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Clinical and Vaccine Immunology, January 2006, p. 152-155, Vol. 13, No. 1
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.1.152-155.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Ultrasensitive Human Immunodeficiency Virus Type 1 p24 Antigen Assay Modified for Use on Dried Whole-Blood Spots as a Reliable, Affordable Test for Infant Diagnosis

Janet C. Patton,1* Gayle G. Sherman,1,2,3 Ashraf H. Coovadia,1 Wendy S. Stevens,2,3 and Tammy M. Meyers1,4

Wits Paediatric HIV Unit, Wits Health Consortium,1 Departments of Molecular Medicine and Hematology,2 Paediatrics, University of the Witwatersrand, Johannesburg, South Africa,4 National Health Laboratory Service, South Africa3

Received 9 July 2005/ Returned for modification 31 August 2005/ Accepted 31 October 2005


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ABSTRACT
 
The ultrasensitive human immunodeficiency virus (HIV) p24 antigen assay was modified for use on pediatric dried whole-blood spots on Whatman no. 1 filter paper. The modified assay was found to be reliable and accurate, making it an affordable tool for pediatric HIV diagnosis in developing countries.


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TEXT
 
In countries with limited resources there is an urgent need for an affordable, reliable diagnostic test for human immunodeficiency virus type 1 (HIV-1) infection in infants in the first months of life. Nucleic acid tests (NAT), the "gold standards" against which other diagnostic tests are measured, are expensive and technically challenging (3, 7, 11), making their use in developing countries problematic.

The HIV-1 p24 antigen (Ag) enzyme-linked immunosorbent assay (ELISA) is a commercially available kit performed with equipment found in most laboratories. The improved ultrasensitive p24 Ag assay performed on plasma, used for pediatric HIV diagnosis, has sensitivities and specificities ranging from 97 to 100%, comparable to HIV DNA and RNA PCR (8, 12, 14, 18). There is, additionally, evidence that the assay is more sensitive to diversity in viral genome than HIV PCR (3, 7, 8, 18).

The advantages of blood collection as dried whole-blood spots (DBS) on filter paper are well documented (9, 10). Importantly, DBS could provide increased access to HIV testing for children in resource-poor countries.

The U.S. Food and Drug Administration-approved Schleicher and Schuell (S&S) Grade 903 paper (10) is currently not widely available in Southern Africa. Whatman no. 1 filter paper, however, can be found in most laboratories on the continent.

The present study examines the performance in terms of precision, reliability, and accuracy of the p24 Ag ELISA on DBS relative to nucleic acid testing for HIV infection in infants. The reliability of Whatman no. 1 filter paper for the collection of whole-blood samples is also investigated.

DBS were from two sources. Ninety-three were from infants born to HIV-infected women attending clinics for routine check-ups at the Coronation Women and Children's Hospital and the Chris Hani Baragwanath Hospital, Johannesburg, South Africa. The HIV status of these infants was determined by qualitative HIV DNA PCR (Amplicor HIV-1 DNA Version 1.5 Assay; Roche Molecular Systems, Inc., Branchburg, NJ). To adequately test the sensitivity of the assay, an additional 48 DBS were made from pediatric anticoagulated blood specimens with a known HIV DNA or RNA PCR (Roche Amplicor HIV-1 Monitor Assay, version 1.5) or NASBA (nucleic acid sequence-based amplification) HIV-1 RNA (Nuclisens; bioMérieux, Boxtel, The Netherlands) result. Of the 48, 34 had an HIV DNA PCR result, and 14 had a known viral load. The age range of the sample population was 34 days to 12 years. (University of the Witwatersrand Human Ethics clearance number M00-01-07).

Four separate drops of blood of unknown volume were collected on Whatman no. 1 filter paper, air dried at ambient temperature for a minimum of 3 h. To simulate local collection conditions, filter papers were stored in individual plastic zip-lock bags at room temperature without a desiccant sachet. Blood spots that contained insufficient blood, or appeared "layered," crusty, clotted, or wetted were discarded (13).

In addition to the reagents supplied with the Ultrasensitive p24 Ag ELISA kit (Perkin-Elmer Life Sciences, Boston, Mass.), a virus-lysing (VL) buffer (30 mM Tris-HCl [pH 7.2], 450 mM NaCl, 1.5% Triton X-100, 1.5% deoxycholic acid [sodium salt], 0.3% sodium dodecyl sulfate, 10 mM EDTA) was used (17).

The stock standard was serially diluted with sterile phosphate-buffered saline (PBS; Roche Diagnostics, Mannheim, Germany) plus 1% bovine serum albumin (BSA), in a range from 12,500 to 3.052 pg/ml.

