Anemia, Thrombocytopenia, and Changes in Biochemical Parameters Occurring in Patients with Uncomplicated Plasmodium falciparum Malaria: Data Analysis from Antimalarial Efficacy-Randomized Trials in Dakar and Kaolack Regions, Senegal.

Background: Artemisinin-based Combination Therapies (ACTs) are widely used in the treatment of uncomplicated malaria. Plasmodium falciparum infection is often accompanied by disturbances of hematological and biochemical parameters. The objective of this study was to evaluate the changes in biochemical and hematological parameters during uncomplicated malaria in patients treated with ACTs. Methods: Data from patient with uncomplicated Plasmodium falciparum malaria were pooled from different open-randomized trial evaluating the efficacy of Artesunate-Mefloquine (ASMQ), Artesunate-Amodiaquine (ASAQ), Artemether-Lumefantrine (AL), and Dihydro-artemisinin-Piperaquine (DHAPQ) combinations. Biochemical (transaminases, creatinine, and bilirubin) and hematological (hemoglobin and platelet levels) parameters were performed at baseline (D0) and at day 7 after treatment (D7). Data were analyzed as both continuous and categorical variables with 95% confidence interval. Risks and trends were calculated using multivariate logistic random effect models. Results: A total of 720 patients with completed biological data were included in the analysis (320 in the AL arm, 160 in the ASMQ arm, 120 in the DHAPQ arm, and 88 in the ASAQ arm). The mean age of the patients was 9.43 ± 9.1 years. Male subjects represented 58.47% (sex ratio was 1.4 for males). The mean hemoglobin level at inclusion (D0) was 9.79 g/dl and anemia (Hb < 11 g/dl) was 71.43% (aOR = 1.16 [0.68 - 1.98]p = 0.57). At D7, hemoglobin level was 9.63 g/dl and anemia was significantly more frequent (78.29% [p = 0.002]). The mean platelet count at day 0 was 154075.5 platelets/mm3 of blood and 339328.7 platelets/mm3 at day 7. Thrombocytopenia was about 53.61% and was associated with malaria (aOR = 3.4 [2.18 - 5.3]p < 10-3). 19.58% of patients had abnormal ALT and 40.28% had abnormal AST at D0. 27.22% of patients had normal bilirubin at D0. Renal function was normal in all patients in the study. Normalization of transaminases was noted between D0 and D7. The percentage of subjects with normal bilirubin increased between D0 and D7. Renal function did not vary significantly between D0 and D7. Conclusion: Results from this analysis showed that subjects with high parasitaemia had a greater risk of anemia and thrombocytopenia. Artemisinin combinations were well-tolerated as no major biological disturbances were noted. The effects of ACTs on hematologic and biochemical parameters were not different.

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.

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.

Sero-epidemiological evaluation of Plasmodium falciparum malaria in Senegal.

BACKGROUND: In Senegal, a significant decrease of malaria transmission intensity has been noted the last years. Parasitaemia has become lower and, therefore, more difficult to detect by microscopy. In the context of submicroscopic parasitaemia, it has become relevant to rely on relevant malaria surveillance tools to better document malaria epidemiology in such settings. Serological markers have been proposed as an essential tool for malaria surveillance. This study aimed to evaluate the sero-epidemiological situation of Plasmodium falciparum malaria in two sentinel sites in Senegal. METHODS: Cross-sectional surveys were carried out in Velingara (south Senegal) and Keur Soce (central Senegal) between September and October 2010. Children under 10 years old, living in these areas, were enrolled using two-level, random sampling methods. P. falciparum infection was diagnosed using microscopy. P. falciparum antibodies against circumsporozoite protein (CSP), apical membrane protein (AMA1) and merozoite surface protein 1_42 (MSP1_42) were measured by ELISA method. A stepwise logistic regression analysis was done to assess factors associated with P. falciparum antibodies carriage. RESULTS: A total of 1,865 children under 10 years old were enrolled. The overall falciparum malaria prevalence was 4.99% with high prevalence in Velingara of 10.03% compared to Keur Soce of 0.3%. Symptomatic malaria cases (fever associated with parasitaemia) represented 17.37%. Seroprevalence of anti-AMA1, anti-MSP1_42 and anti-CSP antibody was 38.12, 41.55 and 40.38%, respectively. The seroprevalence was more important in Velingara and increased with age, active malaria infection and area of residence. CONCLUSION: The use of serological markers can contribute to improved malaria surveillance in areas with declining malaria transmission. This study provided useful baseline information about the sero-epidemiological situation of malaria in Senegal and can contribute to the identification of malaria hot spots in order to concentrate intervention efforts. TRIAL REGISTRATION NUMBER: PACTR201305000551876 ( http://www.pactr.org ).

Usefulness of MALDI-TOF Mass Spectrometry for Routine Identification of Candida Species in a Resource-Poor Setting.

BACKGROUND: Identification of fungal clinical isolates is essential for therapeutic management. In resource-limited settings, identification mostly relies on biochemical tests whose sensitivity and specificity are known to be insufficient for identification of closely related or newly described species. MALDI-TOF has been shown in favored countries to be a reliable and powerful tool for microorganism identification, including yeasts. The aim of this study was to compare MALDI-TOF with routine identification procedures in a resource-poor context. METHODS: A total of 734 clinical specimens (502 vaginal swabs, 147 oral swabs, 61 bronchoalveolar lavage fluids and 24 stool samples) have been tested in the mycology unit of Fann Hospital, Dakar, Senegal. Strains isolated from culture were identified by both conventional phenotypic methods (germ tube formation and biochemical panels) and MALDI-TOF Saramis/VITEK MS, bioMérieux, France. In addition to comparing the final identification, we determined the time of obtaining the results and the cost for both approaches. RESULTS: Overall, 218 (29.7 %) samples were positive for Candida. MALDI-TOF MS enabled the identification of 214 of the 218 strains isolated (98.1 %) at species level. Phenotypic approach yielded identification for 208 strains (95.4 %). Congruence between the tests was observed for 203 isolates. A discrepancy was observed for one isolate identified as Candida krusei with the phenotypic approach and Candida tropicalis with the MALDI-TOF. In addition, ten isolates identified at genus level by phenotypic methods were identified as C. glabrata (n = 8), C. tropicalis (n = 1) and C. parapsilosis (n = 1) by MALDI-TOF. The turnaround time for identification was <1 h using the MALDI-TOF compared to our routine procedures (48 h). The overall cost (reagents + expendables) per isolate was at 1.35 for the MALDI-TOF MS. CONCLUSION: MALDI-TOF clearly outperformed the diagnosis capacities of phenotypic methods by reducing the delay of results and giving accurate identification at species level. Moreover, this approach appears to be cost-effective and should be implemented especially in resource-poor context.

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

BACKGROUND: Malaria 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. METHODS: An 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. RESULTS: Overall, 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. CONCLUSION: In 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 REGISTRATION: PACTR 201305000552290.

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

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.

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.

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

BACKGROUND: In 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. METHODS: This 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. RESULTS: Mutations 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. CONCLUSION: The 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.

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

BACKGROUND: Prompt 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. METHODS: The 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. RESULTS: Overall, 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. CONCLUSION: Artesunate-mefloquine (25 mg/Kg mefloquine) is efficacious and well-tolerated for the treatment of uncomplicated P. falciparum malaria in adult patients.

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.