Molecular epidemiology of non-falciparum Plasmodium infections in three different areas of the Ivory Coast

Malaria is a major public health concern, affecting the tropical regions of America, Africa, Eastern Mediterranean, South East Asia, and Western Pacific. Approximately 241 million cases and 619,000 deaths were reported in 2021 [1]. Of the four species of Plasmodium infecting humans, Plasmodium falciparum is the deadliest species as well as the most prevalent in Africa, whereas the incidence of Plasmodium vivax infection is higher outside Africa [2]. Besides these two main species, Plasmodium ovale and Plasmodium malariae have long been widespread in tropical Africa and other major malaria-endemic areas worldwide [3].

Previous studies have reported that in the humid forest and savanna areas of West and Central Africa, high prevalence of both these species is common in children, often reaching 15–40% for P. malariae and 4–10% for P. ovale. In these studies, thin blood smears were carefully examined by trained microscopists because parasitaemia is usually low [4, 5]. In areas with a long rainy season and/or perennial or semi-perennial Anopheles breeding sites, most P. ovale and P. malariae infections are associated with P. falciparum [6, 7]. In previous studies conducted in Côte d'Ivoire, most malaria cases were due to P. falciparum (95–99%), followed by P. malariae (3–4.2%) and P. ovale (0.3–0.7%) [8, 9].

These figures reported for P. malariae and P. ovale appear to be underestimated because of the low parasitaemia during the infections they induce and the presence of mixed infections with the main parasite species [6, 10]. The advent of polymerase chain reaction (PCR)-based molecular diagnostic methods has revolutionised the detection of Plasmodium pauci-infections [11]. In addition, molecular genotyping has led to the division of P. ovale into two distinct subspecies: P. ovale curtisi, the classical type, and P. ovale wallikeri, the alternative type [12]. The persistence of asymptomatic parasitaemia in the population could constitute a reservoir and threaten the achievement of malaria elimination [13, 14]. In addition, P. ovale may be responsible for distant relapses from dormant (hypnozoite) forms in the liver [15] and may even cause severe clinical disease in naive travellers [16].

The objective of the study was to determine the molecular epidemiology of plasmodial infections caused by P. malariae and P. ovale in Côte d’Ivoire.

DBS from 360 patients were used for molecular analysis. The distribution of samples collected was 89 (25%) in Grand Bassam, 129 (36%) in Abengourou and 142 (39%) in San Pedro.

The sex ratio of the study was 0.84 (27/32), similar to that found in studies performed in the Ivory Coast, which were 0.98 and 0.89 [20, 21]. The average age was 8.9 years with 64.4% (38/59) of participants aged between 6 and 10 years. This could be explained by the fact that this study took place in a school setting, however the number of children aged 0–5 (pre-schoolers) who are generally more affected by malaria [1], was very low. A similar result was observed in a study performed in Burkina Faso, with a mean age of 8.1 years [22].

The study observed high malaria prevalence in the rainy season compared to the dry season. The observations have been reported in all malaria infections. In the rainy seasons, the malaria vector prevalence increases due to increases in the breeding sites. Thus, increasing malaria transmission in the rainy seasons [23]. In a study carried out in Burkina Faso, P. malariae and P. ovale had higher proportions at the end of the rainy part of the study period [22].

The fever observed in 11.9% of the participants (mean axillary temperature 36.4 °C) could be explained by the fact that this study population was largely asymptomatic in 64.4% (38/59) of cases. These schoolchildren were apparently healthy; however, when present, their symptoms were dominated by headache 20.3% (12/59), abdominal pain 13.6% (8/59) and pale conjunctiva 8.5% (5/59). Similarly, several other studies have also reported various clinical signs at inclusion but in different proportions than those in this study, including chills, headache, myalgia, anorexia, and asthenia [24]. According to the work of Li et al. [25] in China, most patients with P. malariae and P. ovale had fever at the time of examination (> 37.5 °C) and a history of fever of 2–5 days.

In this study, after removing the cases of P. falciparum mono-infection, a significantly higher prevalence of P. malariae 66.1%(39/59) was observed compared to P. ovale 15.2%(9/59). However, both P. malariae and P. ovale were sometimes involved in co-infection with P. falciparum. In addition, a study in the Democratic Republic of Congo reported that most P. malariae and P. ovale infections were associated with P. falciparum and that the prevalence of mono-infections of these species was only 1.0 and 0.6%, respectively [26]. Similar findings were observed in other studies in Tanzania and Senegal [27, 28]. In addition, most of these non-falciparum infections were found in asymptomatic individuals, who were, therefore, less likely to seek medical treatment, demonstrating the importance of accurately identifying each species independently. In addition, the need to actively detect and target asymptomatic carriers of infection may allow interventions to reduce transmission of non-falciparum malaria, as asymptomatic carriage appears to be more common with P. malariae and P. ovale [29, 30]. In addition, previous studies have shown that the use of molecular methods, such as PCR, considerably facilitates an increase in the sensitivity of detection of P. malariae and P. ovale [3, 24, 28], which would provide real data on the epidemiology of these species.

Furthermore, typing of P. ovale subspecies showed a predominance of P. o. curtisi compared to P. o. wallikeri (18.8%). This is the first study with epidemiological data of these two P. ovale subspecies in Côte d’Ivoire. Similar trends have been observed in Africa, China, and in places where clinical isolates have been reported to be more closely related to P. o. curtisi than P. o. wallikeri [25, 31, 32]. In contrast, a study in Canada reported a predominance of P. o. wallikeri [33]. However, in this study in Canada it was found that imported malaria cases were associated with a higher proportion of P. o. curtisi. Moreover, P. o. curtisi was mainly implicated in malaria cases reported in travellers returning to France [34], Italy [35], and China [36]. Mixed infections with P. o. curtisi and P. o. wallikeri have been reported in Bangladesh and Gabon [31]. The predominance of P. o. curtisi in this study could be explained by the use of the primer pair used for P. ovale, namely rOVA1/rOVA2. Indeed, this primer pair has a limitation of preferentially binding or amplifying the P. ovale curtisi [18]. In order to avoid this limitation, the rOVA1WC/ rOVA2WC primer pair, which binds to both P. o. wallikeri and P. o. curtisi without binding to other human malaria parasites, is an alternative [19].

Plasmodium falciparum remains the most widespread malaria species in Côte d’Ivoire, but P. malariae and P. ovale are endemic with a low rate. However, no study has reported the presence of the subspecies Plasmodium ovale. Microscopy does not often reliably distinguish between P. falciparum, P. malariae, and P. ovale in Côte d’Ivoire, where all three species are frequently found both in mono-infection and more often in co-infection. Misdiagnosis of P. malariae as P. ovale, and vice versa, is common, which leads to inappropriate treatment or no treatment at all, given that many individuals are asymptomatic carriers of these species. Thus, P. ovale and P. malariae may remain important causes of morbidity in these areas and constitute reservoirs of the parasite. In addition, P. ovale, due to the presence of hypnozoites, may be responsible for distant relapses and even severe disease. Because P. malariae and P. ovale infections usually have low parasitaemia and occur as mixed infections with P. falciparum and P. vivax, molecular detection methods provide a useful tool for the diagnosis of the disease. Molecular detection methods provide an accurate estimate of malarial epidemiology.