According to the WHO data, Plasmodium falciparum is responsible for 99.7% of all malaria cases in sub-Saharan Africa  and as much as 100% malaria cases in the CAR .
The results of the present study suggest otherwise.
In 2018, the WHO reported of 1,367,986 suspected malaria cases in the CAR, of which 972,119 (71.1%) were confirmed as Plasmodium falciparum malaria. Infections caused by Plasmodium other than falciparum or mixed infections were not found . The WHO reports that samples were examined by light microscopy or RDTs. A total of 117,267 malaria cases malaria cases were not confirmed by any diagnostic method . If, however, the molecular biology PCR methods had been used, the results could have been surprising.
The data on the prevalence of different species of malaria in the Central African Republic (CAR), collected in independent researches is insufficient to define the epidemiological situation in this country. Decades of conflicts and political instability disrupted research field in many directions. The results of another studies which had been conducted in Africa have also indicated that the rates of infections with other Plasmodium species are higher than the WHO suggests.
More accurate assessment of malaria epidemiology in CAR could be obtained by analyzing the data gathered in the neighboring countries with similar climate conditions. The overall malaria prevalence in Cameroon is 29% . In the group of children aged 6 months up to 5 years old malaria was reported in 30% in 2011 but there were differences within urban and rural zones with malaria prevalence at the level 20.6% and 37.1% respectively . The eastern region of Cameroon, i.e. the area with the largest CAR refugee population (due to recurrent ethnic and political conflicts) is considered the main cause of high malaria transmission in Cameroon , suggesting a higher malaria prevalence rate in the much poorer CAR. The studies which were conducted in Cameroon using molecular biology methods, confirmed the presence of four species of Plasmodium: P. falciparum, P. vivax, P. ovale and P. malariae [7–10], with a definite predominance of P. falciparum, responsible for 95% of infections . P. vivax was found in 5.6% patients (which constituted 38.6% of all PCR positive samples) , P. ovale and P. malariae were found in a very low percentage, as a co-infection predominantly . These results were consistent with the results obtained by the authors of the present research task. The data published by Kwenti et al. proves unequivocally that P. falciparum is the unique cause of malaria in Cameroon , which is a conclusion contrary to the results of the above-mentioned studies.
Another study on the prevalence of malaria in the neighboring region was carried out by Tsumori et al.  in the Republic of Congo, adjoining to the north with the CAR and it involved patients from both urban and rural areas presenting with fever. The study demonstrated that malaria infection rates were found to be different between the methods used. Microscopic examination revealed that 37% of the subjects living in urban areas were infected with malaria, whereas PCR tests were positive in 42% of the sample. In the group coming from rural areas, RDTs were positive in 59% cases and PCR tests in 72% of the subjects . The studies conducted in the Republic of Congo indicate that P. falciparum was responsible for a vast majority of malaria cases in the region [13, 15, 16], but cases caused by P. vivax , P. ovale and P. malariae have also been reported and should not be neglected .
According to the results of the present study conducted in Dzanga Sangha, P. falciparum was responsible for 94.8% of all malaria cases; however, the percentage distribution of other Plasmodium species was significantly higher (P. malariae 11.1%, P. ovale 9.8%, P. vivax 0.7%) than the 0.3% reported in the 2019 WHO report. This fact is important in the context of the treatment of malaria patients and highlights the necessity to introduce more accurate diagnostic methods and to revise malaria treatment protocols (introduction of a drug regimen targeting the latent forms of Plasmodium residing in the liver).
In a routine malaria diagnostics in rural areas in CAR the role of PCR is none, but in the context of the epidemiological studies is crucial. The PCR was essential to establish the presence of the different Plasmodium species and was a comparative method for RDTs false-negative sensitivity and try to find the factors responsible for it.
A study by Berzosa et al.  conducted in Equatorial Guinea confirmed the presence of P. falciparum alone in 69.2% of all RDT positive samples, and in 27,8% with other than P. falciparum species as a co-infection, whereas PCR tests were positive for P. falciparum in 97% cases and in 1,9% PCR tests also revealed infections with P. malariae, P. ovale and P. vivax. A study which was conducted in Senegal and was based on microscopic examination of blood samples taken from study participants demonstrated that apart from P. falciparum (45.1%) infections, P. ovale and P. malariae infections were also detected (together, they accounted for 5.6% malaria cases) . The epidemiology of malaria and prevalence of different species of Plasmodium in CAR can be similar to these described in the surrounding countries mentioned above.
