Search

Contains fulltext : 167216.pdf (publisher's version ) (Open Access), BACKGROUND: An effective malaria transmission-blocking vaccine may play an important role in malaria elimination efforts, and a robust biological assay is essential for its development. The standard membrane-feeding assay (SMFA) for Plasmodium falciparum infection of mosquitoes is considered a "gold standard" assay to measure transmission-blocking activity of test antibodies, and has been utilized widely in both non-clinical and clinical studies. While several studies have discussed the inherent variability of SMFA within a study group, there has been no assessment of inter-laboratory variation. Therefore, there is currently no assurance that SMFA results are comparable between different studies. METHODS: Mouse anti-Pfs25 monoclonal antibody (mAb, 4B7 mAb), rat anti-Pfs48/45 mAb (85RF45.1 mAb) and a human polyclonal antibody (pAb) collected from a malaria-exposed adult were tested at the same concentrations (6-94 mug/mL for 4B7, 1.2-31.3 mug/mL for 85RF45.1 and 23-630 mug/mL for human pAb) in two laboratories following their own standardized SMFA protocols. The mAbs and pAb, previously shown to have strong inhibition activities in the SMFA, were tested at three or four concentrations in two or three independent assays in each laboratory, and percent inhibition in mean oocyst intensity relative to a control in the same feed was determined in each feeding experiment. RESULTS: Both monoclonal and polyclonal antibodies dose-dependently reduced oocyst intensity in all experiments performed at the two test sites. In both laboratories, the inter-assay variability in percent inhibition in oocyst intensity decreased at higher levels of inhibition, regardless of which antibody was tested. At antibody concentrations that led to a >80 % reduction in oocyst numbers, the inter-laboratory variations were in the same range compared with the inter-assay variation observed within a single laboratory, and the differences in best estimates from multiple feeds between the two laboratori

Contains fulltext : 172790.pdf (publisher's version ) (Open Access), BACKGROUND: Between 2009 and 2012, malaria cases diagnosed in a Medecins sans Frontieres programme have increased fivefold in Baraka, South Kivu, Democratic Republic of the Congo (DRC). The cause of this increase is not known. An in vivo drug efficacy trial was conducted to determine whether increased treatment failure rates may have contributed to the apparent increase in malaria diagnoses. METHODS: In an open-randomized non-inferiority trial, the efficacy of artesunate-amodiaquine (ASAQ) was compared to artemether-lumefantrine (AL) for the treatment of uncomplicated falciparum malaria in 288 children aged 6-59 months. Included children had directly supervised treatment and were then followed for 42 days with weekly clinical and parasitological evaluations. The blood samples of children found to have recurring parasitaemia within 42 days were checked by PCR to confirm whether or not this was due to reinfection or recrudescence (i.e. treatment failure). RESULTS: Out of 873 children screened, 585 (67 %) were excluded and 288 children were randomized to either ASAQ or AL. At day 42 of follow up, the treatment efficacy of ASAQ was 78 % before and 95 % after PCR correction for re-infections. In the AL-arm, treatment efficacy was 84 % before and 99.0 % after PCR correction. Treatment efficacy after PCR correction was within the margin of non-inferiority as set for this study. Fewer children in the AL arm reported adverse reactions. CONCLUSIONS: ASAQ is still effective as a treatment for uncomplicated malaria in Baraka, South Kivu, DRC. In this region, AL may have higher efficacy but additional trials are required to draw this conclusion with confidence. The high re-infection rate in South-Kivu indicates intense malaria transmission. Trial registration NCT02741024.

Item does not contain fulltext

Item does not contain fulltext, Gametocytes are the specialized form of Plasmodium parasites that are responsible for human-to-mosquito transmission of malaria. Transmission of gametocytes is highly effective, but represents a biomass bottleneck for the parasite that has stimulated interest in strategies targeting the transmission stages separately from those responsible for clinical disease. Studying targets of naturally acquired immunity against transmission-stage parasites may reveal opportunities for novel transmission reducing interventions, particularly the development of a transmission blocking vaccine (TBV). In this review, we summarize the current knowledge on immunity against the transmission stages of Plasmodium. This includes immune responses against epitopes on the gametocyte-infected erythrocyte surface during gametocyte development, as well as epitopes present upon gametocyte activation in the mosquito midgut. We present an analysis of historical data on transmission reducing immunity (TRI), as analysed in mosquito feeding assays, and its correlation with natural recognition of sexual stage specific proteins Pfs48/45 and Pfs230. Although high antibody titres towards either one of these proteins is associated with TRI, the presence of additional, novel targets is anticipated. In conclusion, the identification of novel gametocyte-specific targets of naturally acquired immunity against different gametocyte stages could aid in the development of potential TBV targets and ultimately an effective transmission blocking approach.

