Molecular epidemiology of Plasmodium species prevalent in Yemen based on 18 s rRNA

Abstract

Background: Malaria is an endemic disease in Yemen and is responsible for 4.9 deaths per 100,000 population per year and 43,000 disability adjusted life years lost. Although malaria in Yemen is caused mainly by Plasmodium falciparum and Plasmodium vivax, there are no sequence data available on the two species. This study was conducted to investigate the distribution of the Plasmodium species based on the molecular detection and to study the molecular phylogeny of these parasites.

Methods: Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA) gene was amplified using nested PCR. From the 86 positive blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods.

Results: Malaria was detected by PCR in 86 samples (16.8%). The majority of the single infections were caused by P. falciparum (80.3%), followed by P. vivax (5.8%). Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of P. falciparum.

Conclusions: Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.

Figure 1

Neighbor-Joining (NJ) tree, constructed based on the nucleotide sequences of 18 s rRNA, displaying the relationships of 17 sequences representing 13 P. Falciparum isolates and 4 sequences representing 4 P. Vivax isolates. Bootstrap support of more than 80% is indicated. Bold-type represents reference sequences for Plasmodium species from GenBank.

Figure 2

Neighbor-Joining (NJ) tree, constructed based on the nucleotide sequences representing the different types of 18 s rRNA (A, O or S type) from GeneBank and 13 sequences representing 13 P. falciparum isolates and 4 sequences representing 4 P. Vivax isolates from this study. Bootstrap support of more than 90% is indicated. Bold-type represents reference sequences for Plasmodium species from GenBank.