Molecular evidence of Plasmodium vivax mono and mixed malaria parasite infections in Duffy-negative native Cameroonians

Abstract

The malaria parasite Plasmodium vivax is known to be majorly endemic to Asian and Latin American countries with no or very few reports of Africans infected with this parasite. Since the human Duffy antigens act as receptors for P. vivax to invade human RBCs and Africans are generally Duffy-negative, non-endemicity of P. vivax in Africa has been attributed to this fact. However, recent reports describing P. vivax infections in Duffy-negative Africans from West and Central parts of Africa have been surfaced including a recent report on P. vivax infection in native Cameroonians. In order to know if Cameroonians living in the southern regions are also susceptible to P. vivax infection, we collected finger-prick blood samples from 485 malarial symptomatic patients in five locations and followed PCR diagnostic assays with DNA sequencing of the 18S ribosomal RNA gene. Out of the 201 malaria positive cases detected, 193 were pure P. falciparum, six pure P. vivax and two mixed parasite infections (P. falciparum + P. vivax). The eight P. vivax infected samples (six single + two mixed) were further subjected to DNA sequencing of the P. vivax multidrug resistance 1 (pvmdr1) and the P.vivax circumsporozoite (pvcsp) genes. Alignment of the eight Cameroonian pvmdr1 sequences with the reference sequence showed high sequence similarities, reconfirming P. vivax infection in all the eight patients. DNA sequencing of the pvcsp gene indicated all the eight P. vivax to be of VK247 type. Interestingly, DNA sequencing of a part of the human Duffy gene covering the promoter region in the eight P. vivax-infected Cameroonians to identify the T-33C mutation revealed all these patients as Duffy-negative. The results provide evidence of single P. vivax as well as mixed malaria parasite infection in native Cameroonians and add knowledge to the growing evidences of P. vivax infection in Duffy-negative Africans.

Figure 1. Map of Africa (A) highlighting Cameroon and (B) Map of Cameroon showing the sampling location sites.

Figure 2. Gel picture showing bands of P. vivax mono (lanes 2–7) and mixed infection (lanes 9 and 10).

Lanes 1, 8 and 11 were loaded with 100P. vivax and P. falciparum, respectively.

Figure 3. DNA sequence alignment of the eight Cameroonian P. vivax isolates with the reference DNA sequence of P. vivax SAL-1 strain.

Figure 4. DNA sequence alignment of Cameroonian P. falciparum isolates from the mixed infection cases with the reference DNA sequence of P. falciparum 3D7 strain.

Figure 5. DNA sequence alignment and associated chromatogram of the DNA sequence covering the promoter region of the Duffy gene in eight Cameroonians harboring the P. vivax infection.

The T-33C SNP is indicated in blue and the representative chromatogram showing a clear peak of “C” is shown below the alignment.

Figure 6. DNA sequence alignment of Cameroonian P. vivax isolates from the mixed infection cases with the reference DNA sequence of P. vivax SAL-1 isolate.

The Single Nucleotide Polymorphism (SNP) in Pv7 is indicated in red and the representative chromatogram showing a single clear peak of “C” is shown below.