There is need for more evaluations of non-invasive tests in order to broaden the reach of testing programs and to perform large scale epidemiological studies. In this study, three different human immunodeficiency virus (HIV) enzyme linked immunosorbent assays (ELISAs) and one line immunoassay were evaluated to detect HIV antibodies in oral fluid samples. Specimens were collected, after informed consent was obtained, with the Oracol (MMD, Worcester, England) device. A total IgG quantitation test was performed to demonstrate the quality of the sample. Assessment of a modified protocol of the Vironostika(R) HIV Ag/Ab, Enzygnost(R) Anti-HIV 1/2 Plus Genscreen HIV-1/2 Version 2 and a line immune confirmatory assay the INNO-LIA HIV I/II score was done, using oral fluid specimens of 325 HIV positive and negative individuals. For the ELISAs, the addition of an extra internal oral fluid control was evaluated as well as different cut-offs, time between sampling and testing and the effect of drinking water just before sampling. Finally, the confirmatory test and some testing algorithms and combination of tests were discussed. The results obtained from the oral fluid specimens were compared with the gold standard on paired serum specimens. Firstly, there was no significant difference observed between the use of the kit controls and the oral fluid controls. New protocols and calculation of cut-offs were defined for two of the three ELISAs. High sensitivities and specificities were obtained with all three ELISAs without any statistical difference between the three tests. Secondly, no statistically significant difference was observed when samples were stored for different time periods between sampling and testing, meaning that a period of seven days at room temperature before testing is still acceptable. Thirdly, drinking water before sample collection did not interfere with the testing, although lower optical densities were observed. None of the positive samples were missed. In addition, the line immunoassay INNO-LIA HIV I/II score test is a promising test for confirmation of reactive oral fluid specimen, but more samples need to be validated in order to adapt the interpretation rules specifically for oral fluid specimens. Different choices/algorithms adapted for the purpose of testing can be proposed. In conclusion, it can be said that the commercial ELISAs with adapted protocol and cut-off values are suitable tools for making HIV test performance accessible to people. With this non-invasive sampling method, more eligible individuals can and will be selected for further HIV test on blood
