Background: Dried blood spots (DBS) have been shown to work well in HIV RNA and DNA assays for the diagnosis of infant infection. However, nucleic acid assays are typically expensive, technically challenging, and prone to contamination. We optimized the ultra-sensitive p24 antigen (Up24 Ag) enzyme-linked immunosorbent assay (ELISA) -based assay to work with DBS.

Methods: DBS were collected on Whatman 903 paper, air dried, stored with a desiccant in individual zip-lock bags at room temperature or at 4^oC (Vietnam), and shipped to the University of North Carolina laboratory. The final optimized procedure used a modified elution buffer using TritonX100 and Tris found in the Perkin Elmer Up24 Ag kit and an enhanced specimen diluent. After adding the enhanced diluent and Triton/Tris/PBS buffer to 2 DBS punches (6-mm), the specimens were rocked at room temperature for 2 hours or overnight at 4C. The manufacturer's package insert was followed thereafter, except that positive and negative controls also consisted of DBS. Results from the Up24 antigen assay were compared with infant diagnoses by DNA PCR for specimens from the United States, Vietnam, and South Africa, or with HIV RNA results for specimens from Malawi and the Dominican Republic.

Results:  A total of 411 DBS from HIV-exposed infants were tested:  227 presumed subtype B from the United States (167) and Dominican Republic (60), 109 presumed subtype C from Malawi (9) and South Africa (100), and 75 presumed subtype A/E from Vietnam. Overall, sensitivity and specificity were 86.7 % and 100%, respectively. The 10 false-negative specimens were from infants receiving antiretrovirals for prophylaxis (n = 3) or treatment (n = 7). When these were excluded from the analysis, the sensitivity and negative predictive value (NPV) increased to 100%. DBS stored at room temperature for as long as 18 months still gave reliable results.

Conclusions:  The Up24 Ag assay was modified for use with DBS for the diagnosis of vertical HIV infection. DBS stored as long as 18 months gave reliable results and subtypes B, C, and A/E were consistently detected. Infected infants who are receiving antiretrovirals for prophylaxis or treatment, however, may not always be detected in this assay.