Prevalence of the dhfr and dhps Mutations among Pregnant Women in Rural Burkina Faso Five Years after the Introduction of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine.

BACKGROUND: The emergence and spread of drug resistance represents one of the biggest challenges for malaria control in endemic regions. Sulfadoxine-pyrimethamine (SP) is currently deployed as intermittent preventive treatment in pregnancy (IPTp) to prevent the adverse effects of malaria on the mother and her offspring. Nevertheless, its efficacy is threatened by SP resistance which can be estimated by the prevalence of dihydropteroate synthase (dhps) and dihydrofolate reductase (dhfr) mutations. This was measured among pregnant women in the health district of Nanoro, Burkina Faso. METHODS: From June to December 2010, two hundred and fifty six pregnant women in the second and third trimester, attending antenatal care with microscopically confirmed malaria infection were invited to participate, regardless of malaria symptoms. A blood sample was collected on filter paper and analyzed by PCR-RFLP for the alleles 51, 59, 108, 164 in the pfdhfr gene and 437, 540 in the pfdhps gene. RESULTS: The genes were successfully genotyped in all but one sample (99.6%; 255/256) for dhfr and in 90.2% (231/256) for dhps. The dhfr C59R and S108N mutations were the most common, with a prevalence of 61.2% (156/255) and 55.7% (142/255), respectively; 12.2% (31/255) samples had also the dhfr N51I mutation while the I164L mutation was absent. The dhps A437G mutation was found in 34.2% (79/231) isolates, but none of them carried the codon K540E. The prevalence of the dhfr double mutations NRNI and the triple mutations IRNI was 35.7% (91/255) and 11.4% (29/255), respectively. CONCLUSION: Though the mutations in the pfdhfr and pfdhps genes were relatively common, the prevalence of the triple pfdhfr mutation was very low, indicating that SP as IPTp is still efficacious in Burkina Faso

Increase in the prevalence of mutations associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum isolates collected from early to late pregnancy in Nanoro, Burkina Faso.

BACKGROUND: Pregnant women are a high-risk group for Plasmodium falciparum infections, which may result in maternal anaemia and low birth weight newborns, among other adverse birth outcomes. Intermittent preventive treatment with sulfadoxine-pyrimethamine during pregnancy (IPTp-SP) is widely implemented to prevent these negative effects of malaria. However, resistance against SP by P. falciparum may decrease efficacy of IPTp-SP. Combinations of point mutations in the dhps (codons A437, K540) and dhfr genes (codons N51, C59, S108) of P. falciparum are associated with SP resistance. In this study the prevalence of SP resistance mutations was determined among P. falciparum found in pregnant women and the general population (GP) from Nanoro, Burkina Faso and the association of IPTp-SP dosing and other variables with mutations was studied. METHODS: Blood spots on filter papers were collected from pregnant women at their first antenatal care visit (ANC booking) and at delivery, from an ongoing trial and from the GP in a cross-sectional survey. The dhps and dhfr genes were amplified by nested PCR and products were sequenced to identify mutations conferring resistance (ANC booking, n = 400; delivery, n = 223; GP, n = 400). Prevalence was estimated with generalized estimating equations and for multivariate analyses mixed effects logistic regression was used. RESULTS: The prevalence of the triple dhfr mutation was high, and significantly higher in the GP and at delivery than at ANC booking, but it did not affect birth weight. Furthermore, quintuple mutations (triple dhfr and double dhps mutations) were found for the first time in Burkina Faso. IPTp-SP did not significantly affect the occurrence of any of the mutations, but high transmission season was associated with increased mutation prevalence in delivery samples. It is unclear why the prevalence of mutations was higher in the GP than in pregnant women at ANC booking. CONCLUSION: The high number of mutants and the presence of quintuple mutants in Burkina Faso confirm concerns about the efficacy of IPTp-SP in the near future. Other drug combinations to tackle malaria in pregnancy should, therefore, be explored. An increase in mutation prevalence due to IPTp-SP dosing could not be confirmed

Ex vivo anti-malarial drug susceptibility of Plasmodium falciparum isolates from pregnant women in an area of highly seasonal transmission in Burkina Faso.