Washed erythrocytes were prepared from EDTA-anticoagulated whole blood from a consenting HIV-negative individual. Whole blood was centrifuged (1,500 x g, 10 min), plasma and buffy coats were removed, and red blood cells (RBC) were washed three times with sterile PBS. Equal volumes of serially diluted standards and washed erythrocytes were mixed to give standards ranging from 6,250 to 1.526 pg/ml. Aliquots were dropped onto Whatman no. 1 filter paper and allowed to air dry completely (3 h, room temperature). The blank DBS consisted of RBC mixed with an equal volume of sterile PBS. A negative control, prepared from equal volumes of washed RBC and the negative control supplied with the kit was used to calculate the thresholds for positivity and negativity. A high and a low in-house control (2,000 and 50 pg/ml, respectively) were prepared in a similar fashion from the stock standard.

Preliminary studies to determine the optimal eluting efficiency of different buffers on DBS showed the best recovery (91.3% of the concentration of p24 in the high DBS control) was with the Triton X-100 supplied with the kit in conjunction with the VL buffer (data not shown). This combination was used for all subsequent experiments.

With some modifications, the assay was performed according to the manufacturer's instructions and as described elsewhere (2). Using an ordinary paper punch duplicate 6-mm disks were punched from blanks, standards, controls, and samples DBS, and these substituted for diluted liquid blanks, standards, controls, and plasma samples in the assay. The punch was swabbed with 70% ethanol after each use to prevent cross-contamination.

Briefly, disks were incubated with 25 µl of the VL buffer (10 min, room temperature) (16) and then with 275 µl of the supplied Triton X-100 (0.5%, overnight, 4°C). Tubes were heated to 100°C for 5 min and allowed to cool naturally to room temperature. Aliquots (250 µl) of the boiled eluates were transferred to a 96-well plate coated with HIV-1 p24-specific monoclonal antibody and incubated for 60 min at 37°C. Following the protocol supplied by the manufacturer, bound p24 Ag was detected, the signal was amplified and detected, and the reaction was stopped with 4 N sulfuric acid. The plate was washed (10 cycles, 0.05% Tween 20) after each step. Absorbances were read at 490 nm against a reference wavelength of 630 nm using an ELX 808 Bio-Tek microplate reader with onboard software. Quantification of p24 Ag was by endpoint evaluation from a best-fit four-parameter curve constructed from DBS standards. Four negative control DBS were included on each plate to determine the cutoff baseline for positivity (greater than the average absorption plus five standard deviations), and negativity (less than the average absorption plus three standard deviations) (15).

Assay performance was tested by evaluating assay precision and reliability (intra- and interassay coefficients of variance [CV]), and accuracy in terms of sensitivity and specificity. The effect of time on p24 concentration and the relationship between log10-transformed DBS p24 Ag values (pg/ml) and log10 plasma RNA (copies/ml) were also examined on the small subsample of DBS that had a quantitative plasma viral load value.

The standard curve constructed from a range of DBS standards from 1.526 to 6,250 pg/ml consistently showed a good fit with values for R2 of never less than 0.997. This compares favorably with the fit for the curve constructed from liquid standards (Fig. 1). The presence of any agents likely to elevate DBS absorbances (e.g., heme), found by others attempting to measure DBS p24 Ag using liquid blanks and standards (4), was negated by using DBS blank and standards with incorporated RBC.



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  		FIG. 1. Comparison between the p24 Ag standard curve constructed from a range of standards diluted in a plasma-like matrix ({circ}) and that constructed from standards eluted from dried blood spots (•). The standard range was 1.526 to 6,250 pg/ml for both conditions. Absorbances values ranged from 0.056 to 3.0 nm for the liquid standards and from 0.018 to 2.362 nm for the DBS standards.

Intra-assay variation for 10 replicates of the low- and high-DBS control was 4.8% (range, 45.4 to 53.3 pg/ml for the 50-pg/ml DBS control) and 8.0% (range, 2,064 to 2,636 pg/ml for the 2,000-pg/ml DBS control), respectively. These values rival those reported for the assay performed on plasma (5.3 and 28.4%, respectively, for a low and a high control), showing a high a degree of precision for the assay when performed on DBS. These results additionally support the argument that the volume of plasma in each 6-mm disk excised from the Whatman DBS is relatively constant as has been shown for DBS on S&S paper (10), thus abolishing the need to apply a known volume of blood to the filter paper.

Interassay CV for the low and high DBS controls (n = 9) were 9.0% (range, 43.5 to 60.9 pg/ml for the 50-pg/ml DBS control) and 20.4% (range, 1,707 to 2,571 pg/ml for the 2,000-pg/ml DBS control), respectively. The reported CVs for a low- and a high-plasma sample were 6.4 and 9.9%. As discussed later, the higher DBS values may be due to deterioration of the p24 Ag protein in DBS over time (the assays were performed over 3 months) or the filter paper type or storage conditions used.

