Background and objective In the WHO Universal test and treat strategy, falsepositive HIV blood donors and patients may be unnecessarily put under antiretroviral treatment and false-negative subjects may be lost to follow-up. This study assessed the false positivity rate of the Cameroonian national HIV screening testing algorithm and the benefit of a confirmation test in the enrolment of patients and donors in the HIV care programme. Methods We included initial HIV reactive blood donors and patients in a crosssectional study conducted in two Cameroonian hospitals. Samples were retested according to the Cameroon national algorithm for HIV diagnosis. A positive or discordant sample was retested with the Geenius Bio-Rad HIV 1&2 (Bio-Rad, Marnes-la-Coquette, France) for confirmation. The Geenius HIV-1-positive results with 'poor' profiles were retested for RNA as well as the Geenius indeterminate results. Results Of the 356 participants, 190/225 (84Á4%) patients and 76/131 (58%) blood donors were declared positive with the national algorithm; 257 participants (96Á6%) were confirmed HIV-1-positive. The study revealed that about 34/1000 blood donors and patients are false-positive and unnecessarily put on treatment; 89/1000 blood donors and patients declared discordant could have been included immediately in the HIV care programme if confirmatory testing was performed. The second test of the algorithm had a false-negative rate of 3%. Eleven samples (3Á1%) were Geenius poor positive and NAT negative. Conclusion The universal test and treat strategy may identify and refer more individuals to HIV care if a third rapid confirmatory test is performed for discordant cases.