Diagnosis of Human Immunodeficiency Virus Type 1 Infection in Infants by Use of Dried Blood Spots and an Ultrasensitive p24 Antigen Assay

Abstract

We tested 617 dried blood spots (DBS) from human immunodeficiency virus-exposed infants from five countries using an ultrasensitive p24 antigen assay (Up24). The sensitivity was 94.4% (67/71) and the specificity was 100% (431/431) for infants with DBS specimens <20 months old; DBS older than 30 months demonstrated only 72.2% sensitivity (39/54) (P < 0.001) but displayed 100% specificity (61/61).Several factors reduce diagnosis of human immunodeficiency virus (HIV) in infancy in resource-limited settings. Simple, rapid antibody assays cannot be used because of the presence of maternal antibodies. As a consequence, viral nucleic acids or proteins must be detected. Nucleic acid assays are technologically complex and subject to contamination and require expensive equipment and highly trained technologists, limiting availability to selected centers of excellence in resource-limited countries (1,8,12,15,28,30).The ultrasensitive p24 antigen assay (Up24), an alternative that overcomes the limitations of antibody assays while using an enzyme-linked immunosorbent assay (ELISA)-based platform, reduces the need for expensive equipment and personnel. It has been used successfully with plasma for over 12 years for diagnosis of HIV infection in infants (9,11,13,18,19,26,29,32).Another factor limiting diagnosis of HIV infection in infants is the need for phlebotomy to collect specimens and cold chain maintenance while the specimen is transported to a central laboratory. These have been addressed by adaptation of nucleic acid (2-7, 10, 16, 20, 21, 27, 31), and now Up24 antigen assays, for use with dried blood spots (DBS) (14,22,23).Other investigators have adapted the Up24 assay for use with DBS. Table 1 compares these different extraction methods used for p24 antigen assays with DBS. Li et al. (17), following the manufacturer's instructions for plasma specimens, reported 0% specificity using DBS. Knuchel et al. (14) improved the sensitivity by modifying the elution buffer (25) and increased specificity to 100% by adding a quenching step using hydrogen peroxide after the first wash. Patton et al. (22,23) improved elution by using the previously described buffer (25) and 0.5% Triton X-100. The reported sensitivity and specificity were 98.3 to 98.8% and 100%, respectively. Our objectives were to develop and optimize a simpler method for HIV infant diagnosis using DBS and reagents found in commercially available Up24 test kits as much as possible and to assess the optimized assay performance.

Low-cost tools for diagnosing and monitoring HIV infection in low-resource settings

“…These findings are promising because dried blood spot analysis is common for infant diagnosis in rural areas. 37 Compared with standard or reverse transcription PCR, p24 immunoassays are cheaper (up to US$ 10 in Africa) and less demanding of resources, which makes them a worthwhile option in clinics with limited access to more expensive tests. 11 However, p24 immunoassays are not widely used owing to a lack of validation data.…”

Novel application of Locked Nucleic Acid chemistry for a Taqman assay for measuring diverse human immunodeficiency virus type 1 subtypes

“…Furthermore, Gottlieb (Gottlieb et al, 2002) and Kostrikis (Kostrikis et al, 2002) have shown that HIV DNA levels independently predict disease progression. Together, these data suggest that HIV-1 DNA (and/or HIV-1 RNA) measurements from DBS, already shown to be useful for diagnosis and drug resistance testing (Buckton et al, 2008; Cachafeiro et al, 2009; Ikomey et al, 2009; Kerr et al, 2009), should be further explored as a marker of disease progression- risk in situations where plasma VL assays are impractical because of cost and resource limitations.…”

Analysis of the optimal cut-point for HIV-p24 antigen testing to diagnose HIV infection in HIV-exposed children from resource-constrained settings

“…Since age of testing and duration of storage of DBS may also affect the performance characteristics of Up24 antigen detection in DBS9,13, we examined, using a logistic regression model with 8-fold NCSD as the cut-point, their effect on the sensitivity and specificity of Up24 antigen assay. No significant difference in the median age (p = 0.87) or duration of storage (p=0.246) between the samples with negative and positive PCR results were found.…”

“…Frequent HIV-testing of infants with ongoing exposure to HIV through breast-feeding is also deterred. Evaluation of ultrasensitive p24 (Up24) antigen assays on liquid or dried plasma2–8 or blood spots9–13, that is more feasible for RCS, yielded sensitivities of 84–98% and specificities of 98–100%. More recently, Mwapasa et.…”