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Journal of Clinical Microbiology, October 2007, p. 3416-3418, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01314-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Potential of a Simplified p24 Assay for Early Diagnosis of Infant Human Immunodeficiency Virus Type 1 Infection in Haiti{triangledown}

Erik George,1* Carole Anne Beauharnais,2 Emilio Brignoli,2 Francine Noel,2 Gyrlande Bois,2 Patricia De Matteis Rouzier,2 Martine Altenor,2 Daniel Lauture,2 Marlène Hosty,3 Sapna Mehta,4 Peter F. Wright,4 and Jean W. Pape2

Department of Medicine, Division of International Medicine and Infectious Diseases, Weill Medical College of Cornell University, New York, New York,1 Groupe Haitien d'Etude du Sarcome de Kaposi et des Infections Opportunistes (GHESKIO), Port-au-Prince, Haiti,2 Hopital Immaculeé Conception des Cayes, Les Cayes, Haiti,3 Department of Pediatrics, Division of Pediatric Infectious Diseases, Vanderbilt University School of Medicine, Nashville, Tennessee4

Received 30 June 2007/ Accepted 19 July 2007


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ABSTRACT
 
With global efforts to scale up the prevention of mother-to-child transmission services and pediatric antiretroviral therapy, there is an urgent need to introduce a simple, low-cost infant human immunodeficiency virus test in the field. We postulated that the p24 antigen capture enzyme-linked immunosorbent assay could be simplified by eliminating signal amplification without compromising diagnostic accuracy.


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TEXT
 
In 2006, 530,000 children contracted human immunodeficiency virus type 1 (HIV-1) infection worldwide, and the vast majority resided in low-income countries (11, 14, 29). In high-income countries, prompt diagnosis and early introduction of antiretroviral therapy (ART) have dramatically reduced mortality in children infected with HIV (10). Nucleic acid sequence-based amplification assays, the gold standard for infant HIV diagnosis, are costly and technologically complex, making their use in poor countries unfeasible (3, 4, 6, 20). Lately, improved accuracy of commercially available p24 antigen micro-enzyme-linked immunosorbent assay (ELISA) kits has been demonstrated. With p24 antigen-antibody heat denaturation and more efficient solid-phase antigen capture and buffer systems, coupled with signal amplification ELISA, the lower limit of p24 antigen detection has been reduced from 10 to 0.5 pg/ml (8, 16, 18, 22-25). The ultrasensitive p24 assay, validated in countries where HIV subtypes A, B, C, D, and E predominate (7, 13, 15, 19), has achieved a sensitivity of 92 to 100% in pediatric cohorts at a cost of approximately $7 per test (12, 13, 27, 30).

The objective of this study was to determine the performance of two commercially available p24 antigen assays for testing infants perinatally exposed to HIV-1 in Haiti. The tests were the Vironostika HIV-1 p24 antigen micro-ELISA system assay (bioMérieux, Boxtel, The Netherlands)—with (Up24) and without (Sp24) the addition of the signal amplification system enzyme-linked amplified sorbent test (ELAST; PerkinElmer Life Sciences, Inc., Boston, MA) (17)—and the automated VIDAS HIV p24 II assay (bioMérieux, Marcy l'Etoile, France). The studies were conducted at the GHESKIO Centers in Port-au-Prince, Haiti, and the district hospital Immaculée Conception in Les Cayes, Haiti, between April 2005 and October 2006. Caregiver consent was obtained for routine HIV testing of all subjects. Internal review boards at GHESKIO and the Weill Medical College of Cornell University approved these studies. The reference method was the NucliSENS EasyQ HIV-1 real-time RNA assay (version 1.1; bioMérieux, Boxtel, The Netherlands) (5). In blinded cross-sectional studies, we analyzed 401 frozen plasma samples of 233 HIV-1 subtype B-exposed ART-naïve infants (mean age, 2.8 months old; interquartile range [IQR], 0.2 to 3.6 years old) consecutively enrolled at the study centers. The plasma samples were obtained by venipuncture and stored at –70°C. Heat denaturation was performed on plasma samples prior to solid-phase incubation (100°C, 5 min). The manufacturer's instructions for conducting p24 antigen testing with the respective assays were followed. However, the following modifications to the Up24 and Sp24 assays were made: (i) the entire testing procedure was performed at room temperature (24 to 28°C), except antigen-antibody heat denaturation, and (ii) plasma samples were diluted 1:5 with virus disruption buffer (0.5% Triton X-100 and 2.9% sodium dodecyl sulfate). In addition, the antigen solid-phase incubation time of the Sp24 assay was prolonged to 16 h (9). In the initial comparison, the performance of the Up24 assay versus that of the VIDAS assay for HIV-1 RNA quantification was documented by analyzing 200 plasma samples. In a second comparison, the performance of the Sp24 assay for HIV-1 RNA quantification was documented for another 201 plasma samples. For each assay, readings of optical density (OD) values at 450 nm were recorded to calculate the p24 antigen concentration against a standard curve (the cutoff value was the mean absorbance of three negative controls plus 3 standard deviations).