Item does not contain fulltext

Contains fulltext : 171915.pdf (publisher's version ) (Open Access), Infections with helminths and Plasmodium spp. overlap in their geographical distribution. It has been postulated that helminth infections may influence malarial transmission by altering Plasmodium falciparum gametocytogenesis. This cross-sectional study assessed the effect of helminth infections on P. falciparum gametocyte carriage and on humoral immune responses to sexual stage antigens in Gabon. Schistosoma haematobium and filarial infections as well as P. falciparum asexual forms and gametocyte carriage were determined. The antibody responses measured were to sexual (Pfs230, Pfs48/45) and asexual P. falciparum antigens (AMA1, MSP1, and GLURP). A total of 287 subjects were included. The prevalence of microscopically detectable P. falciparum asexual parasites was higher in S. haematobium-infected subjects in comparison to their uninfected counterparts (47% versus 26%, P = 0.003), but this was not different when filarial infections were considered. Plasmodium falciparum gametocyte carriage was similar between Schistosoma- or filaria-infected and uninfected subjects. We observed a significant decrease of Pfs48/45 immunoglobulin G titer in S. haematobium-infected subjects (P = 0.037), whereas no difference was seen for Pfs230 antibody titer, nor for antibodies to AMA1, MSP1, or GLURP. Our findings suggest an effect of S. haematobium on antibody responses to some P. falciparum gametocyte antigens that may have consequences for transmission-blocking immunity.

Contains fulltext : 172668.pdf (publisher's version ) (Open Access)

Contains fulltext : 167311.pdf (publisher's version ) (Open Access), Sexual differentiation of malaria parasites into gametocytes in the vertebrate host and subsequent gamete fertilization in mosquitoes is essential for the spreading of the disease. The molecular processes orchestrating these transitions are far from fully understood. Here, we report the first transcriptome analysis of male and female Plasmodium falciparum gametocytes coupled with a comprehensive proteome analysis. In male gametocytes there is an enrichment of proteins involved in the formation of flagellated gametes; proteins involved in DNA replication, chromatin organization and axoneme formation. On the other hand, female gametocytes are enriched in proteins required for zygote formation and functions after fertilization; protein-, lipid- and energy-metabolism. Integration of transcriptome and proteome data revealed 512 highly expressed maternal transcripts without corresponding protein expression indicating large scale translational repression in P. falciparum female gametocytes for the first time. Despite a high degree of conservation between Plasmodium species, 260 of these 'repressed transcripts' have not been previously described. Moreover, for some of these genes, protein expression is only reported in oocysts and sporozoites indicating that repressed transcripts can be partitioned into short- and long-term storage. Finally, these data sets provide an essential resource for identification of vaccine/drug targets and for further mechanistic studies.

The current epidemic of artemisinin resistant Plasmodium falciparum in Southeast Asia is the result of a soft selective sweep involving at least 20 independent kelch13 mutations. In a large global survey, we find that kelch13 mutations which cause resistance in Southeast Asia are present at low frequency in Africa. We show that African kelch13 mutations have originated locally, and that kelch13 shows a normal variation pattern relative to other genes in Africa, whereas in Southeast Asia there is a great excess of non-synonymous mutations, many of which cause radical amino-acid changes. Thus, kelch13 is not currently undergoing strong selection in Africa, despite a deep reservoir of variations that could potentially allow resistance to emerge rapidly. The practical implications are that public health surveillance for artemisinin resistance should not rely on kelch13 data alone, and interventions to prevent resistance must account for local evolutionary conditions, shown by genomic epidemiology to differ greatly between geographical regions.

