Background
Malaria surveillance has been integrated as a core intervention in all malaria-endemic countries by the World Health Organization (WHO) since 2018 [
1]. Senegal has made substantial efforts in malaria control through the National Malaria Control Programme (NMCP) using surveillance data to implement control strategies. Most detection strategies for malaria surveillance in peripheral health facilities (health posts) where most cases are diagnosed use
Plasmodium falciparum Histidine Rich Protein 2 rapid diagnosis tests (PfHRP2-RDTs), which are specific for
P. falciparum. However, the PfHRP2-RDT tests could miss low density
Plasmodium infections and non-falciparum species usually responsible for asymptomatic infection [
2]. Similarly, home-based management of malaria in high-transmission areas of the country only targets symptomatic individuals diagnosed by PfHRP2-RDTs. These interventions do not capture these types of infections in the health facilities and the communities leading to inappropriate strategies.
For elimination and eradication purposes, there is a need for accurate diagnosis of all
Plasmodium species. Although
P. falciparum is the most prevalent species in sub Saharan Africa,
Plasmodium malariae,
Plasmodium ovale (P. ovale curtisi and
P. ovale wallikeri) and recently
Plasmodium vivax have been reported [
3,
4].
Plasmodium vivax and
P. ovale have hypnozoites that could be responsible for relapses years after the first infection [
5,
6] and
P. malariae presents chronic infection with long parasite carriage. This chronicity requires complementary strategies to detect and treat these infections, thus the real burden of the non-falciparum species needs to be assessed. This could be achieved by using appropriate tools and strategies for better detection and identification of all
Plasmodium spp responsible for infection.
Using highly sensitive tools such as Next Generation Sequencing (NGS) could overcome the limit of detection of standard malaria diagnosis tools that can miss low density parasitaemia which is often the case in asymptomatic malaria infection at the community level. Sequencing allows the detection of both P. falciparum and non-falciparum species for all levels of parasitaemia and to capture additional cases that are missed by the current surveillance system. The availability of more accurate data could help reveal the true burden of malaria and inform the adjustment of interventions for malaria control, elimination, and ultimately eradication.
Here, a targeted sequencing approach was used to amplify regions of two genes, the small subunit ribosomal gene of the 18S rRNA (ssu) and the mitochondrial cytochrome B (cytb). This enabled assessment of the burden of Plasmodium species among asymptomatic and symptomatic subjects in the community and febrile individuals with a negative HRP2-based RDT in western and eastern areas in Senegal during the transmission season of 2020–2021.
Discussion
An accurate estimation of the prevalence of malaria and the causative species is needed to achieve its elimination. The aim of this study was to determine the frequency of Plasmodium species in different endemic sites in Senegal towards the end of the malaria transmission season. The study population included asymptomatic and symptomatic individuals sampled in the community in Diourbel (Sessene) and Kaolack (Parcelles Assainies) and symptomatic patients with a negative PfHRP2-RDT test at the health post in Tambacounda.
Using a genus-specific PET-PCR, that identifies all
Plasmodium species, followed by targeted deep amplicon sequencing of the
ssu and the
cytb genes to distinguish the different species, we detected
P. falciparum,
P. vivax, and
P. malariae in Senegal, with a rare detection of
P. ovale wallikeri. However, the genes used to identify the
Plasmodium species are not discriminative enough to allow for the detection of variants or the multiplicity of infections. Although circulation of
P. vivax in southern and northern Senegal [
14‐
18] has been debated, here,
P. vivax was identified in eastern (Tambacounda, Bakel) and central (Diourbel and Kaolack) regions of Senegal. In sub-Saharan Africa several studies have reported
P. vivax, including in countries bordering Senegal, e.g. Mali and Mauritania, [
19‐
22]. Most
P. vivax infections (7/13) in this study were identified in Tambacounda, where the samples were collected in the health post of Gabou (Bakel district) near the border with Kayes in Mali where
P. vivax has been observed [
23]. However, differences in patient recruitment methods between sites; community sampling from Kaolack and Diourbel and symptomatic sampling of patients in Tambacounda, do not allow a direct comparison of prevalence. However, this strategy includes two population groups likely to be infected by non-falciparum
Plasmodium that are often not considered in NMCP diagnostic and surveillance strategies.
