Prevalence of Asymptomatic Malaria Infections in the Rural Dzanga Sangha Region, Central African Republic, 2020

According to the World Health Organization 94% of global malaria cases and 94% of global malaria deaths have been reported from Africa. Unfortunately, it is difficult to determine the exact prevalence of disease in some African countries due to a large number of asymptomatic cases. The aim of this study was to assess the prevalence of malaria infections in seemingly healthy children living in the Central African Republic (CAR). CareStartTM Malaria HRP2 Pf Ag RDT targeting Plasmodium falciparum was used to test a group of 500 asymptomatic children aged 1-15 years old (330 settled Bantu and 170 semi-nomadic BaAka Pygmies) inhabiting the villages in the Dzanga Sangha region (south-west CAR) in March 2020. 32.4% of asymptomatic Bantu and 40.6% of asymptomatic Pygmy children had a positive result of malaria RDT. The mean age of the study participants with RDT (+) was 8.7 in Bantu and 7.0 years in Pygmies; the mean body temperature was 36.8oC in both groups; the mean haemoglobin level was 10.6 g/dL and 10.1 g/dL, respectively. Our findings allowed us to demonstrate the high prevalence of asymptomatic malaria infections in south-west CAR. RDTs seem to be a useful tool for the detection of Plasmodium falciparum in areas with limited possibilities of using other diagnostic methods, such as light microscopy and molecular biology.


Introduction
Malaria is a vector-borne parasitic disease. In humans, it is mainly caused by five protozoan species: Plasmodium falciparum, P. vivax, P. ovale (P. ovale curtisi, P. ovale wallikeri), P. malariae and P. knowlesi (a species most often found in South-East Asia whose natural hosts are monkeys) [1,2]. Thanks to the latest developments in molecular diagnostics another two enzootic species of Plasmodium have recently been discovered, these include: P. simium and P. cynomolgi, as yet, however, very little is known of the prevalence and clinical picture of the two species [3,4]. Malaria is most often caused by a bite from an infective female Anopheles mosquito (infection vector) when invasive Plasmodium forms are released into the bloodstream of a human host; but it can also be transmitted through transfusion of Republic each year, all apparently caused by the P. falciparum species (the disease is transmitted throughout the year and in all parts of the country). In fact, the actual number of malaria cases in the CAR may be several times higher than the WHO suggests due to exceptionally high rates of asymptomatic cases [11].
The aim of the study was to assess the prevalence of asymptomatic malaria infections in sub-Saharan Africa on the example of seemingly healthy children living in the rural parts of the Dzanga Sangha region in south-west Central African Republic.

Study population
The study was conducted in March 2020 (the end of dry/warm season with average temperatures ranging between 21   Monasao is a village 50 km north of the Bayanga village (the capital of the Bayanga subprefecture) and 60 km south of the Nola city (the capital of Sangha-Mbaere prefecture), which lies at the altitude of 510 m above sea level. Initially, Monasao was inhabited entirely by the Pygmy tribes. Today, however, the village with its population of around 4,000 people is inhabited by both the settled Bantus and the semi-nomadic BaAka Pygmies. The latter, however, tend to build their huts on the outskirts of the village. The Pygmies survive by hunting, fishing and gathering wild plants in local forests but they are also employed for field work by some Bantu people.
Bantu and Pygmy children were recruited for the study at a health care facility run by the local catholic mission (where RDTs were the only available diagnostic tool for malaria detection) and at a local primary school in Monasao. The inclusion criteria were: age ≤15 years and absence of clinical signs and symptoms of malaria (body temperature ≤37.5 o C). The exclusion criteria were: presence of malaria signs and symptoms (fever ≥37.6 o C, chills, headache, vomiting, diarrhea, joint pain, general weakness), anti-malarial treatment received in the past 28 days and difficulties in venipuncture procedure. All participants had the right to withdraw from the study at any stage. On behalf of the pediatric patients, the consent to participate in the study was given by their parents or legal guardians, who had first been informed of the study purpose and methods. The parents/ guardians provided information on the age and sex of their children. The interviews were conducted in their native language, either in Sango (a language spoken by the Bantu) or Mbenzele (the language used by the BaAka Pygmies). Medical personnel responsible for carrying out the study procedures measured the participants' body weight (using a digital body scale) and body temperature (in the frontal or temporal part of the head) using an electronic thermometer (correct temperature was defined as ≤37.0 o C, low-grade fever: 37.1-37.5 o C, fever: ≥37.6 o C). Next, medical staff performed rapid diagnostic tests for malaria as well as haemoglobin measurements to identify any possible cases of anemia; haemoglobin level was measured using a portable analyzer.

