Presence of additional P. vivax malaria in Duffy negative individuals from Southwestern Nigeria: Short Report

Background Malaria in sub-Saharan Africa (sSA) is thought to be hugely caused by Plasmodium falciparum . Recently, growing reports of cases due to P. ovale , P. malariae , and P. vivax have been significantly reported to play a role in malaria epidemiology in sSA. This in fact is due to the usage of very sensitive diagnostic tools (e.g. PCR) which have highlighted the underestimation of non-falciparum malaria in this sub-region. P. vivax was historically thought to be absent in sSA due to the high prevalence of the Duffy null antigen in individuals residing in this sub-continent. For example, recent studies reporting the detection of vivax malaria in Duffy-negative individuals from Mali, Mauritania, Cameroon to mention a few challenges this notion. Methods Following our earlier report of P. vivax in Duffy-negative individuals, we have collected and assessed RDT and/or microscope malaria positive samples following the conventional PCR method and DNA sequencing to confirm both single/mixed infections as well as the Duffy status of the individuals. Results Amplification of Plasmodium gDNA was possible in 59.9% (145/242) of the evaluated isolates and as expected P. falciparum was the most predominant (91.7%) species identified. Interestingly, four P. vivax isolates were identified either as single (3) or mixed (1 – P. falciparum / P. vivax ) infection. Sequencing results confirmed, all vivax isolates as truly vivax malaria and their Duffy status to be that of the Duffy-negative genotype. Conclusion Identification of more vivax isolates among these Duffy-negative individuals from Nigeria, substantiate the expanding body of evidence on the ability of P. vivax to infect RBCs that do not express the DARC gene. Hence, such genetic-epidemiological study should be conducted at the national level in order to evaluate the actual burden of P. vivax in the country.


Abstract
Background Malaria in sub-Saharan Africa (sSA) is thought to be hugely caused by Plasmodium falciparum . Recently, growing reports of cases due to P. ovale , P. malariae , and P. vivax have been significantly reported to play a role in malaria epidemiology in sSA. This in fact is due to the usage of very sensitive diagnostic tools (e.g. PCR) which have highlighted the underestimation of non-falciparum malaria in this sub-region. P.
vivax was historically thought to be absent in sSA due to the high prevalence of the Duffy null antigen in individuals residing in this sub-continent. For example, recent studies reporting the detection of vivax malaria in Duffy-negative individuals from Mali, Mauritania, Cameroon to mention a few challenges this notion.
Methods Following our earlier report of P. vivax in Duffy-negative individuals, we have collected and assessed RDT and/or microscope malaria positive samples following the conventional PCR method and DNA sequencing to confirm both single/mixed infections as well as the Duffy status of the individuals.
Results Amplification of Plasmodium gDNA was possible in 59.9% (145/242) of the evaluated isolates and as expected P. falciparum was the most predominant (91.7%) species identified. Interestingly, four P. vivax isolates were identified either as single (3) or mixed (1 -P. falciparum / P. vivax ) infection. Sequencing results confirmed, all vivax isolates as truly vivax malaria and their Duffy status to be that of the Duffy-negative genotype.
Conclusion Identification of more vivax isolates among these Duffy-negative individuals from Nigeria, substantiate the expanding body of evidence on the ability of P. vivax to infect RBCs that do not express the DARC gene. Hence, such genetic-epidemiological study should be conducted at the national level in order to evaluate the actual burden of P. vivax in the country.

Background
Malaria is a critical infectious disease of public health importance that provokes considerable mortality in all endemic countries. The tremendous gains seen in cases and mortality reduction is as a result of deliberate intervention strategies [1]. However, the observed benefit has seen a plateau in the last two years especially in Africa where the greatest burden of disease is mostly impacted. In sub-Saharan Africa (sSA), majority (99%) of the infections is thought to be due to Plasmodium falciparum and rarely by P. ovale, P. malariae, with P. vivax not even being considered in the picture as one of the players [1] of malaria infection. With the availability of tools that are more sensitive, the detection of non-falciparum and even vivax human malaria parasites has gained more attention in sSA [2][3][4][5][6].
Hence, the former notion is that, P. vivax originates from Asia and South America and gradually finds its way into Africa through the trade-route corridor. However, there are some current evidences supporting the hypothesis that, P. vivax could have originally evolved from a vivax-like strain detected in non-human primates in Africa [21,22] and from there, dispersed to other continents during the period of human migration. Although, both hypotheses (whether from Africa to Asia or, Asia to Africa) require further validation.
However, it seems likely that there might be an interplay of both hypothesis, in which case, simultaneous occurrence takes place and selective adaptation of the Duffy negative allele in sSA might have resulted in the absence of vivax malaria in the region.
Nonetheless, later re-introduced when individuals expressing the Duffy null allele travels between continents and countries.
The Duffy (gp-FY; CD234) gene is the fourth red blood cell (RBC) gene after thalassemia, sickle cell anaemia and glucose-6-phosphate dehydrogenase (G6PD) associated with resistance to Plasmodium species [23] with particular protection against vivax malaria.
Also known as the Duffy antigen receptor for chemokines (DARC), it is a variable receptor usually expressed on the surface of the red blood cell (RBC) and employed by P. vivax merozoites in gaining access in the RBCs and establishing its erythrocytic infection [24].
The DARC, located on chromosome 1 has two exons, and a single nucleotide substitution from a thymine (T) to a cytosine (C), upstream in the promoter region nullifies the expression of this gene on the RBCs, resulting in the FYO* allele [25]. This FYO* null allele predominates amongst sSA inhabitants as with African-Americans but has a very sparse representation in individuals of other ancestry [26]. Thus, the FYO* null allele has been validated to confer protection against P. vivax infection in this sub-region. Nevertheless, 11 countries in this region (Oboh et al unpublished data) have reported the occurrence of P. vivax, making it more real that vivax malaria might be gradually finding its way into sSA, thus it can be postulated that hidden transmission is occurring in this region. In some of these studies, such as in those conducted in Angola, Cameroon, Kenya, Madagascar, Mali and Mauritania, the Duffy status of the infected individuals was characterized and they were found to be mostly Duffy negative [3,4,[27][28][29][30]. In others, however, the investigators were concerned with the identification of P. vivax without stating the Duffy status of the infected individuals [5, [31][32][33]. Interestingly, all studies were carried out amongst indigenous individuals with little or no travel history to vivax endemic areas, thus ruling out the possibility of imported infection.
In Nigeria, P. falciparum is responsible for > 95% of malaria infection, with P. malariae and P. ovale contributing a meagre < 5% of infection [1,34]. Data implicating P. vivax infection in Nigeria includes its detection in a visiting pregnant female [35], two cases detected by microscopy [36,37], both of which were not confirmed by any molecular technique and our previous study [6] which detected five Duffy negative individuals to be infected with P. vivax isolates and were subsequently confirmed by capillary sequencing.
Thus, as a follow-up to our previous study, we have collected samples from two sites -Oredo and Kosofe in Edo and Lagos state respectively and used the classical PCR method, Employing the Qiagen®kit (the QIAamp DNA Blood Mini Kit; Hilden, Germany), genomic DNA was isolated from all 242 samples and subsequently subjected to nested PCR diagnostic protocol targeting the 18S rRNA to identify all four Plasmodium infecting species using primer pairs as designed earlier [38]. For each PCR run, a negative control (nuclease free water) and positive controls (sequenced confirmed Plasmodium species-for all four species) were added. In addition, a part of the promoter region of Duffy gene (for isolates that are P. vivax positive) was PCR amplified and sequenced in order to determine their Duffy status using protocols and primer sequence detailed in our previous work [6].
Representative isolates of Plasmodium species (P. falciparum, P. vivax, P. malariae and P.