The sensitivity values of the Up24, Sp24, and VIDAS assays that were used to test the plasma of our infant population were 93% (95% confidence interval [CI], 85 to 100%), 91% (95% CI, 83 to 100%), and 95% (95% CI, 89 to 100%), respectively (P = 0.44) (Table 1). The detection of p24 antigen with the Sp24 assay failed for four infants aged 0, 2, and 10 days and 3 months with HIV-1 RNA levels of 3.2, 3.6, 3.1, and 3.2 log10 copies/ml, respectively. The detection of p24 antigen with the Sp24 assay was successful for 11/15 (73%) samples with HIV-1 RNA levels of <4.0 log10 copies/ml and for 30/30 (100%) samples with HIV-1 RNA levels of >4.0 log10 copies/ml. The correlation values (r) for HIV-1 RNA (log10 copies/ml) and p24 antigen (log10 fg/ml) quantification by Spearman's rank analysis were 0.567 for Up24 (R2 = 0.321; P < 0.01; n = 41), 0.590 for Sp24 (R2 = 0.348; P < 0.01; n = 40), and 0.704 for VIDAS (R2 = 0.495; P < 0.01; n = 41). The specificity values for the Up24, Sp24, and VIDAS assays were 99% (95% CI, 98 to 100%), 97% (95% CI, 94 to 100%), and 99% (95% CI, 98 to 100%), respectively (P = 0.10). The diagnostic range of the Sp24 assay was 3.0 to 80 pg/ml. Sp24 intraassay signal variability values (defined as the ratios of extreme OD/mean OD) for five repeats with the same plasma with p24 antigen concentrations of 0, 7, 24, and 59 pg/ml were 14% (95% CI, 0 to 28%), 23% (95% CI, 9 to 37%), 21% (95% CI, 12 to 30%), and 21% (95% CI, 13 to 29%), respectively. Sp24 interassay signal variability values for three consecutive trials of plasma samples with p24 antigen concentrations of 0, 1, 5, 20, and 80 pg/ml were 8% (95% CI, 0 to 18%), 24% (95% CI, 15 to 33%), 22% (95% CI, 14 to 30%), 14% (95% CI, 8 to 20%), and 4% (95% CI, 0 to 10%).


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  		TABLE 1. Diagnostic performance of p24 antigen assays in Haiti

Previous decision analysis modeling, assuming 5% ART availability, has estimated that a pediatric HIV test could benefit at-risk populations in terms of total adjusted life years saved if the test achieves at least 90% sensitivity and specificity (1). We explored the utility of different heat-denaturated p24 antigen assays for infant HIV-1 testing in Haiti. Simplifying the protocol to overnight (16 h) incubation for p24 antigen solid-phase ELISA without added signal amplification attained comparable diagnostic accuracy for the Up24 and the VIDAS assays. The detection of p24 antigen with the Sp24 assay was successful for 30/30 (100%) samples with HIV-1 RNA levels of >4.0 log10 copies/ml (18). The mean HIV-1 RNA level of the infected cohort was 5.7 (IQR, 5.3 to 6.0) log10 copies/ml, approximately 1 log10 copy/ml greater than that observed in adults (21, 26). We concluded that the signal amplification of p24 antigen ELISA was redundant when we tested HIV-exposed infants in the perinatal period, when plasma viral concentrations reach levels of >5.0 log10 copies/ml, generally within weeks of infection regardless of the mode of transmission. Storage, preparation, dilution, and pipetting of reagents for signal amplification were eliminated, reducing the effective processing time by 90 min, minimizing person-to-person variability, and cutting expenses by nearly half. We believe that even greater diagnostic accuracy may be achieved for a relatively older population (the mean age of our cohort was 2.8 months, and the IQR was 0.2 to 3.6 months) if routing testing is conducted at ages of 4 weeks and older and if freeze-thaw cycles and storage time for plasma are minimized (1, 2, 28). Importantly, we set up Sp24 assay testing in the district hospital in Les Cayes, Haiti. The trained local technician obtained accuracy levels comparable with those of the central laboratory in Port-au-Prince, Haiti. The Sp24 assay technique yielded interassay signal variability of 16%. Repeat testing of samples falling close to the cutoff level (an OD within the range of ±0.1 [2 standard deviations]) is crucial. Providers also need to monitor and test HIV-exposed infants over time.

The simplified p24 assay achieved significant diagnostic accuracy in settings with minimal infrastructure. We strongly encourage efforts to accelerate the development of point-of-care diagnostic tests for use in resource-limited settings. In the interim period, the utilization of the low-cost assays, such as the Sp24 assay, will be critical for capitalizing on the momentum in the fight to control the global AIDS pandemic.


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ACKNOWLEDGMENTS
 
The project was supported in part by grants from the Fondation Rodolphe Mérieux and grants TW006896 and TW006901 from the Fogarty International Center. Support was also provided by the President's Emergency Plan for AIDS Relief and by the National Institute of Allergy and Infectious Diseases. During his affiliation with the GHESKIO Centers, Emilio Brignoli was employed by the Fondation Rodolphe Mérieux.

We gratefully acknowledge the assistance of Marie Josette Calixte, Yvette Neptune, Marie-Eugenie Beaulieu, Maryse Thimothee, Myriame Auguste, Johanne Charlemagne, Arnaud Drouin, Massiano St-Cyr, Abdias Marcelin, Paul Albert, and Gabriel Thimothee.


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FOOTNOTES
 
* Corresponding author. Present address: Hudson Infectious Disease Associates PC, 540 North State Road, Briarcliff Manor, NY 10510-1598. Phone: (914) 762-2276. Fax: (914) 762-2894. E-mail: erikgeorge{at}yepmail.net Back

{triangledown} Published ahead of print on 1 August 2007. Back


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Journal of Clinical Microbiology, October 2007, p. 3416-3418, Vol. 45, No. 10
0095-1137/07/$08.00+0     doi:10.1128/JCM.01314-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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