Despite the evidence of the circulation of
P. vivax in sub-Saharan Africa, most control and elimination strategies focus on
P. falciparum.
P. vivax produces hypnozoites that cannot be detected using current diagnostic methods used in endemic areas nor treated with artemisinin-based combination therapy and, therefore, will maintain the transmission. Beside
P. vivax,
P. malariae was the third most prevalent species and only one infection with
P. ovale wallikeri was identified. A few studies have reported the circulation of
P. malariae and
P. ovale in symptomatic individuals in Senegal,
P. malariae being responsible for acute renal failure [
17,
24‐
27]. It has been reported that
P. malariae and
P. ovale are responsible for severe and persistent cases of malaria [
28,
29]. Thus, those undiagnosed, and untreated cases of malaria can develop into severe malaria, leading to hospitalization and possibly death.
The prevalence of non-f
alciparum species was higher than expected both in symptomatic patients and asymptomatic individuals. With current control methods, there is a decrease in malaria caused by
P. falciparum, and this could lead to an increase in malaria caused by other species that are neglected [
30,
31]. This could be explained by the interaction of the species within the host as the presence of one species at a parasite density sufficient to trigger treatment seeking restricts the ability of previous lower density infections of the other species from persisting [
31].
It has been shown that
P. falciparum genetic diversity is a useful metric to estimate malaria transmission in endemic areas [
30,
31]. In this study, the non-falciparum species identified also show a genetic diversity of the
ssu gene suggesting a high frequency of transmission of these species in the areas studied. The genetic diversity is high in areas where transmission is high and low in regions implementing effective control strategies [
32‐
36]. These results are based mainly on
P. falciparum, but application of these approaches could be valuable for the non-
falciparum species estimating the intensity of their transmission in endemic areas.
The positivity rate using PET-PCR was much higher than with RDTs for
Plasmodium spp. infection, which is due to the higher sensitivity of the molecular techniques and to the detection of the non-falciparum species which were missed by PfHRP2-RDT. In Tambacounda, situated in the red zone where malaria transmission is the highest in the country, a high proportion of the infections was missed with the PfHRP2-RDT among febrile patients [
37]. Guidelines for the biological diagnosis of malaria in Senegal are the use of PfHRP2-RDTs in health posts receiving most of malaria cases, as well as consultation departments [
38]. Microscopy is available in health centres and hospital laboratories and the LAMP technique in used in the northern zone, which is in the process of eliminating malaria [
38]. In Senegal, control strategies are directed against
P. falciparum which is thought to be responsible for 99% of malaria cases based on data from health facilities and home-based management where symptomatic cases are diagnosed using PfHRP2-RDTs that detect only
P. falciparum. In addition, the non-falciparum species may be more difficult to treat because of relapses [
5,
6].
Current diagnosis strategy leads to an underestimation of non-falciparum species, which are reported in some health facilities using other diagnostic methods or during research studies. Thus, there is an urgent need to establish the prevalence of all species of Plasmodium circulating in the country to inform a better management of these undiagnosed malaria cases in health facilities that rely on only PfHRP2-RDTs for diagnosis. Thus, there is a need for sensitive diagnostic tools that can detect all Plasmodium species circulating in the country for a better management of malaria and for elimination purposes.
Asymptomatic
Plasmodium infection should be investigated for determining the real burden and implement adequate strategies for elimination purposes. However, currently in Senegal no strategy targets the asymptomatic infections. In this study a high proportion of infections detected in individuals recruited in the community were asymptomatic. Most of these infections were not detected with the PfHRP2-RDT as they are often characterized by a low density of parasites that often cannot be detected by microscopy or by the RDTs used in endemic areas for malaria diagnosis [
39,
40]. The prevalence of
Plasmodium infection was much higher in Diourbel than Kaolack, which is in line with the NMCP data from 2021 malaria incidence [
37]. These infections not seen in health facilities would, therefore, go undetected and untreated. In addition, asymptomatic low-density infections due to non-falciparum species, which go undiagnosed, could become chronic and produce gametocytes likely to infect mosquitoes and contribute to the maintenance of malaria transmission [
40‐
42].
NMCP malaria stratification in Senegal is based on PfHRP2-RDTs positivity among febrile patients attending health facilities and symptomatic cases diagnosed with home-based malaria management by the home care provider. While this strategy is less costly it does not consider low density
Plasmodium infection, asymptomatic cases, malaria caused by
P. falciparum with HRP2/3 deletion or malaria due to non-falciparum species [
43‐
45]. To overcome these challenges there is a need to include the screening of asymptomatic
Plasmodium infections and the use of more sensitive diagnostic tools that can detect and identify all
Plasmodium species circulating in the country.
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