Flow-Cytometry Intracellular Detection and Quantification of HIV1 p24 Antigen and Immunocheckpoint Molecules in T Cells among HIV/AIDS Patients.

Abstract

Introduction: HIV p24 antigen-positive T cells measured by flow cytometry (FCM) correlate directly with HIV viral load, inversely with CD₄ ⁺ T cells, and decrease with antiretroviral therapy (ART). However, the sensitivity of FCM assays depends on the protocol of intracellular staining. Therefore, this study aimed to evaluate the diagnostic performance of our FCM protocol for detection of HIV p24-positive T cells and measure the level of immunocheckpoint molecules (PD1 and TIM3) in T cells.

Methods: The study was conducted at the University of Leipzig hospital between January 2020 and November 2020. Viremic and ART-suppressed HIV-positive patients and negative controls were included in this study. HIV1 p24 KC57-, p24 28B7-, PD1-, and TIM3-positive CD₄ and CD₃ T cells were analyzed from whole blood using a BD FACS Canto II flow cytometer equipped with FACSDiva software. HIV1 p24 antigen FCM results were compared with HIV1 RNA viral load results measured by Alinity M assays on the fully automated random-access platform. We analyzed the data using SPSS 20.

Results: The absolute CD₄ ⁺ and CD₄ ⁺:CD₈ ⁺ T-cells ratio showed a significant inverse correlation with HIV1 viral load. Moreover, the absolute CD4⁺ T-cells count showed a significant inverse correlation with p24 KC57-positive CD₄ T cells. The percentage of p24 KC57, p24 28B7, and double-positive CD₄ T cells showed significant correlation with HIV1 viral load. PD1 expressing CD₄ T cells were higher in ART-viremic cases than controls, while TIM3-expressing CD₄ T cells were lower in ART-viremic cases than controls. Sensitivity, specificity, PPV, and NPV of p24 KC57-positive CD₄ T cells were 64%, 82%, 78%, and 69%, respectively, for the diagnosis of HIV infection and 55%, 73%, 40%, and 83%, respectively, for treatment monitoring.

Conclusion: Our protocol showed moderate performance for the diagnosis of HIV infection and treatment monitoring. Therefore, the p24 KC57 but not the p24 28B7 clone could be considered as a simple alternative method for rapid diagnosis of HIV infections and treatment monitoring, particularly in low- and middle-income countries.