Contains fulltext : 154115.pdf (publisher's version ) (Open Access), Extraction and amplification of DNA from dried blood spots (DBS) collected in field studies is commonly used for detection of Plasmodium falciparum. However, there have been few systematic efforts to determine the effects of storage and extraction methods on the sensitivity of DNA amplification. We investigated the effects of storage conditions, length of storage, and DNA extraction methods on amplification via three PCR-based assays using field samples and laboratory controls. Samples stored as DBS for 2 or more years at ambient temperature showed a significant loss of sensitivity that increased with time; after 10 years only 10% samples with parasite densities > 1,000 parasites/muL were detectable by nested polymerase chain reaction (PCR). Conversely, DBS and extracted DNA stored at -20 degrees C showed no loss of sensitivity with time. Samples with low parasite densities amplified more successfully with saponin/Chelex compared with spin-column-based extraction, though the latter method performed better on samples with higher parasite densities stored for 2 years at ambient temperature. DNA extracted via both methods was stable after 20 freeze-thaw cycles. Our results suggest that DBS should be stored at -20 degrees C or extracted immediately, especially if anticipating 2 or more years of storage.

Contains fulltext : 152734.pdf (publisher's version ) (Open Access)

Item does not contain fulltext

Contains fulltext : 152884.pdf (publisher's version ) (Open Access)

Contains fulltext : 153754.pdf (publisher's version ) (Open Access), BACKGROUND: Naturally acquired immunity to clinical malaria is thought to be mainly antibody-mediated, but reports on antigen targets are contradictory. Recognition of multiple antigens may be crucial for protection. In this study, the magnitude of antibody responses and their temporal stability was assessed for a panel of malaria antigens in relation to protection against clinical Plasmodium falciparum malaria. METHODS: Malian children aged two to 14 years were enrolled in a longitudinal study and followed up by passive and active case detection for seven months. Plasma was collected at enrolment and at the beginning, in the middle and after the end of the transmission season. Antibody titres to the P. falciparum-antigens apical membrane protein (AMA)-1, merozoite surface protein (MSP)-1(1)(9), MSP-3, glutamine-rich protein (GLURP-R0) and circumsporozoite antigen (CSP) were assessed by enzyme-linked immunosorbent assay (ELISA) for 99 children with plasma available at all time points. Parasite carriage was determined by microscopy and nested PCR. RESULTS: Antibody titres to all antigens, except MSP-1(1)(9), and the number of antigens recognized increased with age. After malaria exposure, antibody titres increased in children that had low titres at baseline, but decreased in those with high baseline responses. No significant differences were found between antibody titers for individual antigens between children remaining symptomatic or asymptomatic after exposure, after adjustment for age. Instead, children remaining asymptomatic following parasite exposure had a broader repertoire of antigen recognition. CONCLUSIONS: The present study provides immune-epidemiological evidence from a limited cohort of Malian children that strong recognition of multiple antigens, rather than antibody titres for individual antigens, is associated with protection from clinical malaria.

Contains fulltext : 154024.pdf (publisher's version ) (Open Access)

Contains fulltext : 152197.pdf (Publisher’s version ) (Open Access)

Item does not contain fulltext

Contains fulltext : 154582.pdf (publisher's version ) (Open Access)

Contains fulltext : 154184.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, OBJECTIVES: Pfs48/45 and Pfs230 are Plasmodium falciparum sexual stage proteins and promising malaria transmission-blocking vaccine candidates. Antibody responses against these proteins may be naturally acquired and target antigens may be under selective pressure. This has consequences for the future evaluation of vaccine immunogenicity and efficacy in populations naturally exposed to malaria. METHODS: We determined naturally acquired antibody responses to the recombinant proteins Pfs48/45-10C and Pfs230-230CMB in children from three malaria endemic settings in Ghana, Tanzania and Burkina Faso. We also examined genetic polymorphisms in the P. falciparum gene pfs48/45. RESULTS: Antibody prevalence was 1.1-18.2% for 10C and 6.7-18.9% for 230CMB. In Burkina Faso we observed evidence of an age-dependent acquisition pattern for both 10C (p < 0.001) and 230CMB (p = 0.031). Membrane feeding assays on a separate dataset demonstrated an association between functional transmission reducing activity and antibody prevalence for both 10C (p = 0.017) and 230CMB (p = 0.049). 17 single nucleotide polymorphisms were found in pfs48/45 (from 126 samples), with 5 non-synonymous SNPs in the Pfs48/45 10C region. CONCLUSIONS: We conclude there are naturally acquired antibody responses to both vaccine candidates which have functional relevance by reducing the transmissibility of infected individuals. We identified genetic polymorphisms, in pfs48/45 which exhibited geographical specificity.

