False Positivity in Anti-HIV Tests: Causes and Determination of a Reliable Cut-Off Value

Background Currently, fourth-generation assays detecting HIV-1/2 antibodies and p24 antigen are widely used as screening tools for human immunodeficiency virus (HIV). However, false-positive (FP) results remain a significant issue additional confirmatory testing. This study aimed to identify factors associated with false positivity and to determine an optimal cut-off index (COI) value with high sensitivity and specificity by evaluating the relationship between COI values in screening tests and confirmed HIV infection. Methods Between January 2024 and January 2025, a total of 36,046 patients were screened for anti-HIV. The serum samples were tested for anti-HIV using a chemiluminescence immunoassay (Cobas E601, Roche Diagnostics, Germany) with the Elecsys® HIV combi PT kit (Roche Diagnostics, Germany). Reactive samples were referred to a reference laboratory for confirmation testing. The optimal COI value for predicting true HIV infection was determined using receiver operating characteristic (ROC) curve analysis. FP and non-reactive patients were evaluated for various parameters to identify factors contributing to false positivity. Results A total of 36,046 patients were screened for anti-HIV and 122 patients (0.33%) were found to be reactive by ELISA testing. 24 were confirmed as positive and 98 as negative, yielding a false positive (FP) rate of 80.32%. False positivity was found to be positively and moderately associated with having a concurrent infection or receiving a vaccination within the past six months (p<0.05). The median and interquartile range (IQR) of COI values in (FP) cases were 1.75 and 1.66, respectively, while in true-positive (TP) cases, the median and IQR were 458.9 and 384.93, respectively. This difference was statistically significant (p<0.05). The optimal COI value to differentiate TP from FP cases was determined to be 72.085. Conclusion Identifying potential factors that contribute to false positivity in individuals with reactive screening results, along with consideration of COI values before confirmatory testing, may facilitate more accurate clinical decision-making and improve patient counseling during the interim period before definitive results are available.