Semi-nested, multiplex polymerase chain reaction for detection of human malaria parasites and evidence of Plasmodium vivax infection in equatorial guinea

A semi-nested, multiplex polymerase chain reaction (PCR) based on the ampli fication of the sequences of the 18S small subunit ribosomal RNA (ssrRNA) g ene was tested in a field trial in Equatorial Guinea (a hyperendemic focus of malaria in west central Africa). The method uses a primary PCR amplifica tion reaction with a universal reverse primer and two forward primers speci fic for the genus Plasmodium and to mammals (the mammalian-specific primer was included as a positive control to distinguish uninfected cases from inh ibition of the PCR). The second amplification is carried out with the same Plasmodium genus-specific forward primer and four specific reverse primers for each human Plasmodium species. The PCR amplified products are different iated by fragment size after electrophoresis on a 2% agarose gel. Four vill ages from three regions of the island of Bioko (Equatorial Guinea) and two suspected Plasmodium vivax-P, ovale infections from time hospital of Malabo were tested by microscopy and PCR. The PCR method showed greater sensitivi ty and specificity than microscopic examination and confirmed the existence of a focus of P. vivax infections in Equatorial Guinea suspected by micros copic examination. It also provided evidence of several mixed infections, m ainly P. falciparum and P. malariae, the two predominant species causing ma laria in Equatorial Guinea.