Annie Abiola

Assessment of the Molecular Marker of Plasmodium falciparum Chloroquine Resistance (Pfcrt) in Senegal after Several Years of Chloroquine Withdrawal

As a result of widespread antimalarial drug resistance, all African countries with endemic malaria have, in recent years, changed their malaria treatment policy. In Senegal, the health authorities changed from chloroquine (CQ) to a combination of sulfadoxine–pyrimethamine (SP) plus amodiaquine (AQ) in 2003. Since 2006, the artemisinin combination therapies (ACTs) artemether–lumefantrine (AL) and artesunate plus amodiaquine (AS/AQ) were adopted for uncomplicated malaria treatment. After several years of CQ withdrawal, the current study wished to determine the level of CQ resistance at the molecular level in selected sites in Senegal, because the scientific community is interested in using CQ again. Finger prick blood samples were collected from Plasmodium falciparum-positive children below the age of 10 years (N = 474) during cross-sectional surveys conducted in two study sites in Senegal with different malaria transmission levels. One site is in central Senegal, and the other site is in the southern part of the country. All samples were analyzed for single nucleotide polymorphisms (SNPs) in the P. falciparum CQ resistance transporter gene (Pfcrt; codons 72–76) using polymerase chain reaction (PCR) sequence-specific oligonucleotide probe (SSOP) enzyme-linked immunosorbent assay (ELISA) and real-time PCR methods. In total, the 72- to 76-codon region of Pfcrt was amplified in 449 blood samples (94.7%; 285 and 164 samples from the central and southern sites of Senegal, respectively). In both study areas, the prevalence of the Pfcrt wild-type single CVMNK haplotype was very high; in central Senegal, the prevalence was 70.5% in 2009 and 74.8% in 2010, and in southern Senegal, the prevalence was 65.4% in 2010 and 71.0% in 2011. Comparing data with older studies in Senegal, a sharp decline in the mutant type Pfcrt prevalence is evident: from 65%, 64%, and 59.5% in samples collected from various sites in 2000, 2001, and 2004 to approximately 30% in our study. A similar decrease in mutant type prevalence is noted in other neighboring countries. With the continued development of increased CQ susceptibility in many African countries, it may be possible to reintroduce CQ in the near future in a drug combination; it could possibly be given to non-vulnerable groups, but it demands close monitoring of possible reemergence of CQ resistance development.

Monitoring the efficacy and safety of three artemisinin based-combinations therapies in Senegal: results from two years surveillance

BackgroundMalaria remains a major public health problem in developing countries. Then in these countries prompt access to effective antimalarial treatment such as Artemisinin based-Combination Therapies (ACT) proves to be an essential tool for controlling the disease. In Senegal, since 2006 a nationwide scaling up program of ACT is being implemented. In this context it has become relevant to monitor ACT efficacy and provide recommendations for the Senegalese national malaria control program.MethodsAn open randomized trial was conducted during two malaria transmission seasons (2011 and 2012) to assess the efficacy and safety of three combinations: dihydro-artemisinin-piperaquine (DHAPQ), artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ). The primary end point of the study was represented by a PCR adjusted adequate clinical and parasitological response (ACPR) at day 28. Secondary end points included: (i) a ACPR at days 35 and 42, (ii) a parasite and fever clearance time, (iii) ACTs safety and tolerability. The 2003 WHO’s protocol for antimalarial drug evaluation was used to assess each outcome.ResultsOverall, 534 patients were randomized selected to receive, either ASAQ (n = 180), AL (n = 178) or DHAPQ (n = 176). The PCR adjusted ACPR at day 28 was 99.41% for the group ASAQ, while that was 100% in the AL and DHAPQ groups (p = 0.37). The therapeutic efficacy was evaluated at 99.37% in the ASAQ arm versus 100% in AL and DHAPQ arm at day 35 (p = 0.37). At day 42, the ACPR was 99.27% in the ASAQ group versus 100% for both AL and DHAPQ groups, (p = 0.36). No serious adverse event was noted during the study period. Also a similar safety profile was noted in the 3 study groups.ConclusionIn the context of scaling up of ACTs in Senegal, ASAQ, AL and DHAPQ are highly effective and safe antimalarial drugs. However, it’s remains important to continue to monitor their efficacy.Trial registrationPACTR 201305000552290.