BACKGROUND: Ex vivo assays are usually carried out on parasite isolates collected from patients with uncomplicated Plasmodium falciparum malaria, from which pregnant women are usually excluded as they are often asymptomatic and with relatively low parasite densities. Nevertheless, P. falciparum parasites infecting pregnant women selectively sequester in the placenta and may have a different drug sensitivity profile compared to those infecting other patients. The drug sensitivity profile of P. falciparum isolates from infected pregnant women recruited in a treatment efficacy trial conducted in Burkina Faso was determined in an ex vivo study. METHODS: The study was conducted between October 2010 and December 2012. Plasmodium falciparum isolates were collected before treatment and at the time of any recurrent infection whose parasite density was at least 100/µl. A histidine-rich protein-2 assay was used to assess their susceptibility to a panel of seven anti-malarial drugs. The concentration of anti-malarial drug inhibiting 50% of the parasite maturation to schizonts (IC(50)) for each drug was determined with the IC Estimator version 1.2. RESULTS: The prevalence of resistant isolates was 23.5% for chloroquine, 9.2% for mefloquine, 8.0% for monodesethylamodiaquine, and 4.4% for quinine. Dihydroartemisinin, mefloquine, lumefantrine, and monodesethylamodiaquine had the lowest mean IC(50) ranging between 1.1 and 1.5 nM respectively. The geometric mean IC(50) of the tested drugs did not differ between chloroquine-sensitive and resistant parasites, with the exception of quinine, for which the IC(50) was higher for chloroquine-resistant isolates. The pairwise comparison between the IC(50) of the tested drugs showed a positive and significant correlation between dihydroartemisinin and both mefloquine and chloroquine, between chloroquine and lumefantrine and between monodesethylamodiaquine and mefloquine. CONCLUSION: These ex vivo results suggest that treatment with the currently available artemisinin-based combinations is efficacious for the treatment of malaria in pregnancy in Burkina Faso. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00852423

Research Article (New England Journal of Medicine) Four artemisinin-based treatments in African pregnant women with malaria

Background: Information regarding the safety and efficacy of artemisinin  combination treatments for malaria in pregnant women is limited, particularly among women who live in sub-Saharan Africa.Methods: We conducted a multicenter, randomized, open-label trial of treatments for malaria in pregnant women in four African countries. A total of 3428 pregnant women in the second or third trimester who had falciparum malaria (at any parasite density and regardless of symptoms) were treated with artemether–lumefantrine, amodiaquine–artesunate, mefloquine–artesunate, or dihydroartemisinin– piperaquine. The primary end points were the polymerase-chain-reaction (PCR)–adjusted cure rates (i.e., cure of the original infection; new infections during follow-up were not considered to be treatment failures) at day 63 and safety outcomes.Results: The PCR-adjusted cure rates in the per-protocol analysis were 94.8% in the artemether–lumefantrine group, 98.5% in the amodiaquine– artesunate group, 99.2% in the dihydroartemisinin–piperaquine group, and 96.8% in the mefloquine–artesunate group; the PCR-adjusted cure rates in the intention-to-treat analysis were 94.2%, 96.9%, 98.0%, and 95.5%, respectively. There was no significant difference among the amodiaquine–artesunate group, dihydroartemisinin–piperaquine group, and the mefloquine–artesunate group. The cure rate in the artemether–lumefantrine group was significantly lower than that in the other three groups, although the absolute difference was within the 5-percentage-point margin for equivalence. The unadjusted cure rates, used as a measure of the  post-treatment prophylactic effect, were significantly lower in the artemether–lumefantrine group (52.5%) than in groups that received amodiaquine–artesunate (82.3%), dihydroartemisinin–piperaquine (86.9%), or mefloquine–artesunate (73.8%). No significant difference in the rate of serious adverse events and in birth outcomes was found among the treatment groups. Drug-related adverse events such as asthenia, poor appetite, dizziness, nausea, and vomiting occurred significantly more frequently in the mefloquine–artesunate group (50.6%) and the amodiaquine–artesunate group (48.5%) than in the dihydroartemisinin–piperaquine group (20.6%) and the artemether–lumefantrine group (11.5%) (P<0.001 for comparison among the four groups).Conclusions: Artemether–lumefantrine was associated with the fewest adverse effects and with acceptable cure rates but provided the shortest posttreatment prophylaxis, whereas dihydroartemisinin–piperaquine had the best efficacy and an acceptable safety profile. (Funded by the European and Developing Countries Clinical Trials Partnership and others; ClinicalTrials.gov number, NCT00852423.

