Prevalence of non-Plasmodium falciparum species in southern districts of Brazzaville in The Republic of the Congo

Although Plasmodium falciparum infection is largely documented and this parasite is the main target for malaria eradication, other Plasmodium species persist, and these require more attention in Africa. Information on the epidemiological situation of non-P. falciparum species infections is scarce in many countries, including in the Democratic Republic of the Congo (hereafter Republic of the Congo) where malaria is highly endemic. The aim of this study was to determine the prevalence and distribution of non-P. falciparum species infections in the region south of Brazzaville. A cross-sectional survey was conducted in volunteers living in rural and urban settings during the dry and rainy seasons in 2021. Socio-demographic and clinical parameters were recorded. Plasmodium infection in blood samples was detected by microscopic analysis and nested PCR (sub-microscopic analysis). Of the 773 participants enrolled in the study, 93.7% were from the rural area, of whom 97% were afebrile. The prevalence of microscopic and sub-microscopic Plasmodium spp. infection was 31.2% and 63.7%, respectively. Microscopic Plasmodium malariae infection was found in 1.3% of participants, while sub-microscopic studies detected a prevalence of 14.9% for P. malariae and 5.3% for Plasmodium ovale. The rate of co-infection of P. malariae or P. ovale with P. falciparum was 8.3% and 2.6%, respectively. Higher rates of sub-microscopic infection were reported for the urban area without seasonal fluctuation. In contrast, non-P. falciparum species infection was more pronounced in the rural area, with the associated risk of the prevalence of sub-microscopic P. malariae infection increasing during the dry season. There is a need to include non-P. falciparum species in malaria control programs, surveillance measures and eradication strategies in the Republic of the Congo.

cases in 85 malaria endemic countries worldwide, an increase from 227 million in 2019, mainly in countries in the WHO African Region [1]. Also, malaria deaths increased by 12% compared with 2019, to an estimated 627,000; an estimated 47,000 (68%) of the additional 69,000 deaths were due to service disruptions during the COVID-19 pandemic [1].
Among the Plasmodium species infecting humans in Africa, Plasmodium falciparum is the major cause of malaria cases (99.7%) [2], with a minor but underestimated prevalence of other Plasmodium species [3]. Plasmodium vivax is the predominant parasite in the WHO region of Americas, causing 75% of malaria cases [4]. Although few studies have identified P. vivax infections in Mali and Nigeria [5][6][7], this Plasmodium species remains non-endemic in West and Central Africa because of the lack of the Duffy antigen receptor for chemokines, which is essential for erythrocyte invasion by the parasite [8]. Plasmodium ovale infection represents < 1% of all malaria infections worldwide [9,10]. Despite its low prevalence, however, P. ovale infections are directly linked to the danger of relapses after months or even years due to the presence of hypnozoites [11,12], which may cause severe disease and even death [13]. The global distribution of Plasmodium malariae is sparse and variable, but frequently coendemic with P. falciparum [14,15]. A low prevalence of P. malariae infection is commonly reported with asymptomatic cases, however patients may occasionally develop severe symptoms [16]. In addition, the recrudescence of P. malariae blood-stage parasites can occur for long periods, even after the infected person has left endemic regions [17,18].
Despite the decrease in malaria-related morbidity since 2016, the burden of malaria in the Democratic Republic of the Congo (hereafter Republic of the Congo) remains high, with P. falciparum as the most prevalent malariacausing species. A systematic review of the epidemiology revealed that 37% of Congolese living in urban areas tested positive for microscopic P. falciparum infection compared to 59% in the peri-urban areas, with these populations showing 11% and 20% submicroscopic infections, respectively [19]. However, recent information on the epidemiology of non-Plasmodium falciparum species, mainly P. malariae and P. ovale, is limited. Taking into account the new malaria elimination strategies based on the interruption of local transmission of human Plasmodium [20], intensification of malaria species surveillance has been strongly recommended. Therefore, the aim of the present study was to determine the prevalence and the distribution of P. malariae and P. ovale infection, based on multiple community surveys in rural and urban settings in Brazzaville, the capital of the Republic of the Congo and home to > than 50% of the Congolese population.