Malaria screening and blood sampling procedures
Immuno-chromatographic rapid diagnostic test (RDT) targeting Pf HRP2 (Plasmodium falciparum histidine-rich protein 2) was used for the detection of P. falciparum in vitro, in a whole blood sample (CareStart TM Malaria HRP2 Pf Ag, Access Bio, USA). To perform the test a drop of capillary blood (5 µl collected from the test tube with a calibrated pipette provided by the manufacturer) is added into a sample well from which the specimen migrates through the nitrocellulose membrane (paper chromatography) by capillary action.
Next, 2 drops of buffer assay attached to the test are added into the buffer well. The test pad is pre-coated with colloidal gold nanoparticles conjugated with specific antibodies. After the blood sample has been absorbed by the membrane, P. falciparum HRP2 antigens bind to the anti-HRP2 antibodies (if the test is positive). The reaction leads to the formation of antigenantibody complexes which the test is able to detect. The interpretation of the test will depend on the presence or absence of colour bands in the control and test areas (the presence of the color line in the test area confirms the presence of antigens in the blood sample). Thus, the presence of two bandsin the control area and the test area indicates a positive result of the test. The presence of only one band in the control area within the result window indicates a negative result. The test is invalid and the interpretation of the test not possible if the line in the control area does not appear or it only appears in the test area. The results can be read after 20 minutes. The RDT sensitivity (the ability of the test to correctly identify patients who have malaria, true positive rate) is 98% and its specificity (the ability of the test to correctly identify patients who do not have malaria, true negative rate) is 97.5% (according to the manufacturer's specifications) [12].
Asymptomatic malaria was defined as the presence of P. falciparum on RTD test with no clinical signs of the disease (body temperature ≤37.5 o C, no headache, chills, joint pain, weakness, vomiting or diarrhea).

Haemoglobin measurements
Haemoglobin level was measured using a portable DiaSpect TM analyzer (EKF Diagnostics, United Kingdom) by examining a peripheral blood sample from a finger prick.
The definition of anaemia was based on the WHO criteria, where haemoglobin level of ≥11 g/dL is regarded as normal, 10.0-10.9 g/dL as mild, 7.0-9.9 g/dL as moderate, and <7.0 g/dL is regarded as severe anaemia.

Statistical Methods
The statistical analysis was performed using StatSoft Inc. (2014) STATISTICA (data analysis software system) version 12.0. www.statsoft.com and Microsoft Excel. Quantitative variables were characterized by the arithmetic mean, standard deviation, median, minimum and maximum value (range), and 95% CI (confidence interval). The variables of the qualitative type were presented in terms of counts and percentages (percentage). The Shapiro-Wilk test was used to examine if quantitative variables were normally distributed in a population. Levene's test (the Brown-Forsythe test) was used to check the assumption of equal variances. The Student's t-test or Mann-Whitney U test (under conditions when the Student's t-test is not applicable or for variables measured on an ordinal scale) were used to determine if the differences between the two study groups (subjects with a positive RDT result vs. subjects with a negative RDT result) were statistically significant. Chi-square tests of independence were used for qualitative variables (respectively using Yates correction for cell counts below 10, checking Cochran conditions and Fisher's exact test). In all calculations, the level of significance was set at p = 0.05.

Ethical Approval
The

Characteristics of the study participants
In the group of Bantu children (n = 330), a total of 107 subjects (32.4%) tested positive on mRDT; the mean age of the children with mRDT (+) was 8.7 years and their mean body weight was 22.5 kg. The mean body temperature of the tested children, both with mRDT (+) and with mRDT (-) was 36.8 o C. Of all the Bantu children with mRDT (+), 45.8% were girls and 54.2% were boys. We have not observed any statistically significant correlations between age, body weight, body temperature, sex and the mRDT result (Table 1).   (Table 2).

Prevalence of asymptomatic malaria
RDT results showed that the overall prevalence of asymptomatic malaria in the study group (n = 500) was 35.2%; 32.4% in Bantu children (n = 330) and 40.6% in Pygmy children (n = 170).

Bantu children
No statistically significant correlation was found between age, body weight, body temperature, haemoglobin level and the presence of asymptomatic malaria in 150 Bantu females (Table 3). Similarly, no statistically significant correlation was found between the age, body weight, body temperature, haemoglobin level and the presence of asymptomatic malaria in 180 Bantu males (Table 4). whereas the presence of low-grade fever was associated with a higher probability of mRDT (+). Multivariate regression analysis demonstrated that the variables had no statistically significant effect on the probability of obtaining a positive mRDT result (Table 5).

Pygmy children
In the group consisting of Pygmy females (N = 81), the mean age of the girls with  (Table 6). No statistically significant correlations were found between age, body weight, body temperature, haemoglobin level and the presence of asymptomatic malaria in 89 Pygmy males (Table 7). Regression analysis was used to determine the influence of such variables as sex, age, body weight, body temperature and haemoglobin level on the result of the mRDT in the group of Pygmy children. The univariate regression analysis demonstrated that older age and higher body weight were associated with a lower probability of a positive result on mRDT test. The multivariate regression analysis indicated that only higher body weight was associated with a lower probability of a positive mRDT result (Table 8).