Contains fulltext : 152237.pdf (Publisher’s version ) (Closed access)

Contains fulltext : 152284.pdf (publisher's version ) (Open Access)

Contains fulltext : 155200.pdf (publisher's version ) (Open Access), The potential use of ivermectin as an additional vector control tool is receiving increased attention from the malaria elimination community, driven by the increased importance of outdoor/residual malaria transmission and the threat of insecticide resistance where vector tools have been scaled-up. This report summarizes the emerging evidence presented at a side meeting on "Ivermectin for malaria elimination: current status and future directions" at the annual meeting of the American Society of Tropical Medicine and Hygiene in New Orleans on November 4, 2014. One outcome was the creation of the "Ivermectin Research for Malaria Elimination Network" whose main goal is to establish a common research agenda to generate the evidence base on whether ivermectin-based strategies should be added to the emerging arsenal to interrupt malaria transmission.

Contains fulltext : 152908.pdf (publisher's version ) (Open Access)

Contains fulltext : 152883.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 154558.pdf (publisher's version ) (Open Access), A longitudinal study was conducted in a low endemic area in northern Tanzania to examine the influence of the alpha-thalassaemia trait on malaria incidence and antibody responses to malaria apical membrane antigen-1 (AMA-1) and merozoite surface protein1-19 (MSP-119). Out of 394 children genotyped for alpha-thalassaemia trait, 4.1% (16 of 394) and 30.7% (121 of 394) were homozygous and heterozygous, respectively. During the 1 year follow-up, four incidents of malaria cases were detected without an evident association with alpha-thalassaemia. Being heterozygous or homozygous for alpha-thalassaemia was associated with an increased prevalence of antibodies to AMA-1 [odds ratio (OR): 1.83, 95% confidence interval (CI): 1.07-3.12, p = 0.027] and MSP-1 (OR: 2.04, 95% CI: 1.16-3.60, p = 0.013) after adjustment for age and reported bednet use. The observed association between alpha-thalassaemia and malaria antibody responses may reflect longer-term differences in antigen exposure or differences in antibody acquisition upon exposure in this low endemic setting.

Contains fulltext : 153165.pdf (publisher's version ) (Open Access), Direct skin feeding experiments are sensitive assays to determine human infectiousness to mosquitoes but are rarely used in malaria epidemiological surveys. We determined the infectiousness of inhabitants of a malaria hypoendemic area in Senegal. Gametocyte prevalence by microscopy was 13.5% (26 of 192). Of all individuals who were gametocyte positive, 44.4% (11 of 25) infected >/= 1 Anopheles arabiensis mosquito and 10.8% (54 of 500) of mosquitoes became infected. Of all individuals who were gametocyte negative by microscopy, 4.3% (7 of 162) infected >/= 1 mosquito and 0.4% (12 of 3240) of mosquitoes became infected. The 18.2% (12 of 66) of all mosquito infections was a result of submicroscopic gametocyte carriage and two individuals without asexual parasites or gametocytes by microscopy were infectious to mosquitoes. When infectivity and local demography was taken into account, children 5-14 years of age contributed 50.8% of the human infectious reservoir for malaria. Adults and submicroscopic gametocyte carriers may contribute considerably to onward malaria transmission in our setting.

Item does not contain fulltext, The sexual stage Pfs48/45 antigen is a well-established lead candidate for a transmission blocking (TB) vaccine because of its critical role in parasite fertilization. We have recently produced the carboxy-terminal 10C-fragment of Pfs48/45 containing three known epitopes for TB antibodies as a chimera with the N-terminal region of GLURP (R0). The resulting fusion protein elicited high titer TB antibodies in rodents. To increase the relatively low yield of correctly folded Pfs48/45 we have generated a series of novel chimera truncating the 10C-fragments to 6 cysteine residues containing sub-units (6C). All constructs harbor the major epitope I for TB antibodies. One of these sub-units (R0.6Cc), produced high yields of correctly folded conformers, which could be purified by a simple 2-step procedure. Purified R0.6Cc was stable and elicits high titer TB antibodies in rats. The yield, purity and stability of R0.6Cc allows for further clinical development.

