Study site
The study was conducted in the Kedougou region (Figure 1) located in southeast Senegal at the bordering area with Guinea, Mali and Gambia
between isohyets 1,200 and 1,300Â mm. The climate is Sudano-Guinean with a single rainy
season from May to November. The landscape consists of wooded grassland or woodland
and dense gallery forest. The fauna is diverse with herbivorous, insectivorous, rodents,
and monkeys.
Figure 1. Map of Kedougou region showing villages where the majority of samples were obtained
(blue star) and origin of the four Plasmodium vivax infections (red circle).
Malaria remains highly prevalent in Kedougou region and transmission is highly seasonal
occurring during the rainy season (May–November). In 2014, the Senegalese National
Malaria Control Programme reported 25.55% confirmed clinical malaria cases of which
2.73% turned into severe disease 16].
Population and study design
As part of a genetic diversity study of P. falciparum isolates in concurrent malaria-arbovirus infections from patients presenting with
acute febrile illness (AFI) in Kedougou, a molecular diagnostic was conducted to discriminate
P. falciparum from other Plasmodium species. This revealed a unique case of P. vivax infection in a patient from Ninefesha village. Therefore, additional samples from
Ninefesha and nearby villages were retrieved from the sera collection and screened
to survey P. vivax infections in Kedougou. A total of 263 sera from patients presenting with AFI between
July 2009 and July 2013 in the Kedougou region of Senegal were included in this study.
Sera were withdrawn from a collection established as part of a monitoring programme
of arboviruses in Kedougou region. AFI was defined as ‘any patient older than 1 year
with a fever (temperature 38°C) lasting for less than 2 weeks, exhibiting two or
more of the following symptoms: headache, myalgia, eye pain, arthralgia, cough, nausea/vomiting,
diarrhoea, jaundice, bleeding and neurological signs’.
Ethical clearance
The study objectives, benefits and risks were explained in French language or local
dialects to all participants before inclusion. Written informed consent was obtained
from all adults participants and from the parents, or legal guardians of children.
The study was examined and approved by the Senegalese National Health Research Committee.
Molecular detection of Plasmodium species
The detection of Plasmodium spp. genomic DNA (gDNA) in frozen serum samples has been reported earlier 17], 18]. Genomic DNA isolation of Plasmodium parasites was performed using QIamp DNA Mini Kit (Qiagen, Hilden, Germany) according
to manufacturer’s instructions. DNA extracted from blood samples of known microscopically
confirmed P. falciparum, P. malariae and P. ovale-infected patients were used as positive controls. P. vivax gDNA was kindly donated by Dr Ambroise Ahouidi (Le Dantec Hospital, Dakar).
Qualitative detection of Plasmodium parasite DNA was based on nested PCR with primers targeting the Plasmodium spp. 18S small sub-unit ribosomal RNA (18S ssrRNA) gene as described previously 19]. The primary PCR amplification was performed with Plasmodium genus-specific rPLU5 and rPLU6 primers pairs 20], 21] and 1.5 µl of template DNA in a total volume of 25 µl using the GoTaq Green Master
Mix protocol (Catalogue no M7113, Promega) according to manufacturer’s recommendations.
The nested reaction was performed for the specific detection of Plasmodium species using previously described primers pairs rFAL1 and rFAL2 for P. falciparum, rVIV1 and rVIV2 for P. vivax, rOVA1 and rOVA2 for P. ovale and rMAL1 and rMAL2 for P. malariae20], 21]. The genus-specific primary PCR products (1 µl) were used as the template in the
species-nested PCR amplification under the same conditions. Nested PCR results were
scored as categorical variable (presence vs. absence of amplification). PCR cycling
reaction and amplification conditions were as described by Snounou and Singh 19].
Purification of PCR products, DNA sequencing and analysis
In order to determine the sequences’ identity of the P. vivax amplified DNA, the specific bands were extracted from the gel and purified using
the QIAquick Gel Extraction Kit as described by the manufacturer (Qiagen
®
). The purified PCR products were then sent to COGENICS for sequencing. For each DNA
fragment, sequencing was performed from both the 3? and 5? directions (2× coverage).
Consensus sequences were generated from the forward and reverse sequences of each
sample and used in a BLASTn algorithm against the Genbank database for similarity
profile determination.
Sequence alignment and phylogenic trees were performed using Mega 6.06 software. Plasmodium spp. small sub-unit rRNA (SSU RNA) sequences were obtained from Plasmodb version
13.0 and Genbank. Plasmodium vivax: P. vivax Sal1_U030779.1, P. vivax_HF945443.1, P. vivax_HF945441.1; P. falciparum: JQ627152.1; P. ovale: JF894411.1; P. malariae: GU815531.1. Sequences were analysed by the Neighbour Joining method using 100 bootstrap
replicates.