Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy

In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon.


Introduction
In the early 1980s, sulfadoxine-pyrimethamine (SP) has been adopted for treatment of malaria cases when chloroquine treatment failed in many Sub-Saharan African countries [1]. However, soon after its introduction, resistance to SP has gradually emerged and spread widely from Asia to Africa [2,3]. Nevertheless, SP is currently recommended by the World Health Organization (WHO) as intermittent preventive treatment for malaria in pregnant woman (IPTp) and children (IPTi) in malaria-endemic areas; it is also used as partner molecule of artemisinin derivatives [4,5]. It is known that the presence of specific mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthetase (Pfdhps) genes encoding for proteins involved in the folate biosynthesis pathway of P. falciparum is related to SP resistance [6][7][8]. Molecular studies document the prevalence of these mutations in parasite populations across the African continent [9]. The Pfdhfr double mutations N51I-S108N and C59R-S108N confer intermediate levels of resistance while triple mutation N51I-C59R-S108N increases the parasite strains level of resistance in Africa [1]. The quadruple mutant N51I-C59R-S108N-I164R shows highest ex vivo resistance to pyrimethamine so far [6]. Similarly, an amino acid change at codon 437 on Pfdhps enzyme is due to the key mutation associated with sulfadoxine resistance. Additional changes at positions 540, 581, and 613 appear to increase its level [10]. The presence of quintuple mutation, composed of the combination of the Pfdhfr triple mutation (N51I-C59R-S108N) and the Pfdhps double mutation (437-540 or 437-581), increases the risk of therapeutic failure to SP [11]. However, the complexities of the evolutionary pressures that lead to the evolution of drug resistance are not well understood. Microbial systems that allow heterologous expression of malarial proteins provide the way to investigate patterns of evolution that can inform on the more complex factors that influence the evolution of drug resistance in clinical settings [12]. Indeed, preventive therapy based on SP use protects against parasite infection but induces the genesis of gametocytes and can promote the spread of resistant parasites [13]. In Gabon, IPTp-SP for pregnant women and artesunate-SP for uncomplicated malaria treatment are recommended by Malaria National Control Programme (MNCP) for malaria prevention and treatment, respectively. Five years after its introduction, the coverage with IPTp-SP two doses reached 60% [14]. Moreover, SP is still widely sold in pharmacies and distributors in remote areas from the country, despite the introduction of artemisinin combinations therapy (ACTs) for uncomplicated malaria treatment.
At the beginning of the year 2000 before IPT-p-SP implementation, previous studies describing the distribution of drug resistance molecular markers (dhfr and dhps genes) have been carried out in different areas of the country. In the southeast area, in the Haut-Ogooué Province, at Franceville, the proportion of isolates carrying a mutation at codon 108 was of 52%, while mutations at codons 51 and 59 as well as triple mutation were not frequent [15]. In contrast, at the same period and near Franceville, in Bakoumba, the triple mutant DHFR genotype was frequently detected (71.8%) whereas 64.3% combined at least three DHFR and one DHPS mutations [16].
In other regions, such as in the north of Gabon, at Oyem, similar investigations were not performed. In this area, regardless of the implementation of new strategies for malaria control, malaria prevalence remains high [17]. At the time of the study period, malaria prevalence tends to decrease; however, it was above 40% up to now. In this town surrounded by the forest and with a low level of urbanization, malaria transmission is perennial and children are frequently infected and constitute the main reservoir of parasites. Among many factors, the presence of resistant parasites could contribute to maintaining malaria burden in this region. Use of genetic information, for the early detection of resistance and monitoring of drug resistant malaria, is a helpful epidemiological tool. Indeed, molecular markers of resistance have emerged as epidemiologic tools to investigate antimalarial drug resistance even before becoming clinically evident. In this area, a previous study reported a high prevalence of pfcrt and pfmdr1 drug resistance molecular markers associated with amodiaquine resistance, a partner molecule of artemisinin derivatives in ACTs [18]. This is the first study that reports baseline information on the characteristics and implications of antimalarial drug resistance, in the north of Gabon, with the aim to provide a data baseline on drug resistance. Thus, the prevalence of mutations on Pfdhfr and Pfdhps genes was compared in isolates collected in 2005 at the time of IPTp-SP introduction and three years later at Oyem in north of Gabon.

Study Site.
Participants have been recruited through two different cross sectional studies carried out in 2005 and in 2008 at the regional hospital of Oyem (Centre Hospitalier Regional d'Oyem (CHRO)). The hospital of Oyem is the main hospital of the north of the country, a regional hospital. The majority of the population of the Oyem city which accounts for more than 35000 inhabitants and that of the surrounding villages receive healthcare in this hospital. CHRO is one of the five sentinel sites selected for malaria survey by the Malaria National Control Programme (MNCP). At Oyem, malaria prevalence remains above 40% among febrile children for a decade, and infection is predominantly caused by P. falciparum [17].

Patients and Samples.
Isolates were collected from febrile children aged less than 11 years [19]. Patients who had positive blood smears and P. falciparum monoinfection were selected. The oral consent of the parents or legal guardians was also obtained. Blood was spotted on filter paper for further genetic analysis. Demographic data and medical history were reported.
2.3. Malaria Diagnosis. P. falciparum infection diagnosis was done by microscopic examination according to Lambaréné's method [20]. Smears were read using a light microscope with the 100x objective. Parasitaemia was expressed as a number of parasites per microliter of blood and parasite species was identified in the matched thin blood smears. Smears were considered as negative if the examination read under 100 of oil immersion fields did not reveal any parasites. Malaria case was defined as a patient with an axillary temperature >37.5 ∘ C and Plasmodium falciparum infection without WHO criteria of severity.