Until 2010, patients with suspected malaria were administered the recommended treatment but were rarely offered diagnostic tests, which was largely due to limited testing capabilities in the region. Since 2010, however, owing to the WHO support, and its recommendations for pre-treatment diagnosis of malaria in every symptomatic patient, RDTs and microscopic examinations have become more common . RDTs in particular are now widely used in the region, and their sales increased from 46 million in 2008 to 320 million in 2013 globally .
In the rural areas of Africa, such as Dzanga Sangha in the south-western parts of the CAR, RDTs are often the only available diagnostic method for malaria detection. Unfortunately, the number of RDTs distributed by non-governmental organizations (NGOs), as part of the global malaria program is often insufficient. The author’s(EB-S) observations made during the study period have led to the conclusion that malaria diagnosis and treatment protocols applicable in the CAR leave much to be desired. As an example, a vast majority of medical interventions in the Dzanga Sangha region are performed by people without a medical background; the use of expired medications and RDTs or random drug use is common. Additionally, medications and RDTs are rarely stored properly and a large number of patients purchases medications at local street markets.
The results of the research task conducted in the group of BaAka Pygmies have demonstrated significant differences between the results of RDTs and PCR tests. Therefore the question should be raised on the accuracy and usefulness of RDTs in a situation, where the initiation of anti-malaria treatment depends entirely on the interpretation of the RDTs results, regardless of the patient’s condition or the severity of clinical signs and symptoms. On the other hand, the decision to administer anti-malarial treatment on the basis of non-specific symptoms, which may be the manifestation of many other diseases, e.g. bacterial infections of the respiratory and urinary tract or cosmopolitan and tropical diseases of viral etiology, is not always justified and may lead to overdiagnosis of malaria [21, 22]. Primary healthcare providers in sub-Saharan Africa often prescribe anti-malarial treatment in a combination with other antibiotics to treat all kinds of health problems a patient may have, without performing the basic diagnostic tests [21, 23].
There are four main possible causes of false-negativity of the RDTs. Lower sensitivity of RDTs (estimated at 88–90% by their manufacturer) compared to PCR tests is attributable to several factors. One of the limitations of an RDT is its detection limit which has been estimated at between 200 and 2000–5000 parasites/µl, which corresponds to 200 infected erythrocytes per microliter . In cases of very low parasitemia, RDT is likely to give a false negative result. This conclusion is also supported by the work of Djallé et al. conducted in Central African Republic where the sensitivity of three different RDTs was proved to correlate with an intensity of the parasitemia in the blood sample. The sensitivity was 95% in a high parasitemia level (> 500 parasites/ul), but in low parasitemia (100 parasites/ul) the sensitivity was lower than 70%. Many researchers have addressed the issue of malaria parasite transmission on the sensitivity of RDTs. The authors agree, that in areas with low malaria transmission, where low parasitemia ≤ 200/µl is more common, the sensitivity of RDTs is lower than in areas with high transmission rates [25–30]. This applies to approximately half of P. falciparum infections worldwide .
Upon comparison of patients with a positive vs. patients with a negative RDT result we concluded that patients aged < 5, weighing up to 10 kg were more likely to have a positive RDT result (64.8% vs. 25.2%). The conclusion is consistent with the WHO data , according to which malaria is most prevalent in children < 5 years old. The results were quite the opposite in the group of adults aged over 18 years old (20.1% vs. 56.7%).
In the group of patients with an RDT-positive result, there were significantly more patients with body temperature > 39 ͦ C (26.0% vs 8.7%). It can be assumed that these patients had a higher level parasitemia with P. falciparum and therefore the HRP2–specific RDT was positive in their case [24, 32], or that the elevated body temperature in the youngest subjects could have been a result of their hyper-reaction to infection with P. falciparum given their lack of immunity, (with age, people living in endemic areas acquire immunity to P. falciparum malaria naturally) [19, 33–35]. Premunition or otherwise a partial immunity observed in the asymptomatic or poor-symptomatic individuals with the detectable presence of malaria parasites in the blood, inhabiting endemic areas is gained with repeated contacts/infections with Plasmodium spp. It is considered as a protective factor against the severe manifestation of the disease . Another cause of the false-negative RDTs might be deletion of Pfhrp2 gene which results in the lack of the antigen HRP2-protein which is a target for the RDTs HRP2.
