Preliminary study of malaria incidence in Nouakchott, Mauritania

Background Malaria is one of the main motives for outpatient consultation and hospitalization in Mauritania. However, its incidence remains unclear because of diagnostic problems and insufficient epidemiological data. Methods Between April and August 2007, a study on malaria incidence was carried out in Nouakchott city. A total of 237 febrile outpatients, from all Nouakchott districts, attending the two main hospitals of the city were investigated. Finger prick and blood dried filter paper samples were performed to prepare thick and thin films and nested-PCR for malaria parasite species identification and density. The accuracy of diagnosis of 'presumptive malaria', assigned by clinicians and based on fever and other malaria suggestive symptoms, was assessed. Entomological investigations based on morphological and molecular characterization of Anopheline species were conducted in Dar Naïm district. Results Malaria prevalence rate was 25.7% (61/237), the majority of positive blood slides as well as nested-PCR products were due to Plasmodium vivax 70.5% (43/61) and Plasmodium ovale 24.6% (15/61). Two malaria patients, both with P. vivax, have never travelled out of Nouakchott and seem likely to have been autochthonous (3.3%). Of the 237 individuals included in the survey, 231(97.5%) were clinically diagnosed and treated as malaria cases. 26.4% of clinically diagnosed cases were positive for Plasmodium using microscopic examination and PCR. Thus, false positive cases constituted 73.6% (170/231) of the clinically diagnosed malaria cases. The search for mosquito vectors in Dar Naïm district allowed morphological and molecular identification of Anopheles arabiensis and Anopheles pharoensis. Conclusion This study demonstrates that, during the hot and dry season, Plasmodium species responsible of recurrent malaria (P. vivax and P. ovale) are the dominant species in Nouakchott city and autochthonous malaria cases exist but are rare. Clinical diagnosis of malaria has a very low positive predicted value. The systematic use of microscopy-based diagnosis and/or rapid diagnostic tests should be considered to appropriately manage malaria and non-malaria cases.


Background
Malaria is the most deadly vector-transmitted human disease in the world. It causes nearly a million deaths each year, mostly in Africa [1]. This disease and the relevant mortality are due to one of four Plasmodium species transmitted by the bite of an infected female Anopheles species mosquito. Mauritania is located in north-western Africa, between latitudes 15° and 27° North and longitudes 5°a nd 17° West. The country is largely arid, with two-thirds of its areas covered by the Sahara desert and the remainder belongs to the Sahelian zone. It has a low rainfall that increases from North to South and only one rainy season from July to October [2]. Consequently, the country is geographically divided into three climatic areas: the Saharan region in the north with an annual rainfall less than 100 mm, the Sahelian zone characterized by an annual rainfall of 100-300 mm, and the southern area, along the Senegal River valley, with an annual rainfall of 300-500 mm. In Mauritania, malaria is the main motive of outpatient consultation and hospitalization after diarrhoea and respiratory infections [2,3].
According to the World malaria report of 2008, the disease is increasing since the number of reported cases reached 188,025 in 2006 against only 26,933 in 1990 [1]. The main factors affecting this increase are the development of a hydro-agricultural project in the south and the reappraisal of the oases in the north [2]. However, the precise malaria incidence is still unknown since, in most health centers, febrile episodes are often considered as malaria, based only on a clinical diagnosis and the parasitological confirmation is rarely sought by the physician. On the other hand, research describing the situation of this parasitic disease in the country is relatively recent and still limited [3][4][5]. These have described the four Plasmodium species with a predominance of P. falciparum in the southern and south-eastern endemic areas of the country. Furthermore, researches about mosquito species responsible of malaria transmission in the country have mainly focused on the inventory of Anopheline fauna [6]. Nouakchott, capital city of Mauritania has, during the last three decades, undergone rapid and profound demographic changes due to the rural exodus following the drought of 1970s. The city has struggled to cope with the pace and the extension of informal urbanization. Poor housing, lack of sanitation and drainage of surface water and the development of peri-urban orchards, with multiple crops, have resulted in extensive mosquito-favourable environment, encouraging the appearance of vector-borne diseases. This work describes malaria situation in Nouakchott. The main goals were: a) to assess the real contribution of malaria to febrile episodes, b) to determine the Plasmodium species involved and their relative importance, c) to evaluate the impact of "presumptive diagnosis" on the accuracy of malaria incidence, and d) to identify the mosquito vector.
The results should be valuable for the national strategy against malaria in Mauritania.

