improvement in the early diagnosis of PHI, as demonstrated by the evaluation of these tests on seroconversion panels [2–4], their use for large-scale screening purposes needs further assessment. Further evaluation of their sensitivity to the different phases of the viral infection and immune response should be carefully evaluated, together and separately, before replacing classic HIV antibody tests with these new tests. In the meantime, considering their disputable sensitivity for p24 antigen detection and maybe also for HIV antibodies that could appear in early seroconversion, these tests should not be used as sole diagnostic tests. In particular, these tests should not replace p24 antigen tests for confirming an HIV antibody-positive result obtained with a screening assay.

Marie-Luce Delforgea

Nicole Dolléb

Philippe Hermansc

Corinne Liesnarda

References

1. Van Binsbergen J, Keur W, Siebelink A. et al. Strongly enhanced sensitivity of a direct anti-HIV-1/-2 assay in seroconversion by incorporation of HIV p24 ag detection: a new generation Vironostika HIV Uniform II. J Virol Methods 1998, 76: 59–71.
2. Laperche S, Maniez-Montreuil M, Courouce AM. Les tests de dépistage combiné de l'antigène p24 et des anticorps anti-VIH dans l'infection précoce à VIH-1. Transfus Clin Biol 2000, 7 (Suppl. 1) : 18S–24S.
3. Weber B, El Hadji Mbargane Fall, Berger A, Doerr HW. Reduction of diagnostic window by new fourth-generation HIV screening assays. J Clin Microbiol 1998, 36: 2235–2239.
4. Doung Ly T, Edlinger C, Vabret A, Morvan O, Greuet B. Contribution of combined detection assays of p24 antigen and anti-HIV antibodies in diagnosis of primary HIV infection by routine testing. J Clin Microbiol 2000, 38: 2459–2461.
© 2002 Lippincott Williams & Wilkins, Inc.