Item does not contain fulltext, BACKGROUND: Artemisinin combination therapy effectively clears asexual malaria parasites and immature gametocytes but does not prevent posttreatment malaria transmission. Ivermectin (IVM) may reduce malaria transmission by killing mosquitoes that take blood meals from IVM-treated humans. METHODS: In this double-blind, placebo-controlled trial, 120 asymptomatic Plasmodium falciparum parasite carriers were randomized to receive artemether-lumefantrine (AL) plus placebo or AL plus a single or repeated dose (200 microg/kg) of ivermectin (AL-IVM1 and AL-IVM2, respectively). Mosquito membrane feeding was performed 1, 3, and 7 days after initiation of treatment to determine Anopheles gambiae and Anopheles funestus survival and infection rates. RESULTS: The AL-IVM combination was well tolerated. IVM resulted in a 4- to 7-fold increased mortality in mosquitoes feeding 1 day after IVM (P < .001). Day 7 IVM plasma levels were positively associated with body mass index (r = 0.57, P < .001) and were higher in female participants (P = .003), for whom An. gambiae mosquito mortality was increased until 7 days after a single dose of IVM (hazard rate ratio, 1.34 [95% confidence interval, 1.07-1.69]; P = .012). Although we found no evidence that IVM reduced Plasmodium infection rates among surviving mosquitoes, the mosquitocidal effect of AL-IVM1 and AL-IVM2 resulted in 27% and 35% reductions, respectively, in estimated malaria transmission potential during the first week after initiation of treatment. CONCLUSIONS: We conclude that IVM can be safely given in combination with AL and can reduce the likelihood of malaria transmission by reducing the life span of feeding mosquitoes. CLINICAL TRIALS REGISTRATION: NCT0160325.

Item does not contain fulltext, BACKGROUND: Methylene blue (MB) has been shown to be safe and effective against falciparum malaria in Africa and to have pronounced gametocytocidal properties. METHODS: Three days of treatment with artesunate (AS)-amodiaquine (AQ) combined with MB was compared with AS-AQ treatment in a randomized controlled phase IIb study; the study included 221 children aged 6-59 months with uncomplicated falciparum malaria in Burkina Faso. The primary end point was gametocyte prevalence during follow-up, as determined by microscopy and real-time quantitative nucleic acid sequence-based amplification (QT-NASBA). RESULTS: The gametocyte prevalence of Plasmodium falciparum at baseline was 3.6% (microscopy) and 97% (QT-NASBA). It was significantly lower in the AS-AQ-MB than in the AS-AQ group on day 7 of follow-up (microscopy, 1.2% vs 8.9% [P < .05]; QT-NASBA, 36.7% vs 63.3% [P < .001]). Hemoglobin values were significantly lower in the AS-AQ-MB group than in the AS-AQ group at days 2 and 7 of follow-up. Vomiting of the study medication occurred significantly more frequently in the AS-AQ-MB group. CONCLUSIONS: The combination of MB with an artemisinin-based combination therapy has been confirmed to be effective against the gametocytes of P. falciparum. MB-based combinations need to be compared with primaquine-based combinations, preferably using MB in an improved pediatric formulation. Clinical Trials Registration. NCT01407887.

Contains fulltext : 153121.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, OBJECTIVES: The development of drugs to reduce malaria transmission is an important part of malaria eradication plans. We set out to develop and validate a combination of new screening assays for prioritization of transmission-blocking molecules. METHODS: We developed high-throughput assays for screening compounds against gametocytes, the parasite stages responsible for onward transmission to mosquitoes. An existing gametocyte parasitic lactate dehydrogenase (pLDH) assay was adapted for use in 384-well plates, and a novel homogeneous immunoassay to monitor the functional transition of female gametocytes into gametes was developed. A collection of 48 marketed and experimental antimalarials was screened and subsequently tested for impact on sporogony in Anopheles mosquitoes, to directly quantify the transmission-blocking properties of antimalarials in relation to their effects on gametocyte pLDH activity or gametogenesis. RESULTS AND CONCLUSIONS: The novel screening assays revealed distinct stage-specific kinetics and dynamics of drug effects. Peroxides showed the most potent transmission-blocking effects, with an intermediate speed of action and IC50 values that were 20-40-fold higher than the IC50s against the asexual stages causing clinical malaria. Finally, the novel synthetic peroxide OZ439 appeared to be a promising drug candidate as it exerted gametocytocidal and transmission-blocking effects at clinically relevant concentrations.

Contains fulltext : 152039.pdf (publisher's version ) (Open Access)

Item does not contain fulltext, BACKGROUND: The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42. METHODS: DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure. RESULTS: Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72-76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia. CONCLUSIONS: Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence.

Item does not contain fulltext

Item does not contain fulltext

Item does not contain fulltext, BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.

Item does not contain fulltext