A high percentage of infections caused by P. falciparum parasites with a Pfhrp2 gene deletion has been reported from sub–Saharan Africa, in the Indian subcontinent and in some countries of South America (Peru). Pfhrp2 gene is responsible for the production of HRP2 protein, which is an antigen detected by the P. falciparum-specific RDTs. The rate of such infections has been estimated at approximately 5% in sub-Saharan Africa [37–39], up to 40% in Peru [40, 41] and even over 80% in Eritrea depending on the region. As suggested by the WHO, the use of RDTs targeting HRP2 protein for the diagnosis of malaria is questionable when the percentage of P. falciparum species with a Pfhrp2 deletion is over 5% in a given area [43, 44]. The threshold of 5% was assumed to have an important impact on public health, and the number of undetected cases was lower than when tests targeting other less sensitive P. falciparum antigens were used . There are no data available on the prevalence of species with Pfhrp2 deletion in the Central African Republic; however, in the neighboring Democratic Republic of Congo, the rates of P. falciparum species with the Pfhrp2 gene deletion have been estimated at 6.4% , the results may be similar in other countries of the region, including the CAR. The authors of the present research task did not undertake the analysis of exon 2 for the assessment of the Pfhrp2/3 deletion. Further studies to evaluate the prevalence of the Pfhrp2/3 gene deletion seem necessary in the context of determining the usefulness of RDTs targeting HRP2 protein in Central Africa.
In the study by Mensah–Addai et al. which was carried out in Ghana, the issue of genetic variability of exon 2 of the Pfhrp2 gene, which may result in lower sensitivity of RDTs using the P. falciparum HRP2 protein antigen was raised .
Furthermore the existence of anti-HRP2 antibodies forming the immune-complexes with HRP2 antigen in P. falciparum infected individuals may be another cause of the false-negative RDTs results .
Amoah et al. tested blood samples taken from patients living in Rwanda who presented with clinical symptoms of malaria using three methods: RDTs, microscopy examination and PCR; of all RDT-negative samples 24.4% tested positive when examined by light microscopy and 44.1% were positive when PCR tests were used . The differences between the results obtained by using different methods were less significant in comparison to the results of the present study, yet, they clearly show that a large proportion of RDTs are false-negatives. In Gambia, where the epidemiological situation has improved significantly over the past twenty years, owing to the introduction of malaria control measures [26, 27], Mwesigwa et al. screened a group of symptomatic and asymptomatic patients using two diagnostic methods: RDTs and PCR. The results of the study demonstrated that 62% of the RDT-negative samples were found to be positive when PCR method was used .
In order to reduce the number of false negative RDT results, in countries with a high prevalence of P. falciparum species with a Pfhrp2 deletion or regions with high rates of malaria parasites other than P. falciparum, RDTs detecting pLDH are additionally used  (they are, however, less sensitive in cases of low parasitemia ≥ 200–1000 parasites / µl than RDTs directed against HRP2 protein  or aldolase). Both enzymes are produced by all malaria species found in humans  and have a short half-life of 2–4 days. Therefore, they can be found in the blood of individuals with an active malaria infection . Nevertheless, the WHO recommends restricting the use of RDTs that detect both HRP2 and pLDH proteins to regions where infections by one species of malaria are expected , presumably for a cost-effective reasons. According to the author’s observations, RDTs other than those targeting HRP2 specific for P. falciparum were not available in the Dzanga Sangha region.
In recent years, there has been a lot of debate on the usefulness of RDTs in malaria diagnosis and control. In some countries, microscopic examination of thick blood films remains the gold standard for diagnostics of malaria [52, 53], in other countries PCR tests are the recommended detection method in the epidemiological studies or research [32, 49], in others both methods are in use [13, 25, 48, 54]. It is difficult to decide which of these methods are optimal and should be recommended for malaria diagnosis.