Acute SARS-CoV-2 infection has been associated with false-positive HIV screening tests. The underlying mechanism is unclear, and for clinical cases, evidence beyond a temporal connection is missing. However, several experimental studies point toward SARS-CoV-2 spike/HIV-1 envelope (Env) cross-reactive antibodies (Abs) as a cause. Here, we present the first case of an individual with convalescent SARS-CoV-2 infection testing false positive in both an HIV screening and confirmatory test. Longitudinal sampling showed that the phenomenon was temporary but lasted for at least 3 months before waning. After excluding a multitude of common determinants for assay interference, we further show by antibody depletion studies that SARS-CoV-2-spike-specific Abs did not cross-react with HIV-1 gp120 in the patient sample. No additional case of HIV test interference was identified in a cohort of 66 individuals who presented to a post-COVID-19 outpatient clinic. We conclude the SARS-CoV-2-associated HIV test interference to be a temporary process capable of disturbing both screening and confirmatory assays. The assay interference is short-lived and/or rare but should be considered by physicians as a possible explanation for unexpected HIV diagnostic results in patients with a recent SARS-CoV-2 infection.

Here, we present an optimized enzyme-linked immunosorbent assay (ELISA) protocol for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike-specific immunoglobulin G (IgG) in plasma or serum samples. The protocol offers a rapid and reliable method, delivering results in less than 6 hours. On day 1, 96-well plates are coated with a concentration of 5 µg/mL of SARS-CoV-2 spike protein in 0.1 M sodium hydrogen carbonate and left to incubate overnight at 4 °C. Subsequently, the plates undergo thorough washing and are blocked with a buffer containing bovine serum albumin for one hour at room temperature. Diluted samples are added to the wells, followed by a two-hour incubation period. After another round of washing, a horseradish peroxidase-labeled antibody directed against the crystallizable fragment region of IgG is added, and the plates are incubated for one hour. The plates are then washed and developed using 3,3′,5,5′-tetramethylbenzidine-substrate for 30 minutes. The reaction is stopped by the addition of 1 N sulfuric acid. The optimized protocol demonstrated excellent performance, exhibiting a specificity of 99.5% and a sensitivity of 98.1% for the detection of SARS-CoV-2 spike-specific IgG in human plasma or serum samples.