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HIV prevalence in Cambodia among pregnant women was 1.7–2.7% in 2000.1 Standard methods for the early diagnosis of HIV infection in infants born to HIV-seropositive mothers are DNA-polymerase chain reaction (PCR), RNA detection, and viral cultures.2 DNA-PCR and RNA detection are the preferred standard techniques for early diagnosis.2 However, certain HIV-1 variants might not be detected by these techniques, although if at least two different regions of the HIV-1 genome are targeted, improved sensitivity would be achieved. Non-molecular tests such as viral cultures, another standard technique, would not be affected by these concerns. However, these techniques are costly and cumbersome; they require viable cells, specialized procedures and technicians, and 3–4 weeks for results. In resource-poor countries, the high cost of these standard diagnoses precludes their use, and most infants wait 18 months for classic HIV serologic tests.
The HIV-1 p24-antigen boosted ELISA assay has been described as an alternate low-cost and easy-to-perform method for detecting HIV-1 infection. This assay involved heat-mediated immune complex dissociation and tyramide signal amplification of p24 antigen ELISA. Five studies reported the use of this test as a diagnosis tool for pediatric HIV infection with excellent sensitivities (88.7–100%) and specificities (94.7–100%).3–7 In particular, HIV-1-CRF01_AE, the major subtype in Southeast Asia, was detected by a similar boosted method.7 In this study, we tested a boosted-p24 assay previously described for the diagnosis of HIV-exposed infants8 to evaluate the feasibility of implementing such a system in a resource-poor country.
This study used leftover specimens not individually identifiable that were collected for routine analysis and that would have been otherwise discarded. The National Ethical Committee has been informed in compliance with the current criteria for exemption from the Investigational Device Exemptions.9 All assays were retrospectively performed on frozen plasma samples (donated by J.-M. Reynès) representing 169 blood samples collected, from May 1999 to March 2004, from 147 Cambodian infants (age: 1–24 months; median age: 4 months). All tested infants were born to HIV-1–seropositive mothers or were HIV-1 serology–positive orphans proposed for adoption. At the time of blood collection, no breastfeeding was reported. Each blood sample had previously been tested by in-house DNA-PCR of the gag and pol genes in duplicates using described primers10,11 and by viral cultures.12 Viral culture supernatants were collected at days 7, 10, 14, 17, and 21 and assayed for HIV infection by HIV-1 p24 core profile ELISA (Perkin-Elmer, Boston, MA). Because most tested infants were orphans, no blood samples could be taken from the mother as positive controls. A sample was declared positive when at least one molecular technique (DNA-PCR on one viral gene) and viral cultures were both positive, and negative when all three techniques were negative. When molecular technique(s) and viral cultures were discordant, a second and/or third sample of the same infant was retested. In that case, if confirmation of the positive test(s) was obtained, HIV infection was diagnosed. HIV infection was diagnosed when two consecutive blood samples of the same infant are positive.2 HIV infection can be excluded when two or more samples taken from infant at age 
1 month are negative.2 Although the counseling to mothers recommended at least two blood samples taken at a month interval to establish a diagnosis, only 18 infants (12%) came back for a second confirmatory test. Among them, 14 were diagnosed HIV negative and 4 were HIV positive. The low levels of second testing could be caused by the high cost of the tests; alternatively, in the absence of overt infection or already declared infection, families might have not seen the necessity for a confirmation test.
Retrospective analyses, using HIV-1 p24 core profile ELISA and ELAST ELISA amplification system (Perkin-Elmer; donated by I. Cabruja and S. Guibert), were performed in duplicates on heat-denatured plasma samples according to the manufacturer’s instruction (designated Boosted p24). Three negative and seven positive controls, included in the kit, were used per 96-well plate. Kinetic and endpoint readings were carried out using the Quanti-Kin Detection System software (RILAB; donated by M. Giacomini and S. Bertone). Both readings gave identical qualitative diagnostic results; therefore, kinetic reading seems to be unnecessary for future assays. Readings were performed on a LP400 ELISA reader (Diagnostics Pasteur).
Classic HIV-1 p24 core profile ELISA assays are routinely used in our laboratory for the detection of viral infection in viral culture supernatants. The detection limits for classic ELISA and Boosted p24 assays were, respectively, 10 and 2 pg/mL.
When previous and recent retrospective assays were compared, all of them displayed 100% specificity (Table 1
). The sensitivities were as follows: pol-DNA-PCR, 100%; gag-DNA-PCR, 93.5%; Boosted p24, 91.3%; viral cultures, 91.1%.
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When viral cultures and DNA-PCR were previously performed, three discordant results were observed (Table 2, infants 1–3). In these cases, the Boosted p24 assays were more sensitive than viral cultures. However, in another two previously validated positive cases, the Boosted p24 assays were negative (Table 2, infants 5–6).
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), showing probable cases of genetic heterogeneity in the gag region. These results were confirmed on a second sample from infant 3. Infants 4–6 did not come back for a second testing.
A greater number of samples could be more easily processed with Boosted p24 assay (up to 42 samples) compared with DNA-PCR (eight samples; Table 3). When considering the costly technical apparatus, specialized technical formation, and longer manipulation time required for DNA-PCR, the final costs of the two techniques would most likely be similar. Whatever the criteria, viral cultures are much more expensive and have longer turnaround time for results.
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According to our data on assay sensitivity, DNA-PCR on two HIV-1 genes should be the preferred techniques for early diagnosis; however, only specialized laboratories can perform these tests. Similarly, although RNA detection using real-time RT-PCR has been reported to have an excellent performance and to be a low-cost alternative test,14 it requires a specialized laboratory. Therefore, a greater accessibility could be achieved if ELISA-user laboratories were to perform Boosted p24 assays and in parallel, for confirmation, send a blood sample to a central laboratory for DNA-PCR or RNA detection tests, using for instance dried blood spots.10 This could easily be set up in resource-poor countries. However, before implementation, a prospective study is recommended to validate our observations and estimate the feasibility and costs of such a system with local laboratories.
Received April 19, 2006. Accepted for publication August 19, 2006.
Acknowledgments: We are grateful to R. Sutthent and C. Watcharapin for technical help and to J.-M. Reynès and Y. Henin for critical reading of the manuscript. The authors thank Perkin-Elmer and their representatives I. Cabruja and S. Guibert for the gift of the HIV-1 p24 core profile ELISA and ELAST ELISA amplification kits and M. Giacomini and S. Bertone for the gift of the Quantikin Detection System software and RS 232 cable. We acknowledge L. Chartier for help in statistics.
* Address correspondence to Marie Nguyen, Institut Pasteur, PFID, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15, France. E-mail: marie{at}pasteur.fr 
Authors’ addresses: Janin Nouhin, Institut Pasteur du Cambodge, 5 Boulevard Monivong, BP 983, Phnom Penh, Cambodia. Marie Nguyen, Institut Pasteur, PFID, 25-28, rue du Docteur Roux, 75724 Paris Cedex 15, France, Telephone: 33-1-45-68-84-97, Fax: 33-1-45-68-89-49, E-mail: marie{at}pasteur.fr.
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  J. C. Patton, A. H. Coovadia, T. M. Meyers, and G. G. Sherman Evaluation of the Ultrasensitive Human Immunodeficiency Virus Type 1 (HIV-1) p24 Antigen Assay Performed on Dried Blood Spots for Diagnosis of HIV-1 Infection in Infants Clin. Vaccine Immunol., February 1, 2008; 15(2): 388 - 391. [Abstract] [Full Text] [PDF]
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