Efficacy and tolerability of a new formulation of artesunate-mefloquine for the treatment of uncomplicated malaria in adult in Senegal: open randomized trial

BackgroundPrompt treatment of malaria attacks with arteminisin-based combination therapy (ACT) is an essential tool for malaria control. A new co-blister tablet of artesunate-mefloquine (AM) with 25 mg/kg mefloquine has been developed for the management of uncomplicated malaria attacks. This non-inferiority randomized trial, was conducted to evaluate the efficacy and safety of the new formulation of AM in comparison to artemether-lumefantrine (AL) for the treatment of acute uncomplicated Plasmodium falciparum malaria in adults in Senegal.MethodsThe study was carried out from September to December 2010 in two health centres in Senegal. The study end points included (i) PCR corrected adequate clinical and parasitological response (ACPR) at day 28, (ii) ACPR at days 42 and 63, (iii) parasites and fever clearance time, (iv) incidence of adverse events and patients biological profile at day 7 using the WHO 2003 protocol for anti-malarial drug evaluation.ResultsOverall, 310 patients were randomized to receive either AM (n = 157) or AL (n = 153). PCR corrected ACPR at day 28 was at 95.5% in the AM arm while that in the AL arm was at 96.7% (p = 0.83). Therapeutic efficacy was at 98.5% in the AM arm versus 98.2% in the AL group at day 42 (p = 1). At day 63, ACPR in the AM and AL arms was at 98.2% and 97.7%, respectively (p = 0.32). The two treatments were well tolerated with similar biological profile at day 7. However, dizziness was more frequent in the AM arm.ConclusionArtesunate-mefloquine (25 mg/Kg mefloquine) is efficacious and well-tolerated for the treatment of uncomplicated P. falciparum malaria in adult patients.

Safety, Immunogenicity and Efficacy of Prime-Boost Vaccination with ChAd63 and MVA Encoding ME-TRAP against Plasmodium falciparum Infection in Adults in Senegal

Malaria transmission is in decline in some parts of Africa, partly due to the scaling up of control measures. If the goal of elimination is to be achieved, additional control measures including an effective and durable vaccine will be required. Studies utilising the prime-boost approach to deliver viral vectors encoding the pre-erythrocytic antigen ME-TRAP (multiple epitope thrombospondin-related adhesion protein) have shown promising safety, immunogenicity and efficacy in sporozoite challenge studies. More recently, a study in Kenyan adults, similar to that reported here, showed substantial efficacy against P. falciparum infection. One hundred and twenty healthy male volunteers, living in a malaria endemic area of Senegal were randomised to receive either the Chimpanzee adenovirus (ChAd63) ME-TRAP as prime vaccination, followed eight weeks later by modified vaccinia Ankara (MVA) also encoding ME-TRAP as booster, or two doses of anti-rabies vaccine as a comparator. Prior to follow-up, antimalarials were administered to clear parasitaemia and then participants were monitored by PCR for malaria infection for eight weeks. The primary endpoint was time-to-infection with P. falciparum malaria, determined by two consecutive positive PCR results. Secondary endpoints included adverse event reporting, measures of cellular and humoral immunogenicity and a meta-analysis of combined vaccine efficacy with the parallel study in Kenyan adults.We show that this pre-erythrocytic malaria vaccine is safe and induces significant immunogenicity, with a peak T-cell response at seven days after boosting of 932 Spot Forming Cells (SFC)/106 Peripheral Blood Mononuclear Cells(PBMC) compared to 57 SFC/ 106 PBMCs in the control group. However, a vaccine efficacy was not observed: 12 of 57 ME-TRAP vaccinees became PCR positive during the intensive monitoring period as compared to 13 of the 58 controls (P = 0.80). This trial confirms that vaccine efficacy against malaria infection in adults may be rapidly assessed using this efficient and cost-effective clinical trial design. Further efficacy evaluation of this vectored candidate vaccine approach in other malaria transmission settings and age-de-escalation into the main target age groups for a malaria vaccine is in progress.

Prevalence of molecular markers of drug resistance in an area of seasonal malaria chemoprevention in children in Senegal