The public health impact and cost-effectiveness of the R21/Matrix-M malaria vaccine: a mathematical modelling study

Background The R21/Matrix-M vaccine has demonstrated high efficacy against Plasmodium falciparum clinical malaria in children in sub-Saharan Africa. Using trial data, we aimed to estimate the public health impact and cost-effectiveness of vaccine introduction across sub-Saharan Africa. Methods We fitted a semi-mechanistic model of the relationship between anti-circumsporozoite protein antibody titres and vaccine efficacy to data from 3 years of follow-up in the phase 2b trial of R21/Matrix-M in Nanoro, Burkina Faso. We validated the model by comparing predicted vaccine efficacy to that observed over 12–18 months in the phase 3 trial. Integrating this framework within a mathematical transmission model, we estimated the cases, malaria deaths, and disability-adjusted life-years (DALYs) averted and cost-effectiveness over a 15-year time horizon across a range of transmission settings in sub-Saharan Africa. Cost-effectiveness was estimated incorporating the cost of vaccine introduction (dose, consumables, and delivery) relative to existing interventions at baseline. We report estimates at a median of 20% parasite prevalence in children aged 2–10 years (PfPR2–10) and ranges from 3% to 65% PfPR2–10. Findings Anti-circumsporozoite protein antibody titres were found to satisfy the criteria for a surrogate of protection for vaccine efficacy against clinical malaria. Age-based implementation of a four-dose regimen of R21/Matrix-M vaccine was estimated to avert 181 825 (range 38 815–333 491) clinical cases per 100 000 fully vaccinated children in perennial settings and 202 017 (29 868–405 702) clinical cases per 100 000 fully vaccinated children in seasonal settings. Similar estimates were obtained for seasonal or hybrid implementation. Under an assumed vaccine dose price of USD 3, the incremental cost per clinical case averted was USD 7 (range 4–48) in perennial settings and USD 6 (3–63) in seasonal settings and the incremental cost per DALY averted was USD 34 (29–139) in perennial settings and USD 30 (22–172) in seasonal settings, with lower cost-effectiveness ratios in settings with higher PfPR2–10. Interpretation Introduction of the R21/Matrix-M malaria vaccine could have a substantial public health benefit across sub-Saharan Africa. Funding The Wellcome Trust, the Bill & Melinda Gates Foundation, the UK Medical Research Council, the European and Developing Countries Clinical Trials Partnership 2 and 3, the NIHR Oxford Biomedical Research Centre, and the Serum Institute of India, Open Philanthropy

Longitudinal estimation of Plasmodium falciparum prevalence in relation to malaria prevention measures in six sub-Saharan African countries.