Study areas
The study was conducted in rural areas of the Goma Tsé-Tsé District (Ntoula and Djoumouna villages) in the Department of Pool and in the urban area of Brazzaville Department (Mayanga in the West Quarter of Madibou District) of the Republic of the Congo (Fig. 1). The two departments have a tropical humid climate divided in two seasons: a short dry season (June to September) and a long rainy season (October to May). The geo-location of these zones is 4.36° S, 15.15° E, and the average altitude is 217 m a.s.l [21]. Djoumouna is a village located 25 km from Brazzaville. It is characterized by the presence of a gallery forest bordering the Djoumouna River. The locality is surrounded by four rivers (Lomba, Kinkoue, Loumbangala and Djoumouna rivers) which supply water to a series of fish farming ponds [22] that can serve as potential foci of malaria vectors. The population of Djoumouna village comprises about 800 inhabitants, with agriculture as the main economic activity. Ntoula is a neighboring village of Djoumouna and irrigated by two rivers. This village has around 635 inhabitants who mainly depend on agriculture and fishing for their livelihoods. Mayanga is an urban area located in the 8th Madibou District in the south of Brazzaville. With a population of about 28,422 inhabitants, Mayanga is characterized by the presence of market gardening sites (Agni-Congo 1 and 2 and the Groupement Jean Felicien Mahouna). Irrigation water is supplied by three rivers (Djoué, Laba and Matou rivers). Several public and private services are established in Mayanga, such as health centers and primary and secondary schools.

Study design
A cross-sectional survey was carried out from March to September 2021, covering both the rainy and dry seasons. Individuals aged at least 1 year who lived in the study areas for at least 3 months were included in the study. Volunteers from a random sample of households were recruited in each area. Participants who decided to withdraw their consent during the study were excluded from the analysis. Socio-demographic and clinical data of the participants were recorded in a well-structured collection sheet (age, gender, temperature, bed net use etc.). The axillary temperature was determined to define fever in the participants. Fever was diagnosed when the axillary temperature was ≥ 37.5 °C [23,24]. Bed net use was defined based on the answers of participants, and on the observation of bed nets in each household. After an examination by a clinician (diagnosis of fever, headaches, vomiting, nausea etc.), blood samples (3 ml) were collected in EDTA tubes by a nurse and the tubes then transported to the "Centre de Recherches sur les Maladies Infectieuse-Christophe Merieux" (CeRMI) for further investigations, including thick and thin blood smears for microscopic examination. Participants presenting a positive blood smear were treated by the medical staff with artemisinin-based combination therapy (ACT) in accordance to WHO and national malaria program policies [25]. Hemoglobin level was determined performed using CYANHemato, a fully automated bench-top hematology cell counter (Cypress Diagostic, Hulshout, Belgium).

Microscopic screening of Plasmodium infections
Plasmodium infection was screened by microscopy using the Lambaréné method [26]. In brief, 10 µl of whole blood of each sample was distributed on a microscope slide over a rectangular area of 10 × 8 mm. The contour of the rectangle was drawn on a piece of paper placed under the slide before the whole blood sample was spread. The slides were air dried and stained for 20 min with 20% Giemsa solution (Giemsa R-Solution [Merck, Darmstadt, Germany], Titrisol buffer pH 7.2 [Merck]). Each slide was read by two microscopists. When no agreement was reached on a specific slide, a third microscopist was asked to decide. Positive slides of each parasite species served as the quality controls of the microscopy studies. The number of parasites and the number of highpower fields (HPF) were counted, and parasitemia per microliter was calculated using the microscope factor of 708 as follows: where N is the number and para represents parasites.

