Digital PCR Reveals High PvDBP 1 but not PvEBP/DBP 2 and PvRBP 2b Copy Number in Plasmodium vivax From Duffy-Negative Individuals in Central Africa

Abstract

BACKGROUND: Vivax malaria, once thought rare in Duffy-negative Africans, is now reported in various parts of Africa, suggesting alternate invasion mechanisms and parasite adaptability to Duffy-null (DN) cells. One hypothesis is that copy number variation (CNV) of genes involved in erythrocyte invasion may impact parasite invasion capability and/or host immune evasion, particularly in Duffy-negative individuals. METHOD: Using novel digital polymerase chain reaction (PCR), we assessed CNV of 3 key erythrocyte-binding genes of Plasmodium vivax isolated from DN individuals in 3 ecoepidemiological zones of Cameroon. For a subset of samples, we compare digital PCR (dPCR) results with quantitative PCR (qPCR) and PCR diagnostic approaches. RESULTS: PvDBP1 duplications were detected in approximately 92% of DN P. vivax samples, compared to approximately 10% of the samples with multicopy PvEBP/DBP2 and PvRBP2b. A significant positive correlation was detected between PvDBP1 CNV and parasite load among the samples. Both Malagasy- and Cambodian-type PvDBP1 duplications were detected. About one-third of the samples harbored both duplication types and these samples were exclusively in northern highlands of Cameroon, suggesting either polyclonal infections or a third duplication type. CONCLUSIONS: Multicopy PvDBP1 across all study sites may imply significant parasite adaptability and improved parasite invasion, potentially influencing parasitemia. This was confirmed by the significantly higher parasitemia observed in samples with Cambodian type and those with both duplication types compared to ones with no duplication. Furthermore, our data showed CNV analysis by qPCR may not be as precise as dPCR, particularly for low-parasitemia DN P. vivax samples. Predominantly low PvEBP/DBP2 and PvRBP2b CNV raises questions to their role in parasite adaptation to invading DN erythrocytes.