Of the 141 DBS assayed for p24 Ag, 83 were from children diagnosed as HIV infected and 58 HIV uninfected on the basis of HIV-1 DNA or RNA PCR or NASBA HIV-1 RNA tests. The results for the p24 Ag ELISA on DBS were 81 positive, 59 negative, and one result regarded as "indeterminate" by the criteria suggested by Schupbach et al. (15). With no false-positive values, the diagnostic specificity for the p24 Ag assay was 100%. Since the vast majority of HIV infections in South Africa are by subtype C virus (18, 20), this result supports the finding of others that the assay is sensitive to viral subtype C (3, 4, 5, 8, 17).

A single value that tested positive by PCR was not detected by the p24 Ag ELISA giving the assay a diagnostic sensitivity of 98.8%, which equals that for HIV DNA PCR performed on plasma and DBS (8, 12, 14, 18). The indeterminate result fell in the "gray" area between diagnostically positive and diagnostically negative.

The relationship between log10 DBS p24 Ag values (pg/ml) and log10 plasma RNA (copies/ml) was tested on the small subsample of DBS that had a known HIV viral load. The Pearson correlation coefficient and analysis of variance indicate a significant positive correlation between log10 DBS p24 Ag and log10 plasma RNA (Fig. 2, r = 0.79, P < 0.001). Although only on a small sample, this compares well with the correlation found between log10 plasma p24 Ag and log10 plasma RNA (r = 0.74, P < 0.0001 (G. Stevens, N. Rekhviashvilli, L. E. Scott, and W. Stevens, Abstr. 2nd IAS Conf. HIV Pathog. Treatment, abstr. 475, 2003).



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  		FIG. 2. Correlation between log10 plasma HIV RNA copy number and log10 DBS p24 Ag concentration, respectively, in the small subsample with a known viral load (n = 14). The Pearson correlation coefficient of 0.79 indicates a significant positive correlation (P < 0.001) between the two measures of HIV infection. The 95% confidence intervals are indicated by the broken lines.

Using the same 14 DBS the effect of time on p24 Ag concentration in DBS in our storage conditions was examined (Table 1). Within 6 weeks of collection, the DBS showed 100% specificity and sensitivity in complete agreement with the HIV RNA results. When re-assayed 6 weeks later, 2 of the 14 DBS remained negative and 2 had slightly higher p24 Ag values. Of the remaining 10 DBS, 4 that had previously tested positive were negative 6 weeks later, and 6 DBS had p24 Ag concentrations substantially lower than when initially measured (mean decrease ± the standard deviation, 69.85% ± 33.15%). This observation agrees with the work of Behets et al. (1), who found in Zaire that HIV-1 antibody concentrations in S&S 903 DBS were stable for 6 weeks but declined steadily thereafter. Possible causes for the protein decrease are currently being investigated.


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  		TABLE 1. Effect of time on the stability of p24 Ag protein in DBS

In a recent study undertaken in the Democratic Republic of the Congo, de Baets and coworkers found the ultrasensitive p24 Ag assay to be 100% sensitive and specific using a known volume of plasma dried on S&S 903 paper (5). Our results, using 6-mm disks punched from an unknown volume of whole blood dried on Whatman no. 1 filter paper show that blood collection and processing can be further simplified with little loss of sensitivity or specificity, with the proviso that the assay is performed within a 6-week window.

For reasons of expense and technical complexity most vertically exposed babies in developing countries do not have access to an HIV diagnosis until at least 12 months of age, when as few as 15% present for testing (6, 19). We report here a simple, inexpensive method for accurate testing for HIV-1 infection in infants using ordinary, easily obtained equipment.


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ACKNOWLEDGMENTS
 
We are grateful to the patients and staff that participated in this study. We thank Jörg Schüpbach (Swiss National Center for Retroviruses, Zurich, Switzerland) for support, advice, and the kind donation of the p24 Ag kits used in this study.

This study was supported by grants from the Elizabeth Glaser Pediatric AIDS Foundation, the Bristol-Myers Squibb Secure the Future Initiative, the National Health Laboratory Services, and the NIH CIPRA Project program.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department Molecular Medicine and Hematology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Rd., Parktown 2193, Johannesburg, South Africa. Phone: 27-11-4898496. Fax: 27-11-4845812. E-mail: janet.patton{at}nhls.ac.za. Back


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Clinical and Vaccine Immunology, January 2006, p. 152-155, Vol. 13, No. 1
1071-412X/06/$08.00+0     doi:10.1128/CVI.13.1.152-155.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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