Results And Discussions
Between December 2016 and January 2017, we were able to collect a total of two hundred and forty-two samples from both study areas, with majority of the samples (171) being from Oredo in Edo State. The mean age group from both localities are almost the same, 25 years in Kosofe and 26 years in Oredo. As with the mean age, the ratio of male to female is almost same (1:1.2) Table 1.  ovale /falciparum -6, P. falciparum/P. vivax -1) infections Fig. 1 The gDNA of the identified P. vivax isolates were re-extracted and amplified twice following our earlier [6] protocol in order to be sure of their status. Gel documentation of all newly identified isolates is presented in Fig. 2.
Authentication of the PCR results were followed by sequencing (in the forward and reverse direction) the 18S rRNA of the identified P. vivax and representative of other species.
Sequences were edited and aligned (using BioEdit) with their respective reference strain: P. vivax with the Sal-1 strain (accession number U03079.1), P. falciparum with the 3D7 strain (accession umber XR_002273095.1), P. ovale (accession number L48987.1) and P. malariae (accession number NG_011626. 30). Surprisingly, all sequences showed perfect homology (100% similarity) with their references as expected (Fig. 3-for P. vivax). This is not unexpected as cases of P. vivax have been identified in many countries in sSA [3-5, 29, 33, 39-42] including Nigeria [6], where it was thought to be absent due to the nonexpression of the DARC gene on the RBC of majority of the population.Thus, this is adding to the growing evidence of the proposed gradual incursion of P. vivax into sSA sub-region.
In order to discern the Duffy status of those P. vivax infected patients, a portion of the DARC gene (precisely the promoter region covering the T33C point mutation) was amplified and sequenced follwing previous protocol [43]. Unanticipatedly, all four additional P. vivax isolates carried a single cytosine (C) peak at the 33 rd nucleotide position upstream (Fig. 4). Thus, confirming that none of them expressed the Duffy gene on their RBCs and as such are Duffy negative.
The identification of more P. vivax isolates among these Duffy-negative individuals from Nigeria, substantiate the expanding body of evidence of the ability of P. vivax to infect RBCs that do not express the DARC gene. Although a very recent findings point to another receptor on the reticulocyte-transferrin receptor 1 as a specific P. vivax receptor [44], which is being proposed to be an alternate route of entry into the RBCs, this however is subject to further verification. The above hypothesis is one of the proposition being made to support the observation of P. vivax in sSA [45]. Another which relies on the first conjecture (assuming it is agreed that P. vivax at least possesses alternative invasion pathway), is that the Duffy positive carriers in northern part of Africa and the Afro-Asiatic populations of Sudan, Somalia [20,46] and Ethiopia [47,48] serve as reservoir to effect transmission to Duffy negative individuals in those areas as well as other countries (in sSA) through migration. Albeit, this particular hypothesis at play here is yet to be determined. One thing is clear here however, that the true epidemiological situation of P.
vivax in sSA in particular and Africa in general is yet to be ascertained.

Conclusion
It is pertinent to carry out more genetic-epidemiological studies in other areas (for example this study covers only two states out of the thirty-six in Nigeria) of the country as with other sSA countries. This will aid in putting in place appropriate control strategies to combat the menace of malaria infection in this most affected population (Africa) and also prevent further spread of P. vivax in Africa.

Consent for publication
Not applicable

Availability of data
The datasets generated in this study are included in the manuscript except the sequences which will be submitted to Genbank (……) and acession made availble.

Competing interest
The authors declare that they have no competing interests.

Funding
This work was graciously funded by the Department of Science and Technology, and