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To determine to what extent HIV-1 group O strains are present in different African countries.A total of 14 682 samples of sera from a range of patients from 12 different African countries were tested. All the sera were tested with an enzyme-linked immunosorbent assay (ELISA) using a combination of V3 peptides from ANT-70 and MVP-5180. Samples reactive in ELISA were retested in a line immunoassay (LIA-O). Samples reactive in ELISA were also retested with an in-house Western blot to determine the presence of antibodies to gp120 of HIV-1 ANT-70. Polymerase chain reaction was performed on HIV-1 group O and group O indeterminate sera.Of all the sera samples tested, only 19 sera had antibodies to group O V3 peptides exclusively and 46 were indeterminate for group O infection in LIA-O. The highest prevalence of HIV-1 group O infection among HIV-positive sera was observed in Cameroon (2.1%) and neighbouring countries, 1.1% in Nigeria and 0.9% in Gabon. The lowest rates were seen in west Africa: 0.07% in Senegal, 0.14% in Togo, 0.16% in Chad and 0.3% in Niger. Group O sera were observed in almost all the population categories tested. The ANT-70 V3 peptide in LIA-O was reactive with all of the sera considered to be HIV-1 group O antibody positive by LIA, versus 78.9% for the MVP-5180 peptide. Thirteen out of 19 group O samples of sera were tested in PCR. Eight samples were identified as group O by specific group O pol and/or V3 primers; in the remaining five samples no HIV RNA could be detected. Of the indeterminate sera samples, two were identified as group O.In eight of the 12 countries tested, antibodies to group O viruses were identified. Numbers of HIV-1 group O viruses are low. Their presence is not restricted to Cameroon and neighbouring countries but can also be found in west and south-east Africa.