Pfdhfr and Pfdhps Genes
Typing. Parasite nucleic acids were extracted from filter paper using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions and subsequently stored at −20 ∘ C until use.

Ethical Considerations. Department of Parasitology and Mycology (DPM) is committed by the Gabonese Ministry of
Health represented by MNCP to carry out malaria diagnosis and antimalarial drug resistance monitoring throughout the country. Parents and legal guardians were informed about the studies and the consecutive molecular analysis. Their oral consent was obtained prior to inclusion in the study and before sample collection. Each patient with malaria positive blood smears was treated according to national recommendations at the time of the study.
2.6. Data Analysis. All data were entered and cleaned using Epi-info version 3.3.2. Analysis was performed with the Statview 5.0 software. All variables were compared using 2 test or Fisher's exact test. value < 0.05 was considered statistically significant.

Discussion
Occurrence and expansion of P. falciparum mutant genotypes frequency may depend on various factors such as year and location of study and age and clinical status of sampled population. In the present study, the prevalence of mutations on Pfdhfr and Pfdhps genes was compared in isolates collected in 2005 at the time of IPTp-SP introduction and three years later at Oyem, in the north of Gabon. Malaria prevalence there is above 40% and the treatment failures rates after SP administration were of 11.6% among children aged less than 5 years in 2005 [19]. In this area, the proportion of mutant alleles N108 and R59 of Pfdhfr gene in P. falciparum isolates was already high in 2005 and did not vary in 2008. Frequencies of these mutants' alleles were comparable to those found in isolates collected during the same period, between 2005 and 2006, at Lambaréné in the centre of Gabon and Libreville, the capital city of Gabon, two areas where malaria prevalence is different [22,23]. Likewise, during the year 2006, in Kenya, more than 95% of P. falciparum isolates carried dhfr mutant alleles, a proportion that was already around 80% in the 1990s [24]. In contrast, in Iran and Senegal where malaria transmission is lower, during the same period, such high prevalence of dhfr mutant alleles was not found (<90%) [25,26]. Concerning the mutant allele 51I, its frequency was above 75% in Lambaréné (79%) and Libreville (92%) while at Oyem it did not reach 50% (48.3%) [22,23]. However, very quickly, in 2008, this allele was found in more than 95% of P. falciparum isolates collected at Oyem, a proportion comparable to those reported in regions from Congo (88%) and Benin (>90%) [27,28]. Mutation at codon 164, which is associated with an elevated level of pyrimethamine resistance, was not detected. This mutation is rare in Africa [28,29]. The triple dhfr mutation was also frequently detected at Oyem in 2008 (>90%), in a proportion comparable to the one found in Senegal (93%) and Benin (91.8%) in 2011 but higher compared to the ones found in Burkina Faso (54.3%) and Rwanda (78%) during the same period [26,30]. In Iran, where malaria transmission is low and although sulfadoxine/pyrimethamine-artesunate combination has been adopted and recommended as first-line drug treatment the proportion of the triple mutation 108N-59R-437G is constant around 39% [25]. The combination of the triple mutation Pfdhfr and 437G mutation of the gene Pfdhps was found in two-thirds of the isolates analyzed in the present study, while in Senegal 44% of the isolates carried this haplotype [26]. The data obtained during the present study and those from previous investigations underline a different distribution and evolution of SP molecular markers resistance in Gabon. Indeed, while proportions of triple dhfr mutation and I51 mutant allele were already high in Lambaréné and Libreville during the years 2005-2006, at Oyem comparable frequencies were reached three years after in 2008. Nevertheless, between 2005 and 2011 at Libreville, the triple Pfdhfr mutation frequency increased from 92.9% to 100% and multiple mutation from 17.9% to 75.6% [31]. Dhps mutant alleles at codons 581 and 540 as well as the dhfr-dhps quintuple mutation were not found at Oyem while they were detected at Libreville and Lambaréné. The presence of the dhfr-dhps quintuple mutation in P. falciparum isolates is reported to be a good indicator of the abandonment of the SP [9,23,32,33]. These mutations were detected in 4% and 22% of the isolates from Lambaréné and Libreville, respectively. The main limitation of the present study could be the small number of isolates available and analyzed. However, these data provide important information allowing the setup of a database on antimalarial drug resistance molecular markers in Gabon. Indeed, although the number of samples is small, it is possible to draw from the present analyses accurate data and information on the prevalence of mutant parasites circulating in this area as performed by others [34]. These data already represent an "alarm" in this area where malaria transmission is high and suggest at least an increasing drug pressure in this area, related presumably to SP or other antifolates use.

Conclusion
The present study shows that Pfdhfr and Pfdhps mutant allele's frequencies are elevated, probably due to an increased use of sulfadoxine-pyrimethamine at Oyem. Three years after the adoption of WHO recommendation, frequencies of the combination of triple Pfdhfr mutation and Pfdhps 437G allele have risen, although among multiple mutations detected the quintuple mutation was not found. The monitoring of drug resistance molecular markers should be performed regularly and associated with the assessment of SP efficacy in vivo or ex vivo assays for Oyem.