The Effect of Sample Handling on Cross Sectional HIV Incidence Testing Results

Abstract

Objective(s)To determine if mishandling prior to testing would make a sample from a chronically infected subject appear recently infected when tested by cross-sectional HIV incidence assays.MethodsSerum samples from 31 subjects with chronic HIV infection were tested. Samples were subjected to different handling conditions, including incubation at 4°C, 25°C and 37°C, for 1, 3, 7 or 15 days prior to testing. Samples were also subjected to 1,3, 7 and 15 freeze-thaw cycles prior to testing. Samples were tested using the BED capture enzyme immuno assay (BED-CEIA), Vironostika-less sensitive (V-LS), and an avidity assay using the Genetic Systems HIV-1/HIV-2 plus O EIA (avidity assay).ResultsCompared to the sample that was not subjected to any mishandling conditions, for the BED-CEIA, V-LS and avidity assay, there was no significant change in test results for samples incubated at 4°C or 25°C prior to testing. No impact on test results occurred after 15 freeze-thaw cycles. A decrease in assay results was observed when samples were held for 3 days or longer at 37°C prior to testing.ConclusionsSamples can be subjected up to 15 freeze-thaw cycles without affecting the results the BED-CEIA, Vironostika-LS, or avidity assays. Storing samples at 4°C or 25°C for up to fifteen days prior to testing had no impact on test results. However, storing samples at 37°C for three or more days did affect results obtained with these assays.

How can we better identify early HIV infections?

“…The key recent advance in cross-sectional HIV incidence estimation is the development, refinement, and validation of multiassay algorithms (MAAs) [28•,98••,99•,100•,101••,102,103•,104]. MAAs apply a sequential, hierarchical set of assays to classify confirmed seropositive samples into recent versus long-standing infection categories.…”

Cross-protective vaccine efficacy of the bivalent HPV vaccine against HPV31 is associated with humoral immune responses

“…The method of assessing the avidity of the anti-HPV16 antibodies was previously reported. 19,20 We only evaluated HPV16 avidity because of the availability of particles for HPV16 for ELISA assays in our laboratory. In addition, the lower neutralization titers for the cross-related HPV types, would not allow a precise estimation for the ELISA-based avidity levels.…”

Factors Associated with Incorrect Identification of Recent HIV Infection Using the BED Capture Immunoassay

“…26 We previously demonstrated the stability of BED-CEIA results over time in a cohort of HIV-infected Ugandan women. 27 Interestingly, most men in the MACS also had stable BED-CEIA results over a period of several years (Fig. 1C) if they did not become virally suppressed (Fig.…”

“…We recently demonstrated that repeated freeze thaws or prolonged incubation at various temperatures did not impact results obtained with the BED-CEIA; therefore, use of archived samples that may have been thawed previously was not likely to have led to artificially low BED-CEIA results in this study. 27 Studies are underway to determine whether persistent misclassification is explained by a viral factor (e.g., if the gp41 sequence in HIV from participants with persistently low BED-CEIA values differs significantly from the sequence of the peptide target in the BED-CEIA assay).…”