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AIDS:
doi: 10.1097/01.aids.0000232251.66533.ca
Correspondence

Generic screening test for HIV infection

Plantier, Jean-Christophea; Lemée, Véroniquea; Nabias, Renéb; Sire, Jean-Mariec; Simon, Françoisa,c

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aLaboratoire de Virologie Associé au Centre National de Référence du VIH, EA2656, IFR23, CHU Charles Nicolle, Rouen, France

bCentre de Transfusion Sanguine, Hôpital Principal, Dakar, Sénégal

cInstitut Pasteur, Dakar, Sénégal.

Received 15 December, 2005

Accepted 23 February, 2006

More than 30 million individuals infected by HIV worldwide live in poor countries. Major efforts are being made to improve access to antiretroviral drugs in these countries, but the diagnosis and monitoring of HIV-infected patients remains problematical. Alternative methods [1,2] and specific programmes have recently been developed for treatment and resistance monitoring, but there are no screening or confirmatory tools specifically designed for developing countries. In western countries, screening is based on commercial enzyme-linked immunoassays (EIA) and Western blot confirmation, a strategy too costly for developing countries [3,4]. Simple disposable tests are available for use in poor countries, but they are not suitable for large-scale population screening, and financial support from the international community is still required to implement them.

We have developed and validated, on a broad panel of samples, a generic indirect EIA based on antigen peptides representing all known HIV variants including HIV-1 group O and HIV-2, which are endemic in Africa.

The peptides correspond to highly conserved epitopes located in the immunodominant region of the transmembrane glycoprotein gp41/36 of HIV-1 group M (RILAVERYLKDQQLLGIWGCSGKLICTTAV), group O (LALETLIQNQQLLNLWGCKGKLIC) and HIV-2 (TAIEKYLKDQAKLNSWGCAFRQVC), as described elsewhere [5]. The EIA is performed as previously described [5] except for the following modifications: coating is performed on polystyrene microtitre plates (Microwell Maxisorp, Nunc, Fontenay-sous-Bois, France) with 100 μl per well of an equimolar mixture of the three peptides at a final concentration of 2 μg/ml of antigen solution in 0.05 M bicarbonate buffer, pH 9.6, an aluminium foil seal is used for the incubation step at 37°C and the serum sample is tested at a 1: 100 dilution in 0.01 M sodium phosphate buffer, pH 7.4, containing 0.75 M sodium chloride, 10% newborn calf serum, and 0.05% Tween 20. Subsequent steps are performed as previously described [5].

We analysed the specificity and sensitivity on 1230 negative and 223 positive samples (plasma or serum). Negative samples were collected from the blood bank of Dakar Military Hospital, Sénégal, which uses a sensitive commercial EIA screening test for HIV-1/HIV-2 antibodies and HIV-1 p24 antigen (Genscreen Plus HIV Ag-Ab; Bio-Rad, Paris, France). The positive panel corresponded to samples collected in France, positive with the AsXYM HIV1/2gO test (Abbott, Chicago, Illinois, USA), and confirmed by Western blotting. This panel covered a wide range of variants (201 HIV-1 group M, B and non-B subtypes), five group O, 15 HIV-2, and two cases of HIV-1/HIV-2 dual infection.

The cutoff was defined as the absorbance of the negative control plus 0.100. Figure 1a shows the specificity results for the 1230 negative samples. Optical density/cutoff (index) variations ranged from 0.008 to 2.4. Only four samples (0.35%) were considered positive, with values close to the cutoff (index between 1 and 1.3, n = 3) or above the cutoff (index 2.4, n = 1).

Fig. 1
Fig. 1
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The sensitivity results for the 223 positive samples are shown in Fig. 1b. Of the 223 samples, 222 (99.5%) were positive with our test. The reactivity index ranged from 2.3 to 27.6. Only one sample, corresponding to an HIV-1 group M subtype B early seroconversion sample (negative Western blot but positive detection of p24 antigen and RNA) was under the cutoff (index 0.5). Eleven samples (eight HIV-1 group M and three HIV-1 group O) had low reactivity indexes but were largely above the cutoff (indexes between 2.3 and 7.5).

Our generic EIA thus performed well on this panel. It had excellent sensitivity (100%) for chronic infection (n = 222) by divergent strains (including HIV-1 group O and HIV-2) and also excellent specificity (99.65%). The negativity in our test of an early seroconversion sample positive on the AsXYM test based on the immunocapture format is not surprising. Contrary to immunocapture, our indirect format cannot detect IgM isotypes. Similarly, the lack of HIV-1 p24 antigen detection limits the sensitivity of our method during the acute phase of infection, as for all disposable tests currently used in developing countries.

The test is robust; the coated microplates and the peptide solutions are stable at −20°C for at least 2 months and one year, respectively (data not shown). The peptides synthesized by specialized manufacturers are sufficiently stable to be mailed to countries seeking to produce this type of test, the other reagents being universally available. The estimated cost is approximately 20 Euro cents per test.

Since 1997, the World Health Organization has recommended screening and confirmation strategies adapted to the screening objective and to the local prevalence, and based on a combination of different EIA or disposable tests using different antigenic preparations or different methodologies. Our generic test is simple to assemble, and can be used at the screening or confirmation step by all countries with minimal laboratory infrastructures and capable of implementing appropriate quality controls.

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References

1. Braun J, Plantier J-C, Hellot M-F, Tuaillon E, Gueudin M, Damond F, et al. A new quantitative HIV load assay based on plasma virion reverse transcriptase activity for the different types, groups and subtypes. AIDS 2003; 17:331–336.

2. Plantier J-C, Dachraoui R, Lemee V, Gueudin M, Borsa-Lebas F, Caron F, Simon F. HIV-1 resistance genotyping on dried serum spots. AIDS 2005; 19:391–397.

3. Centers for Disease Control and Prevention. Update: serologic testing for HIV-1 antibody – United States. Morb Mortal Wkly Rep 1990; 39:69–72.

4. Mylonakis E, Paliou M, Lally M, Flanigan TP, Rich JD. Laboratory testing for infection with the human immunodeficiency virus: established and novel approaches. Am J Med 2000; 109:568–576.

5. Simon F, Souquiere S, Damond F, Kfutwah A, Makuwa M, Leroy E, et al. Synthetic peptide strategy for the detection of and discrimination among highly divergent primate lentiviruses. AIDS Res Hum Retroviruses 2001; 17:937–952.

© 2006 Lippincott Williams & Wilkins, Inc.

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