Molecular identification of Plasmodium species responsible for malaria reveals Plasmodium vivax isolates in Duffy negative individuals from southwestern Nigeria

Abstract

Background: Malaria in Nigeria is principally due to Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. Plasmodium vivax is thought to be absent in Nigeria in particular and sub-Saharan Africa in general, due to the near fixation of the Duffy negative gene in this population. Nevertheless, there are frequent reports of P. vivax infection in Duffy negative individuals in the sub-region, including reports from two countries sharing border with Nigeria to the west (Republic of Benin) and east (Cameroon). Additionally, there were two cases of microscopic vivax-like malaria from Nigerian indigenous population. Hence molecular surveillance of the circulating Plasmodium species in two states (Lagos and Edo) of southwestern Nigeria was carried out.

Methods: A cross-sectional survey between September 2016 and March 2017 was conducted. 436 febrile patients were included for the present work. Venous blood of these patients was subjected to RDT as well as microscopy. Further, parasite DNA was isolated from positive samples and PCR diagnostic was employed followed by direct sequencing of the 18S rRNA of Plasmodium species as well as sequencing of a portion of the promoter region of the Duffy antigen receptor for chemokines. Samples positive for P. vivax were re-amplified several times and finally using the High Fidelity Taq to rule out any bias introduced.

Results: Of the 256 (58.7%) amplifiable malaria parasite DNA, P. falciparum was, as expected, the major cause of infection, either alone 85.5% (219/256; 97 from Edo and 122 from Lagos), or mixed with P. malariae 6.3% (16/256) or with P. vivax 1.6% (4/256). Only one of the five P. vivax isolates was found to be a single infection. DNA sequencing and subsequent alignment of the 18S rRNA of P. vivax with the reference strains displayed very high similarities (100%). Remarkably, the T-33C was identified in all P. vivax samples, thus confirming that all vivax-infected patients in the current study are Duffy negative.

Conclusion: The present study gave the first molecular evidence of P. vivax in Nigeria in Duffy negative individuals. Though restricted to two states; Edo in South-South and Lagos in South-west Nigeria, the real burden of this species in Nigeria and sub-Saharan Africa might have been underestimated, hence there is need to put in place a country-wide, as well as a sub-Saharan Africa-wide surveillance and appropriate control measures.

Keywords: Duffy negative; Edo; Lagos; Malaria; Plasmodium vivax; Sub-Saharan Africa.

Fig. 1

Flow chart of sample processing. A total of 300 samples were positive by RDT out of which only 135 and 256 turned out positive by microscopy and PCR respectively. From the pool of 136 randomly selected negative samples, 33 more turned out positive by PCR

Fig. 2

Gel documentation of P. vivax species detected in the study area: First well: DNA base pair ladder, well 2: PC-P. vivax positive control, well 3–7: P. vivax samples

Fig. 3

Map of a Africa showing the location of Nigeria with Benin and Cameroon bordering Nigeria to the west and east respectively. b Nigeria pointing to the two states (Lagos and Edo) where study was conducted and c study sites showing the diversity of the Plasmodium species present in each site (grouped site)

Fig. 4

Multiple sequence alignment of 18S rRNA of Nigerian. Plasmodium vivax isolates with the 18S rRNA of P. vivax SAL-1 strain

Fig. 5

Sequence alignment of human Duffy gene of P. vivax patients and its accompanying chromatogram with the reference positive strain (NG_011626.3). The T-33C mutation which depicts Duffy negative is shown in the rectangle enclosing the cytosine