Prevalence of anaemia
The mean haemoglobin level in Bantu children with a positive result on mRDT was 10.3 g/dL, and in those with a negative result of mRDT it was 10.7 g/dL. Children with mRDT (+) had a significantly lower haemoglobin level (p = 0.0399). 13.1% of the study participants with mRDT (+) vs. 19.7% with mRDT (-) had normal haemoglobin level, which confirms a high prevalence of mild to moderate anaemia in the region, regardless of the mRDT result (Table 9).

Discussion
The study aimed to determine the occurrence of asymptomatic malaria infections in children living in the Central African Republic (CAR). According to the official statistics malaria affects approximately 8% of the country's population each year. However, the WHO has indicated that the actual number of malaria cases in the CAR is grossly underestimated and in fact it might be even several times higher than the reports suggest. This is largely due to the fact that a significant proportion of malaria cases in the region are asymptomatic [11].
The epidemiological situation of infectious diseases (malaria, tuberculosis, measles) continues to deteriorate in the Central African Republic, which is largely attributable to the effects of natural disasters (such as devastating floods during the rainy season) that affect the country on a regular basis. The United Nations Office for the Coordination of Humanitarian Affairs (OCHA) has estimated that in 2021 as much as 57% of the CAR residents (2.8 million) will be in need of humanitarian aid, mostly due to food shortages, lack of medical care and a high prevalence of diseases [13]. Of all the problems the country has to face, limited access to health care seems to be the most problematic and challenging. Hulland et al. [14] has pointed out that in many parts of the country (including in the south-west, where the present research project was carried out) it takes longer than 24 hours to reach the nearest medical facility. In such circumstances, it is impossible to generate accurate data on actual morbidity rates in the country. The results of a recent screening that was carried out in the region revealed high prevalence of malaria among the local people. According to the data from the National Committee for Epidemic Control in Chad, 60.5% of 943,000 malaria tests performed between January and September 2020 were positive, and most positive tests were reported from the southern parts of the country, i.e. the region neighbouring to the Central African Republic (Logone oriental, Mandoul, Moyen-Chari) [15]. In countries where medical care is hard to obtain and where advanced laboratory techniques are unavailable, rapid diagnostic tests (RDTs) are often the only available tool for malaria diagnosis [16][17][18][19]. RDTs are immunechromatographic tests which identify malaria antigens (e.g., P. falciparum histidine-rich- supply they can easily be used during field work [21]. RDTs seem to be very useful for detecting Plasmodium falciparum infections, especially in areas where other diagnostic methods, such as light microscopy or molecular biology techniques are not available. The accuracy of RDTs for the diagnosis of uncomplicated P. falciparum infection is equal or superior to routine microscopy (but inferior to expert microscopy) [22]. Many researchers believe that the quality of microscopic diagnosis of malaria in many regions of sub-Saharan Africa is not satisfying [23][24][25]. The number of malaria RDTs used in the public sector of endemic countries has increased from <200,000 in 2005 to 88,000,000 in 2010, and an annual demand for 1.5 billion RDTs has been forecasted globally [22]. The present research project used RDTs targeting Pf HRP2 to detect P. falciparum in vitro, in a whole blood sample (CareStart TM Malaria HRP2 Pf Ag, Access Bio, USA).
The results of this study indicate that malaria is a major public health issue in Central African Republic (CAR) and malaria diagnosis remains a significant challenge for local health care professionals. The most vulnerable groups affected by malaria in hightransmission areas, such as sub-Saharan Africa, are children [26]. In malaria-endemic countries, many Plasmodium falciparum infections are asymptomatic [27]. The asymptomatic carriers do not seek treatment for their infection and, therefore, add up to a reservoir of parasites available for transmission by Anopheles mosquitoes [28]. According to some authors, long term asymptomatic carriage may represent a form of tolerance to the parasite in children building up their immune response. In this way, asymptomatic carriage would protect these children from developing a mild or severe malaria attack, by keeping their immunity effective [29,30]. Conversely, asymptomatic carriage may represent a mode of entry to symptomatic malaria, especially in young children [31].
The results of this study, which was aimed to assess the occurrence of asymptomatic 27.9% of the participants had malaria [32]. Screening tests for malaria with RDTs by Maziarz et al. [33] in northern Uganda in a sample of more than 1,000 seemingly healthy children under 16 years old identified asymptomatic malaria infections in 52.4% participants.
Asymptomatic infections go unnoticed and thus are never treated [34], resulting in anaemia, disturbed concentration and learning difficulties, which eventually lead to school absenteeism [35]. They are prevalent in all parts of sub-Saharan Africa [36][37][38] and pose a real challenge for malaria prevention and control strategies on the continent. Most malaria data are based on national surveys, which are not sufficiently granular to provide local insights [39], they tend to focus on participants in a restricted age range, usually children [40], or on clinical cases [41], which provide limited information about the asymptomatic cases that represent the main reservoir of infection that spawns new clinical cases.
The mean age of asymptomatic children with a positive result on mRDT in our research study was 8.7 years in the Bantu children and 7.0 years in Pygmy children. Pygmy children with mRDT (+) were significantly younger, we also found that older age and higher body weight were associated with a lower probability of a positive result on mRDT in this ethnic group. Similar findings have been reported by Ndamukong-Nyanga et al. [32], where the highest occurrence of malaria was reported among children aged between 6 and 10 years old. According to these authors, children aged >10 years are generally more knowledgeable about malaria and are more likely to take preventive measures against the infection compared to the younger children. The drop in occurrence of malaria with an increase in age could also be related to the acquisition of protective immunity due to repeated infections as children grow older in high transmission areas [42]. These findings have been confirmed by the results of a study conducted in Tanzania which showed a reduction in positive mRDTs among older children [43].
We have not found any statistically significant correlation between sex and the result of the mRDT in our study sample. In the group of Bantu children, boys accounted for 54.2% of children with mRDT (+) and for 54.7% of those with a negative result on mRDT; in the group of Pygmy children, boys accounted for 52.2% of those with a positive result on mRDT and 52.5% of the children with mRDT (-). A study by Teh et al. [27] and Kimbi et al. [42] carried out in Cameroon show different results, they found that malaria occurrence was higher in girls than in boys, probably due to the fact that females spend more time outdoors at dusk and dawn performing household chores, and are therefore more exposed to mosquito bites.
The mean body temperature in asymptomatic Bantu and Pygmy children with mRDT (+) was found to be normal (36. Bantu children with a lower haemoglobin level were found to be more likely to have mRDT (+). These findings are comparable with the results obtained by Teh et al. [27], who found that there were more positive mRDTs among anaemic than non-anaemic children. Common occurrence of anaemia has also been confirmed by a cross-sectional study by Nzombo et al.
The study, which aimed to assess the occurrence of malaria in a sample of more than 300 asymptomatic Tanzanian school-children aged 6-13 years, revealed that their mean haemoglobin level was 10.1 g/dL [44].
The strength of the presented study was the assessment of malaria occurrence in asymptomatic children aged 1-15 years old living in remote and hard-to-reach parts of the Central African Republic. Because asymptomatic individuals are not usually tested for malaria, it is impossible to assess the actual malaria morbidity in the country. The study had its limitations as well. Participants were recruited from among the children population living in the Monasao village or in its close vicinity, which limited the sample size. Also, limiting the sample to pediatric population only (the total population of Bayanga subprefecture has been estimated at 12,000 people) prevented the assessment of malaria occurrence in the adult population, especially in pregnant women and patients with underlying conditions.