Plasmodium species identification by PCR
The parasite's DNA was extracted from 200 µl whole blood samples using the QIAamp DNA mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's Parasiteamia para µl N Para N HPF × Microscope Factor,  (20) instructions. DNA was recovered in 150 µl of elution buffer and stored at − 20 °C until use. The detection of malaria parasite DNA was based on nested PCR amplification of the 18S ribosomal RNA (rRNA) gene in a total reaction volume of 20 µl using the appropriate primers (Additional File 1: Table S1). The first PCR reaction targeted the Plasmodium spp. and was performed in a reaction volume composed of 18 µl of a master mix (2.5 µl of 10× PCR buffer, 1.25 µl of 25 mM MgCl 2 , 0.5 µl of 10 mM dNTPs, 0.5 µl of each 10 µM primer, 0.25 µl of Taq DNA polymerase 5U/µl and 12.5 µl of nuclease-free water) and 2 μl of the extracted DNA. The reaction was carried out under the following cycling conditions: an initial denaturation at 94 °C for 4 min; followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 1 min and extension at 72 °C for 1 min, with a final extension at 72 °C for 4 min. In the second PCR, 19 µl of master mix (containing 13.5 µl of nuclease-free water and the same volume of reagents as described for the first PCR) and 1 µl of primary PCR product were added to the PCR tube, and the reaction was carried out under the same cycling conditions as described for the first PCR, except that the annealing temperature was 58 °C.
PCR products and the 100-bp molecular weight maker were stained with Syber Green solution (1:1, v/v), electrophesed at 100 V for 45 min in a 1.5% agarose gel and visualized on the GelDoc ™ EZ Imager (Bio-Rad Laboratories, Hercules, CA, USA). A sample was considered positive for P. falciparum, P. malaria, P. ovalea or P. Vivax if a 205-bp, 144-bp, 800-bp or 120-bp band was detected, respectively. The known Plasmodium-positive samples from our library and distilled water served as positive and negative controls, respectively, in every set of reactions.

Statistical analysis
Management and tabulation of raw data were carried out using Microsoft Excel (Microsoft Inc., Redmond, WA, USA) version 2016. All statistical analyses were performed using SPSS version 22.1 (SPSS, IBM Corp., Armonk, NY, USA). The normality of data distribution was checked using the Shapiro-Wilk test [27]. Variables were expressed as proportions for categorical variables or as medians (with range)/means (standard deviations [SD}) for continuous variables. The prevalence of Plasmodium spp. infections was determined as the proportion of individuals identified as positive for the presence of parasites (either for all parasite species or for an individual species) in each community. Age was stratified into five groups according to WHO guidelines [28]: 1-4, 5-9, 10-14, 15-19 and ≥ 20 years.
Participants with a negative result in the microscopic examination, but positive in the PCR analysis were classified as the sub-microscopically infected group. Those Table 2 Prevalence of Plasmodium species identified by microscopy and by sub-microscopy

Prevalence of Plasmodium species was compared between the Goma Tsé-Tsé and Madibou districts
The Chi 2 test was used to compare the proportion of cases between the two districts.
N Overall number of participants, n 1 , n 2 total number of participants in Goma Tsé-Tsé and Madibou districts, respectively **Significant difference in prevalence between the two districts according to overall microscopic findings at P < 0.0001 and according to sub-microscopic study at P = 0.0099 Plasmodium species by sub-microscopy study (n 1 = 373; n 2 = 159; (N = 532) Overall positivity, % (n) 59. 8 (1) P. falciparum/P. malariae/P. ovale 1.9 (7) 1.3 (2) 1.7 (9) who had microscopic Plasmodium-positive results were classified as the microscopically infected group. Chi-square tests (or Fisher's exact tests when appropriate) were used to calculate the differences in participant characteristics and prevalences among groups, and to assess associations between independent variables and Plasmodium spp infection. The strength of association between each risk factor and the occurrence of Plasmodium infection was calculated using univariate logistic regression. The relative risks (RR) and 95% confidence intervals (CIs) were calculated to determine the risk factors associated with Plasmodium infection. The level of significance was set at P < 0.05.