BACKGROUND: Plasmodium falciparum prevalence (PfPR) is a widely used metric for assessing malaria transmission intensity. This study was carried out concurrently with the RTS,S/AS01 candidate malaria vaccine Phase III trial and estimated PfPR over ≤ 4 standardized cross-sectional surveys. METHODS: This epidemiology study (NCT01190202) was conducted in 8 sites from 6 countries (Burkina Faso, Gabon, Ghana, Kenya, Malawi, and Tanzania), between March 2011 and December 2013. Participants were enrolled in a 2:1:1 ratio according to age category: 6 months-4 years, 5-19 years, and ≥ 20 years, respectively, per year and per centre. All sites carried out surveys 1-3 while survey 4 was conducted only in 3 sites. Surveys were usually performed during the peak malaria parasite transmission season, in one home visit, when medical history and malaria risk factors/prevention measures were collected, and a blood sample taken for rapid diagnostic test, microscopy, and haemoglobin measurement. PfPR was estimated by site and age category. RESULTS: Overall, 6401 (survey 1), 6411 (survey 2), 6400 (survey 3), and 2399 (survey 4) individuals were included in the analyses. In the 6 months-4 years age group, the lowest prevalence (assessed using microscopy) was observed in 2 Tanzanian centres (4.6% for Korogwe and 9.95% for Bagamoyo) and Lambaréné, Gabon (6.0%), while the highest PfPR was recorded for Nanoro, Burkina Faso (52.5%). PfPR significantly decreased over the 3 years in Agogo (Ghana), Kombewa (Kenya), Lilongwe (Malawi), and Bagamoyo (Tanzania), and a trend for increased PfPR was observed over the 4 surveys for Kintampo, Ghana. Over the 4 surveys, for all sites, PfPR was predominantly higher in the 5-19 years group than in the other age categories. Occurrence of fever and anaemia was associated with high P. falciparum parasitaemia. Univariate analyses showed a significant association of anti-malarial treatment in 4 surveys (odds ratios [ORs]: 0.52, 0.52, 0.68, 0.41) and bed net use in 2 surveys (ORs: 0.63, 0.68, 1.03, 1.78) with lower risk of malaria infection. CONCLUSION: Local PfPR differed substantially between sites and age groups. In children 6 months-4 years old, a significant decrease in prevalence over the 3 years was observed in 4 out of the 8 study sites. Trial registration Clinical Trials.gov identifier: NCT01190202:NCT. GSK Study ID numbers: 114001

Four Artemisinin-Based Treatments in African Pregnant Women with Malaria.

BACKGROUND: Information regarding the safety and efficacy of artemisinin combination treatments for malaria in pregnant women is limited, particularly among women who live in sub-Saharan Africa. METHODS: We conducted a multicenter, randomized, open-label trial of treatments for malaria in pregnant women in four African countries. A total of 3428 pregnant women in the second or third trimester who had falciparum malaria (at any parasite density and regardless of symptoms) were treated with artemether-lumefantrine, amodiaquine-artesunate, mefloquine-artesunate, or dihydroartemisinin-piperaquine. The primary end points were the polymerase-chain-reaction (PCR)-adjusted cure rates (i.e., cure of the original infection; new infections during follow-up were not considered to be treatment failures) at day 63 and safety outcomes. RESULTS: The PCR-adjusted cure rates in the per-protocol analysis were 94.8% in the artemether-lumefantrine group, 98.5% in the amodiaquine-artesunate group, 99.2% in the dihydroartemisinin-piperaquine group, and 96.8% in the mefloquine-artesunate group; the PCR-adjusted cure rates in the intention-to-treat analysis were 94.2%, 96.9%, 98.0%, and 95.5%, respectively. There was no significant difference among the amodiaquine-artesunate group, dihydroartemisinin-piperaquine group, and the mefloquine-artesunate group. The cure rate in the artemether-lumefantrine group was significantly lower than that in the other three groups, although the absolute difference was within the 5-percentage-point margin for equivalence. The unadjusted cure rates, used as a measure of the post-treatment prophylactic effect, were significantly lower in the artemether-lumefantrine group (52.5%) than in groups that received amodiaquine-artesunate (82.3%), dihydroartemisinin-piperaquine (86.9%), or mefloquine-artesunate (73.8%). No significant difference in the rate of serious adverse events and in birth outcomes was found among the treatment groups. Drug-related adverse events such as asthenia, poor appetite, dizziness, nausea, and vomiting occurred significantly more frequently in the mefloquine-artesunate group (50.6%) and the amodiaquine-artesunate group (48.5%) than in the dihydroartemisinin-piperaquine group (20.6%) and the artemether-lumefantrine group (11.5%) (P<0.001 for comparison among the four groups). CONCLUSIONS: Artemether-lumefantrine was associated with the fewest adverse effects and with acceptable cure rates but provided the shortest post-treatment prophylaxis, whereas dihydroartemisinin-piperaquine had the best efficacy and an acceptable safety profile. (Funded by the European and Developing Countries Clinical Trials Partnership and others; ClinicalTrials.gov number, NCT00852423.)

Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

YesBackground. Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings. The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance. Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination.This work was supported by the Flemish Ministry of Sciences (EWI, SOFI project IDIS).This paper has been subject to a correction. Please see Correction file above