Study area
This study was conducted in the city of Nouakchott (18°.11'N; 16°.16'W) at an average altitude of 7 m. The city is situated in a Sahara zone, near the Atlantic coast ( Figure 1), comprises nine districts and houses 743,511 inhabitants corresponding to one fourth of the whole population of the country [7]. The wet season is short, extending from August to September, with little annual variation in the amount of rainfall (50 to 80 mm). The average annual temperatures range from 20.7°C to 33.3°C, and the average relative humidity from 33.4% to79.1%.

Subject and blood sample collection
From April to August, corresponding in most part to the hot and dry season, a total of 237 febrile outpatients attending the two main hospitals of Nouakchott city were included in this study. All the patients had a fever associated with malaria-like symptoms (e.g. headache, vomiting, abdominal pain and diarrhoea). Blood samples were collected by finger-prick and blood dried filter paper samples were performed. An epidemiological record including age and sex of patients, axillary temperature, provenance, clinical symptoms, type of treatment and recent journey was completed for each patient. Of the patients attending the two hospitals, 6.75% had come from areas outside the city, the rest were inhabitants of Nouakchott who had no recent travel history or had never left Nouakchott.

Malaria parasites identification
Identification of Plasmodium sp. was undertaken using microscopy and polymerase chain reaction (PCR). Thin and thick blood films were put on the same slide and stained using standard methods [8]. One experienced laboratory technician examined the stained blood. The parasite density/μl was calculated by counting the number of parasites per 200 white blood cells (WBC), assuming a WBC count of 8,000/μl [9]. Data collected were graded according to parasitaemia into five classes: 1:<50; 2: 50-499; 3: 500-4,999; 4: 5,000-49,999; and 5: ≥ 50,000 [10]. As quality control, all slides were re-read at the 'Laboratoire de Paludologie et Zoologie tropicale' of the IRD (Senegal). Epidemiological and laboratory data were analysed using EpiInfo 6.04 software.
DNA was extracted from filter paper according to DNeasy Blood and Tissue handbook protocol (Qiagen, France). Amplification of genus and species-specific Plasmodium was done by using the primers: rPLU5 (5'-CCTGTTGTT-GCCTTAAACTTC-3'), rPLU6 (5'TTAAAATTGTTGCAGTT Map of Nouakchott city showing the different districts and the location of the two main hospitals  [11]. Each 20 μL reaction mixture for nest-1 amplification contained 5 μL of template DNA, 250 nM of each oligonucleotide primer (rPLU5 & rPLU6), 125 μM dNTP, 10× PCR buffer (500 mM KCl, 100 mM Tris-HCl [pH 8.3], 20 mM MgCl2), and 0.4 Units of HotStar Taq DNA polymerase (Qiagen, France). The reactions were subjected to 25 cycles under the following conditions: initial denaturation at 95°C for 5 min, annealing at 58°C for 2 min, extension at 72°C for 2 min, denaturation at 94°C for 1 min, final annealing at 58°C for 2 min, and final extension at 72°C for 5 min. About 5 μl of the nest-1 amplification products served as the DNA template for each of the 20 μl of second PCR (nest-2) amplification. Nest-2 was performed as nest-1, except that 30 cycles were performed. After amplification, the reaction mixture was loaded on a 1.5% agarose gel, separated by electrophoresis, stained with ethidium bromide and photographed under UV light.

Entomological investigations and sporozoites detection
Entomological investigations were carried out in Dar Naïm District, especially the surrounding area of Zaatar, for the search of malaria vector mosquitoes. This area is characterized by the presence of intense agricultural activity mainly consisting of vegetable crops. Mosquitoes were collected from five houses after indoor insecticide spraying early in the morning. The anophelines were identified to species level using morphological characteristics according to the identification keys of Gillies & De Meillon [12] and Gillies & Coetzee [13]. After identification, all mosquitoes belonging to Anopheles gambiae complex were stored in individual tubes with silica gel and preserved at -20°C in the laboratory. Species identification of the An. gambiae complex was done by PCR according to Scott et al [14]. All collected anopheline mosquitoes were examined by ELISA to detect circumsporozoite protein (CSP) as described by Burkot et al [15] and modified by Wirtz et al [16].