Ethical consideration
This study received ethical approval from the Institutional Ethics Committee of "Fondation Congolaise pour la Recherche Medical" (No. 013/CIE/FCRM/2018), administrative authorizations from the Marien Ngouabi University (No. 247/UMNG.FST.DFD.FD-SBIO) and approval from the major of each study area. Prior to enrollment, participants were informed in writing and orally about the study and its benefits. Before enrollment,

Characteristics of the study population
Of the 773 participants enrolled in this study 573 and 200 were resided in Goma Tsé-Tsé District (District 1) and Madibou District (District 2), respectively ( malariae than in children aged between 6 and 14 years (Fig. 2).

Sub-microscopic Plasmodium spp. infection
Sub-microscopic Plasmodium spp. infections were found in 63.7% (339/532) of all samples ( . The prevalence of sub-microscopic infection was high in all age groups (Fig. 2), with study participants between the ages of 15 and 19 years harboring a highest proportion of sub-microscopic infection (77.3%).

Predictors of microscopic malaria infection
The relationship between participant characteristics and the presence of Plasmodium spp. infections was investigated. Those participants found to be anemic were classified according to their age group following the WHO guideline [29]. Plasmodium falciparum infection was significantly associated with anemia in children aged < 15 years (P = 0.0374 for children aged 1-4 years, P = 0.0001 for 5-to 9-year olds, P < 0.0001 for 10-to 14-year olds), but no relationship was observed between P. malariae infection and the risk of anemia ( Table 3). As shown in Table 4, the rainy season was shown to be a risk factor (RR: 3.1, 95% CI: 1.7-5.7; P = 0.0003) for increased P. falciparum prevalence in Madibou. Plasmodium falciparum infection was associated with gender in Goma Tsé-Tsé (RR: 1.4, 95% CI: 1.1-1.7; P = 0.0072), but no such association was found in Madibou. According to the age group, P. falciparum prevalence for all age group was higher in Goma Tsé-Tsé District than in Mabidou District, with participants aged 5 to 14 years having a higher risk of malaria infection. The presence of fever was significantly (P = 0.0223 [Goma Tsé-Tsé]; P = 0.0306 [Madibou]) associated with P. falciparum infection in the two districts. No association was observed  between the socio-demographic characteristics of the study population and the microscopic prevalence of P. malariae in the two districts ( Table 5).