BackgroundIn sub-Saharan Africa, malaria is the leading cause of morbidity and mortality especially in children. In Senegal, seasonal malaria chemoprevention (SMC) previously referred to as intermittent preventive treatment in children (IPTc) is a new strategy for malaria control in areas of high seasonal transmission. An effectiveness study of SMC, using sulphadoxine-pyrimethamine (SP) plus amodiaquine (AQ), was conducted in central Senegal from 2008 to 2010 to obtain information about safety, feasibility of delivery, and cost effectiveness of SMC. Here are report the effect of SMC delivery on the prevalence of markers of resistance to SP and AQ.MethodsThis study was conducted in three health districts in Senegal with 54 health posts with a gradual introduction of SMC. Three administrations of the combination AQ + SP were made during the months of September, October and November of each year in children aged less than 10 years living in the area. Children were surveyed in December of each year and samples (filter paper and thick films) were made in 2008, 2009 and 2010. The prevalence of mutations in the pfdhfr, pfdhps, pfmdr1 and pfcrt genes was investigated by sequencing and RTPCR in samples positive by microscopy for Plasmodium falciparum.ResultsMutations at codon 540 of pfdhps and codon 164 of pfdhfr were not detected in the study. Among children with parasitaemia at the end of the transmission seasons, the CVIET haplotypes of pfcrt and the 86Y polymorphism of pfmdr1 were more common among those that had received SMC, but the number of infections detected was very low and confidence intervals were wide. The overall prevalence of these mutations was lower in SMC areas than in control areas, reflecting the lower prevalence of parasitaemia in areas where SMC was delivered.ConclusionThe sensitivity of P. falciparum to SMC drugs should be regularly monitored in areas deploying this intervention. Overall the prevalence of genotypes associated with resistance to either SP or AQ was lower in SMC areas due to the reduced number of parasitaemia individuals.

Selection of Antimalarial Drug Resistance after Intermittent Preventive Treatment of Infants and Children (IPTi/c) in Senegal

Our study investigated the possible impact of SP-IPT given to infants and children on the prevalence of SP-resistant haplotypes in the Plasmodium falciparum genes Pfdhfr and Pfdhps, comparing sites with and without IPTi/c. P. falciparum positive samples (N = 352) collected from children < 5 years were analyzed to determine the prevalence of SP resistance-related haplotypes by nested PCR followed by sequence-specific oligonucleotide probe-enzyme-linked immunosorbent assay. The prevalence of the Pfdhfr triple mutant haplotype (CIRN) increased in both groups, but only significantly in the IPTi/c group from 41% to 65% in 2011 (P = 0.005). Conversely, the Pfdhps 437G mutation decreased in both groups from 44.6% to 28.6% (P = 0.07) and from 66.7% to 47.5% (P = 0.02) between 2010 and 2011 in the control and the IPTi/c groups, respectively. A weak trend for decreasing prevalence of quadruple mutants (triple Pfdhfr + Pfdhps 437G) was noted in both groups (P = 0.15 and P = 0.34). During the two cross-sectional surveys some significant changes were observed in the SP resistance-related genes.

Accuracy of HRP2 RDT (Malaria Antigen P.f®) compared to microscopy and PCR for malaria diagnosis in Senegal

Abstract Rapid diagnosis tests (RDTs) allow for the confirmation of malaria diagnosis. In Senegal, RDTs detecting HRP2 have been adopted in 2008 for malaria diagnosis. However, the sustainability of this strategy requires adequate and regular quality control. PCR on DNA extracted in nitrocellulose band of RDTs enable quality control. A RDT (Malaria Antigen P.f®) and a thick smear were performed on patients with suspected malaria. DNA was extracted from the nitrocellulose band of RDTs to which a non-specific PCR and a specific PCR were applied. The results of the RDT were compared with those obtained from the thick smear and the PCR to measure sensitivity, specificity as well as positive and negative predictive values. For 81·6% of the 273 patients involved, the thick smear was positive. Rapid diagnosis tests were positive for 85·7% of the patients. Non-specific PCR was positive on 87·9% of RDTs. Plasmodium falciparum was found in 99·5% of patients and Plasmodium ovale appeared in only 0·4% of patients. Sensitivity of the Malaria Antigen Pf® RDT in relation to thick smear and to PCR was 98·2% and 97·1% respectively. Quality control with PCR on the nitrocellulose band performed several months after it was used confirms its adequate level of sensitivity. The collection and screening of DNA present in already used RDT is a good means of quality control for this tool. It is also a relevant alternative to the molecular approach in the context of a reduction in the transmission of malaria.