Conclusions
The overall occurrence of asymptomatic malaria in children living in the southwestern parts of the Central African Republic is very high. RDTs seem to be an effective method for detecting Plasmodium falciparum infections, especially in areas where the access to medical care is limited and where other diagnostic methods, such as light microscopy or molecular biology are unavailable. The WHO has warned that there may be an increase in malaria-related morbidity and mortality in Africa in the next few years, because the resources which are at the disposal of national health care systems will, in the first place, be allocated to finance the COVID-19 control strategies, rather than managing the spread of tropical diseases.
On the other hand, because COVID-19 related mortality is much lower in Africa than in other parts of the world, it seems very likely that Africa may soon become an popular holiday destination for travellers from developed countries. Given the fact that the Bayanga subprefecture in the south-west of the Central African Republic is one of the most attractive tourist destinations in Africa (it is where the Dzanga Sangha National Park with its famous lowland gorillas is located), the knowledge of the exact occurrence rates of malaria in sub-Saharan Africa seems to be as important for the local health care providers as for the centers for travel and tropical medicine operating in developed countries on other continents.
Author Contributions: K.K. acquired data, performed analysis and drafted the manuscript; E.B-S. and A.L. revised and edited the manuscript. All authors have read, approved and agreed to the published version of the manuscript.
Funding: The project of the study was supported by The Ministry of Science and Higher Education in Poland [Grant No. 556/WIM/2018]. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.

Acknowledgements:
The authors are grateful to Father Wojciech Lula (a catholic mission superior and manager of the healthcare center in Monasao, Central African Republic), medical personnel (a nurse Ms. Marinella) and teaching staff of the primary school in Monasao (a teacher Mr. Simplice Taylor) for their excellent assistance and supervision in patient recruitment, collection of personal data and performing the screening tests.

Conflicts of Interest:
The authors declare no conflict interest.