Comparison of Two Human Immunodeficiency Virus (HIV) RNA Surrogate Assays to the Standard HIV RNA Assay

Abstract

Human immunodeficiency virus (HIV) RNA testing is the gold standard for monitoring antiretroviral therapy in HIV-infected patients. However, equipment and reagent costs preclude widespread use of the assay in resource-limited settings. The Perkin-Elmer Ultrasensitive p24 assay and the Cavidi Exavir Load assay both offer potentially simpler, less costly technologies for monitoring viral load. These assays were compared to the Roche Amplicor HIV-1 Monitor Test, v1.5, using panels of clinical samples (subtype B) from HIV-positive subjects and HIV-spiked samples (subtypes A, C, D, CRF_01AE, CRF_02AG, and F). The Ultrasensitive p24 assay detected 100% of the spiked samples with virus loads of >250,000copies/ml and 61% of the clinical samples with virus loads of 219 to 288,850 copies/ml. Detection rates were improved substantially if an external lysis buffer was added to the procedure. The Cavidi assay detected 54 to 100% of spiked samples with virus loads >10,000 copies/ml and 68% of the clinical samples. These detection rates were also greatly improved with a newly implemented version of this kit. Coefficients of variation demonstrate good reproducibility for each of these kits. The results from the Cavidi v1.0, Cavidi v2.0, and Perkin-Elmer, and the Perkin-Elmer Plus external buffers all correlated well with the results from the Roche Monitor Test (r ‫؍‬ 0.83 to 0.96, r ‫؍‬ 0.84 to 0.99, r ‫؍‬ 0.58 to 0.67, and r ‫؍‬ 0.59 to 0.95, respectively). Thus, the use of these two assays for monitoring patients, together with less-frequent confirmation testing, offers a feasible alternative to frequent HIV RNA testing in resource-limited settings.The human immunodeficiency virus (HIV) pandemic has affected countries worldwide, but the impact on resource-limited countries has been especially devastating. Pressure to lower the cost of antiretroviral therapies (ART) has been critical in fighting this battle. The current challenge is to identify simplified assays for monitoring patients on ART that are less expensive and less technically demanding with respect to facilities and instrumentation (6, 12). In the past, the compromise for using simplified methods has often been reduced sensitivity or poor correlation with the gold standards used in industrialized settings (4, 10). We compare here two commercially available kits that measure HIV-specific proteins, p24 and reverse transcriptase (RT), respectively, and utilize simpler technologies to perform the tests. The first method is the Ultrasensitive HIV p24 enzyme-linked immunosorbent assay (ELISA; HIV-1 p24 ELISA plus the ELAST ELISA Amplification System; Perkin-Elmer Life Sciences, Inc.), and the second is the RT assay (Exavir Load Assay; Cavidi Tech AB, Sweden). Both kits offer less expensive alternatives for detecting HIV.For the Ultrasensitive p24 assay, a standard ELISA format is used for the capture and detection of HIV p24 coupled with a specific signal amplification to increase the assay sensitivity. Heat denaturation of the plasma prior to binding in the ELISA step he...

Establishment of In-House Quantitative Real-Time RT-PCR Assay for HIV-1 Viral Load Measurement: Application to Evaluate Efficacy of ART in Ghanaian Patients in an Urban Setting

“…CRF02_AG is the major driver of HIV infection in Ghana and others in minor proportion of circulation such as CRF06_cpx, sub-subtype A3, CRF09_cpx and subtypes A, G and D had been identified. Studies have shown that RT activity is independent of the HIV-subtype [40][41][42][43][44][45]. This is in agreement with other studies that evaluated an in-house real-time RT-PCR assay that targeted the same Long Terminal Repeat (LTR) region probing for viral RNA.…”

Detection of HIV-1 P24 Gag in Plasma by a Nanoparticle-Based Bio-Barcode-Amplification Method

“…The results of this protein-detection method were highly reproducible, regardless of the approach used to quantify the barcode DNA. Conventional enzyme immunoassays that are accepted and comparable clinically for detecting the same target have sensitivity limits of approximately 5 pg/ml (~0.2 pM), an order of magnitude less sensitive [9,29].…”

“…The model assumes that all the HIV-1 p24 Gag protein in peripheral blood is in intact virions and a mature virus core contains 1000-1500 p24 Gag proteins [9,35]. The accuracy of the ratio of p24 Gag protein to viral genomic RNA is limited by the inaccuracy in the empirical determination of the amount of HIV-1 p24 Gag sufficient for mature-virus particle assembly and is confounded by the presence of HIV-1 p24 Gag in defective or immature virus particles and residual circulating immune complexes, degraded virus particles entangled in the follicular dendritic-cell network in lymphoid tissue and HIV-1 p24 Gag released from productively infected cells or leaked from cells killed by viral or immune-mediated cytotoxicity.…”

“…Commonly used methods rely on the detection of antibodies to HIV-1 in plasma or sera [2,[7][8][9]. Unfortunately, these techniques are of little value in the earliest stages of infection before the development of antibodies against HIV-1 (average infectious-window period of 25 days; 95% confidence interval: 9-41 days) [10][11][12][13][14] and also in neonates because maternal antibodies can persist in the newborn for up to 12 months or more [5,[15][16][17].…”

HIV Diagnostics: Challenges and Opportunities

“…The general consensus from the meeting was that assays should include simple specimen collection methods, use nonrefrigerated reagents, cost under $8 per data point, consist of benchtop or handheld instrumentation priced under US$1000 that run on rechargeable batteries, require minimal training and generate results in less than 2 h [1]. There have been assays adapted for developing countries [105,106], but they currently fall short of these recommendations. Whereas NAT will likely remain limited to clinical and laboratory settings for the time being [107], CD4 + T -cell counting methods using flow cytometry have become simpler, easier to use, less expensive and more accessible in RLS [108,109].…”