Genetic Analysis of Erythrocyte Binding Antigen 175 (EBA-175), ApicalMembrane Antigen (AMA-1) and Merozoite Surface Protein 3 (MSP-3) AllelicTypes in Plasmodium Falciparum Isolates From Rural Area in Senegal

Background: Several of the intended P. falciparum vaccine candidate antigens are highly polymorphic and could render a vaccine ineffective if their antigenic sites were not represented in the vaccine. This study aimed to characterize genetic diversity of vaccine candidate antigens merozoite surface protein-3 (MSP-3), apical membrane antigen-1 (AMA-1) and erythrocyte binding antigen (EBA-175) in P. falciparum isolates from Senegal. Methods: DNA analysis was completed on 170 isolates of P. falciparum collected from Keur Soce in Senegal between 2006 and 2008. Genetic diversity was determined in the three P. falciparum genes by, PCR followed by restriction fragment length polymorphism (RFLP). Results: From 170 samples collected, successful, PCR products were obtained from 135 (79%), 140 (82%) and 128 (75%) for AMA-1, MSP-3 and EBA-175, respectively. The results showed that the EBA-175 gene presented 4 different alleles [EBA-175F_loop (62.3%), EBA-175C_loop (46.1%), EBA-175~400bp (17.6%), EBA-175~360bp (8.4%)]. Regarding the MSP-3 patterns, the analysis revealed the presence of three alleles MSP-3_K1 (49.2%), MSP-3_3D7 (54.2%) and MSP-3~350bp (15%). For AMA-1, the results showed three different alleles AMA-1_K1 (39%), AMA-1_HB3 (33%), AMA-1_3D7 (32%). Conclusion: Characterization of the genetic diversity in Plasmodium isolates from Keur Soce in Senegal in the three genes investigated showed a high degree of polymorphism. These findings are helpful in the formulation of a vaccine considering restricted repertoire populations.

Potential Impact of Seasonal Malaria Chemoprevention on the Acquisition of Antibodies Against Glutamate-Rich Protein and Apical Membrane Antigen 1 in Children Living in Southern Senegal

Seasonal malaria chemoprevention (SMC) is defined as the intermittent administration of full treatment courses of an antimalarial drug to children during the peak of malaria transmission season with the aim of preventing malaria-associated mortality and morbidity. SMC using sulfadoxine–pyrimethamine (SP) combined with amodiaquine (AQ) is a promising strategy to control malaria morbidity in areas of highly seasonal malaria transmission. However, a concern is whether SMC can delay the natural acquisition of immunity toward malaria parasites in areas with intense SMC delivery. To investigate this, total IgG antibody (Ab) responses to Plasmodium falciparum antigens glutamate-rich protein R0 (GLURP-R0) and apical membrane antigen 1 (AMA-1) were measured by enzyme-linked immunosorbent assay in Senegalese children under the age of 10 years in 2010 living in Saraya and Velingara districts (with SMC using SP+AQ [SMC+] since 2007) and Tambacounda district (without SMC (SMC−)). For both P. falciparum antigens, total IgG response were significantly higher in the SMC− compared with the SMC+ group (for GLURP-R0, P < 0.001 and for AMA-1, P = 0.001). There was as well a nonsignificant tendency for higher percentage of positive responders in the SMC− compared with the SMC+ group (for GLURP-R0: 22.2% versus 14.4%, respectively [P = 0.06]; for AMA-1: 45.6% versus 40.0%, respectively [P = 0.24]). Results suggest that long-term malaria chemoprevention by SMC/SP+AQ have limited impact on the development of acquired immunity, as tested using the P. falciparum antigens GLURP-R0 and AMA-1. However, other factors, not measured in this study, may interfere as well.

Profil des cytokines associées à la protection contre les accès palustres au cours de la grossesse en zone hypo-endémique au Sénégal

BACKGROUND Malarial infection in non immune pregnant women is a major risk factor for pregnancy failure. However in malaria endemic areas, intermittent preventive treatment (IPTp) have been adopted to prevent malaria in pregnancy women since 2003 in Senegal. The impact of IPT on the development of immunity is not very well documented. We conducted a prospective study at the Roi-Baudouin maternity hospital of Guediawaye in Senegal to assess IL10, IL12, TNFα and IFNγ cytokines production in pregnant women under IPTp. Cytokines were analyzed in 82 sera at inclusion and delivery. P. falciparum HRP2 antigen was detected in 17% of women included by rapid diagnostic test (RDT). At inclusion the mean of IL10 response was higher in P. falciparum negative women (8 UA) compare to RDT-positive women (7 UA) p=0.069 while in delivery the opposite was found p=0.014. Low production of inflammatory cytokines IL12, IFNγ and TNFα was noted in both groups. Between inclusion and delivery, a significant increase of IL-10 production was noted while a decrease of IFNγ and TNFα cytokine was noted. Thus, IL12 and IFNγ responses may synergistically associate as malaria immune response during pregnancy.