Discussion
Since publication of the systematic review on the malaria situation in the Republic of the Congo in 2016 [19], studies conducted in the country have mostly focused on the epidemiology and diversity of P. falciparum infection [30][31][32]. However, information on the epidemiological situation of non-Plasmodium falciparum species in the country is lacking. The aim of the present study was to evaluate the prevalence of Plasmodium species in two endemic areas for malaria of the Republic of the Congo, namely Goma Tsé-Tsé District, as representative of rural areas in the Republic of the Congo, and Madibou District, which is an urban area. Of 773 participants enrolled in this study, 573 and 200 were living in Goma Tsé-Tsé District and Madibou District, respectively. This vast difference in number of participants reflects the low interest of the population of Madibou to participate in the study, possibly due to the presence of health centers close by. Almost all study participants were sleeping under bed nets (97.4%), suggesting a strict compliance of the inhabitants of these localities to the malaria control measures. The overall prevalence of Plasmodium spp. detected by microscopy was 31.2% in this study. Plasmodium falciparum remained the most prevalent species, in accordance with prevalences reported in many previous studies conducted in African malaria endemic areas [33][34][35][36]. The high prevalence of P. falciparum in the overall study population demonstrates the importance of malaria control strategies specifically against this species. The prevalence of malaria infection was significantly higher in villages  15:209 of Goma Tsé-Tsé District than in Madibou District, which may be related to the different ecological situations in these two areas (rural vs. urban) [37]. The prevalence of Plasmodium spp. in this study was higher than that reported in recent studies in Benin (23.7%) by our research group [33], but similar to the findings of Amoah et al. (32%) [34].
Among the identified non-P. falciparum species in the whole study population, only P. malariae was detected by microscopy at a rate of 1.3% (with 0.8% of dual P. falciparum/P. malariae infection). This prevalence was higher in the rural area of Goma Tsé-Tsé District (1.4%) compared to the urban area of Madibou District (1%), suggesting that this variation in prevalence was probably due to the socio-demographic characteristics of each study area. Suitable containers or conditions for water to pool and persist, deforestation and agriculture have all been associated with an increased risk of malaria transmission [37]. The P. malariae prevalence observed in this study was comparable to that reported in Benin, in which an increase of this prevalence was observed over the past 10 years [33]. A potential explanation could be the differences in the use of bed nets by the population. Almost all participants of this study, compared to less than one quarter of the study population in Benin, slept under a bed net. This difference highlights a heath preoccupation related to this species among the non-Plasmodium falciparum parasites that must be taken into account in the WHO strategic plan for malaria eradication [20].
The rainy season was associated with a higher prevalence of P. falciparum infection in Madibou District. In the villages of Goma Tsé-Tsé District, where the prevalence of P. falciparum was high and almost stable throughout the year, men and children appeared to be at higher risk of getting malaria infection. All of these results suggest that malaria management strategies must be directly applicable to the area they are being used: in urban areas, they should take seasonality into account; in rural areas, they must be permanent, with a particular focus on the sensitization of men and adolescents. Fever was significantly associated with P. falciparum infection in the two districts of this study. We did not observe any association between socio-demographic factors or clinical outcomes with an increased prevalence of P. malariae, probably due to low number of events and, therefore, power issues. However, many studies have reported a significant association of P. malariae infection with severe anemia, probably due to prolonged erythrocyte destruction and bone marrow suppression with a minimal reduction in erythrocyte life-span at a low parasitemia level  [38,39]. Pulmonary complications and renal impairment have also been reported to be associated with P. malariae infection [40]. The pooled proportion of severe complication caused by P. malariae infection was reported to be 2% [16]. Compared to microscopic malaria infection, a higher sub-microscopic infection (63.7%) of Plasmodium spp. was observed, being more pronounced in older participants (> 15 years). These findings are similar to those reported in a previous study from Ghana (66%) [34], and higher than those reported from a study conducted in Benin [33]. This difference could be attributed to geographical zone, including the level of local Plasmodium infection endemicity. Indeed, in Benin, the survey was conducted in villages, while in Ghana, the study covered a mixture of communities. In addition, the sub-microscopic prevalence in our study varied with respect to the study district, being lower (59.8%) in the rural area compared to the urban areas (73%). The high prevalence of sub-microscopic infections observed in the present study, as well as in other studies [19,30], represents a major challenge to malaria control programs in the Republic of the Congo since the management of malaria patients is based on the microscopic detection of parasites. Our findings suggest that despite the best efforts of the national malaria program, malaria transmission will continue to be high because half of infected individuals remain afebrile and will, therefore, not be identified as having malaria and subsequently treated. Among the non-P. falciparum identified by PCR, P. malariae and P. ovale were detected in 14.9% and 5.3% of participants, respectively. In terms of co-infection, participants co-infected with P. falciparum/P. malariae (8.3%) were the most prevalent, followed by participants co-infected with P. falciparum/P. ovale (2.6%). It is notable that although non-P. falciparum parasites were hardly detected by microscopy, the high rates of sub-microscopic detection of these parasites translate into perennial transmission of these species in endemic areas, thereby constituting a major challenge to achieving malaria eradication. The presence of persons with P. ovale and P. malariae co-infected with P. falciparum in this study highlights the impact of those two latter parasites in asymptomatic and chronic malaria infection. This becomes more critical with triple infection, which was detected at a sub-microscopic prevalence of 1.7% in the present study. Plasmodium malariae and P. ovale are rarely associated with severe cases of malaria, but their persistence in the body remains a great challenge for malaria eradication. Although P. malariae does not cause malarial relapse from persistent liver-stage parasites, recrudescence of blood-stage parasites can