Results
This study was carried out to assess the incidence of malaria fever among a set of 237 outpatients with febrile symptoms consulting the two main Nouakchott's hospitals. The median age of the patients was 33.9 years ( Table  1). Most of the patients were males 59.1% (140) while 40.9% (97) were females (χ 2 = 7.48, P = 0.006). The mean axillary temperature overall the patients was 38.9°C. Thick blood film analysis showed that 61 out of the 237 tested individuals were positive for Plasmodium sp. giving a malaria infection rate of 25.7%. The highest number of infections was due to P. vivax with a rate of 70.5% (43/61) followed by P. ovale with 24.6% (15/61) and P. falciparum with a rate of 1.6% (1/61) ( Table 2). No infection with Plasmodium malariae was found. Variation of parasitaemia among the 61 positive patients showed that 22 had parasite density between 50-499 parasites/μL of blood ( Table  2). 18 and 15 malaria patients had a parasite density respectively between 500-4,999 and 5,000-49,999 parasite/μL of blood. The results of Nested-PCR conducted on a DNA extracted from blood samples of 148 patients demonstrated the high prevalence of P. vivax. Indeed an amplification product of 120 bp characterizing this species has been evidenced in 43 of the 148 tested samples.
The 61 positive cases came from all Nouakchott districts, except Sebkha, El Mina and Riadh districts were no malaria infections were found ( Table 3). The highest number of positive cases was recorded in the District of Dar Naïm (40 of 61) and Teyarett (13 of 61). Two of the 61 malaria positive patients, both with P. vivax, had never left Nouakchott and could, therefore, be considered as endemic cases.
The others lived in Nouakchott but had travelled to other regions. Of the 237 feverish cases, who attended the two hospital emergencies, the physicians diagnosed and treated 231 (97.5%) as malaria on clinical grounds (Table  4). A 26.4% (61/231) of clinically diagnosed cases were slide positives for Plasmodium using microscopic examina-

Discussion
This study is one of the few reports addressing malaria situation in Nouakchott city. Starting from 237 feverish outpatients consulting the two main health centers in this Saharan city, results showed a malaria prevalence rate of 25.7% among cases of fever with malaria-like symptoms and identified P. vivax as the dominant species followed by P. ovale. In a previous study, conducted in Nouakchott and based on microscopic examination of blood films from 446 febrile patients, Cortes and al [3] showed a malaria prevalence rate of 18.5%, 68.5% of which were due to P. falciparum and 35.5% to P. vivax. The difference between these results and those obtained by Cortes et al [3] can be explained by the difference in the periods during which the two studies were conducted and/or the different techniques used. In N'Djaména (Chad), a Sahelian town with highly seasonal malaria transmission, Othnigué and al [17] found that among 1,658 patients 30% were positive for Plasmodium (all P. falciparum) using rapid test diagnosis and microscopic examination. Low parasite densities were observed among the 61 malaria patients since 45 of them have less than 5,000 parasites/ μL. It could be that, in the majority of cases, these were relapses, characteristic of vivax malaria and where parasite density is generally lower than during the primary infection. Of the 237 analysed patients, 221 have no recent history of travel outside Nouakchott including two patients (3.3% of the cases diagnosed) that had never traveled out of the city. This result should support the results of Cortes et al [3] suggesting the presence of malaria endemic to Nouakchott. The high rate of malaria infection observed among febrile patients from Dar Naïm District and the identification of An. arabiensis and An. pharaoensis mosquitoes in the same district, strongly support this hypothesis. Indeed, the intensive gardening activities carried out in the areas surrounding this district could create adequate conditions for mosquitoes breeding. The presence of Anopheles spp. has been previously reported in the neighbouring district of Toujounine [18]. However, none of the eight trapped anophelines mosquitoes was found positive with CSP ELISA. The absence of sporozoïtes infection in this case may be due to the small sample size of mosquitoes. Further studies need to be carried out on bionomics of An. arabiensis and An. pharaoensis to determine their exact role in malaria transmission in Nouakchott.
The study also revealed that presumptive malaria was diagnosed as the main health problem for 97% of patients. The high frequency of malaria presumptive diag- * Parasitaemia is considered as the number of parasites per microlitre of blood; ** P = 0.002; *** P. vivax and P. ovale  [17,19]. It is worth remembering that giving anti-malarial drugs to all febrile cases or all presumptive malaria cases will contribute to the appearance of resistant forms [20,4]. Inadequate treatment can lead to severe complications and may results in a high burden and economic costs on health services and households.

Conclusion
During hot-dry season, malaria fever accounts for 25.7% of febrile cases in Nouakchott city and Plasmodium species responsible of recurrent malaria (P. vivax and P. ovale) are the most dominant species. Autochtonous malaria exists but is rare. Clinical diagnosis of malaria has a very low positive predicted value, and its low specificity leads to inappropriate care and high economic coast for a large proportion of patients. Regarding the low specificity of malaria clinical diagnosis in this Saharan urban setting, no malaria treatment should be given unless clinical diagnosis is confirmed by a laboratory-based diagnosis, such as rapid test or microscopy. Anopheles arabiensis may be the most important species involved in the transmission of malaria in Nouakchott, although its occurrence seems to be relatively low. * These were clinically diagnosed and treated as non-malaria fevers