Molecular-based assay for simultaneous detection of four Plasmodium spp. and Wuchereria bancrofti infections

Abstract

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)-based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction-fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85-93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.

Figure 1.

Detection of species-specific DNAs by the five-species multiplex polymerase chain reaction oligonucleotide ligation detection reaction–fluorescent microsphere assay (LDR-FMA). Data represent a summary of five positive-control experiments detecting Plasmodium falciparum, P. vivax, P. malariae, P. ovale, and Wuchereria bancrofti genomic DNAs. Whereas genomic DNAs were added individually, LDR-FMA reactions included oligonucleotide primers and microspheres representing all five species. Numbers in parentheses next to parasite-species designations in the legend ((78), (37), (30), (14), (3)) identify FlexMap™ microspheres (Luminex Corp., Austin, TX). B identifies two blank samples to which no genomic DNAs were added.