Recent studies have shown that transmission of human immunodeficiency virus (HIV) infection from infected but screened HIV antibody sero-negative individuals have continued to be a public challenge. This unprecedented finding may be due to the long pre- seroconvertion / window period or other predisposing factors like resource poor settings especially where there are no availability of ideal HIV p24 antigen /antibody fourth generation panels or biomarkers during early phase of this infection. The current study investigated HIV p24 core antigen status of healthy Long Distant Truck Drivers (LDTDs) of commercial and public vehicles, who were previously screened and documented as HIV antibody- sero-negative males with ages above 20 years in Calabar Municipality, Cross River State, Nigeria. About five milliliters of blood samples were collected from precounseled and consented 400 apparently healthy married and unmarried male participants who were initially screened for HIV 1 and 2 antibodies using DetermineTM HIV-1 and 2 (Inverness Medical Japan company limited ), Stat-Pak HIV-1 and 2 (Chembio Diagnostic System International Inco-operation ,United State of America) and HIV Uni-gold rapid test kit (Trinity Biotech, United State of American ) respectively and repeated for HIV p24 antigens screening using Alere TM HIV-1 and 2 p24 Ag/ Ab Combo test kit-tool ( Inverness Medical Japan company limited, United State America).After data analysis using IBM-SPSS version 26, about 12 (3%), 10 (2.5%) and 9 (2.25%) of participants were reactive to HIV 1 and 2 Determine, Stat-Pak and Unigold test panels respectively, with statistical significant difference between the results according to marital status (p=0.7065) and mean age range in years .While some of the 15 (3.75%), 12 (3.0%) and 10 (2.5%) participants who reacted to HIV 1 and 2 antibodies panels including the 388, 390 and 391 participants respectively who initially tested non-reactive for the three HIV antibody kits become reactive to HIV p24 core antigens with no statistically significant difference between the results of the three HIV 1& 2 antibody test kits and HIV p24 antigen/antibody tests (P=0.901).In the current study, the use of fourth generation AlereTM HIV P24 Core Antigen rapid test kit tool had not only improved detection of HIV infection in the index population but had also demonstrated the discrepancies, limitations and short-coming associated with the routine antibody screening testing panels when done alone in this population at early stage of HIV infection and long HIV window period.