The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum.

BACKGROUND: At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. RESULTS: A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-119, MSP-142, MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. CONCLUSION: Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross-comparisons of results of vaccine trials performed in different centres/with different products; (2) to facilitate standardization and harmonization of immunological assays used in epidemiology research; and (3) to allow optimization and validation of immunological assays used in malaria vaccine development.

Serology describes a profile of declining malaria transmission in Farafenni, The Gambia.

BACKGROUND: Malaria morbidity and mortality has declined in recent years in a number of settings. The ability to describe changes in malaria transmission associated with these declines is important in terms of assessing the potential effects of control interventions, and for monitoring and evaluation purposes. METHODS: Data from five cross-sectional surveys conducted in Farafenni and surrounding villages on the north bank of River Gambia between 1988 and 2011 were compiled. Antibody responses to MSP-119 were measured in samples from all surveys, data were normalized and expressed as seroprevalence and seroconversion rates (SCR) using different mathematical models. RESULTS: Results showed declines in serological metrics with seroprevalence in children aged one to 5 years dropping from 19 % (95 % CI 15-23 %) in 1988 to 1 % (0-2 %) in 2011 (p value for trend in proportions < 0.001) and the SCR dropping from 0.069 year(-1) (0.059-0.080) to 0.022 year(-1) (0.017-0.028; p = 0.004). The serological data were consistent with previously described drops in both parasite prevalence in children aged 1-5 years (62 %, 57-66 %, in 1988 to 2 %, 0-4 %, in 2011; p < 0.001), and all-cause under five mortality rates (37 per 1000 person-years, 34-41, in 1990 to 17, 15-19, in 2006; p = 0.059). CONCLUSIONS: This analysis shows accurate reconstruction of historical malaria transmission patterns in the Farafenni area using anti-malarial antibody responses. Demonstrating congruence between serological measures, and conventional clinical and parasitological measures suggests broader utility for serology in monitoring and evaluation of malaria transmission.

Genetic determinants of anti-malarial acquired immunity in a large multi-centre study.

BACKGROUND: Many studies report associations between human genetic factors and immunity to malaria but few have been reliably replicated. These studies are usually country-specific, use small sample sizes and are not directly comparable due to differences in methodologies. This study brings together samples and data collected from multiple sites across Africa and Asia to use standardized methods to look for consistent genetic effects on anti-malarial antibody levels. METHODS: Sera, DNA samples and clinical data were collected from 13,299 individuals from ten sites in Senegal, Mali, Burkina Faso, Sudan, Kenya, Tanzania, and Sri Lanka using standardized methods. DNA was extracted and typed for 202 Single Nucleotide Polymorphisms with known associations to malaria or antibody production, and antibody levels to four clinical grade malarial antigens [AMA1, MSP1, MSP2, and (NANP)4] plus total IgE were measured by ELISA techniques. Regression models were used to investigate the associations of clinical and genetic factors with antibody levels. RESULTS: Malaria infection increased levels of antibodies to malaria antigens and, as expected, stable predictors of anti-malarial antibody levels included age, seasonality, location, and ethnicity. Correlations between antibodies to blood-stage antigens AMA1, MSP1 and MSP2 were higher between themselves than with antibodies to the (NANP)4 epitope of the pre-erythrocytic circumsporozoite protein, while there was little or no correlation with total IgE levels. Individuals with sickle cell trait had significantly lower antibody levels to all blood-stage antigens, and recessive homozygotes for CD36 (rs321198) had significantly lower anti-malarial antibody levels to MSP2. CONCLUSION: Although the most significant finding with a consistent effect across sites was for sickle cell trait, its effect is likely to be via reducing a microscopically positive parasitaemia rather than directly on antibody levels. However, this study does demonstrate a framework for the feasibility of combining data from sites with heterogeneous malaria transmission levels across Africa and Asia with which to explore genetic effects on anti-malarial immunity.

Glucose-6-phosphate dehydrogenase polymorphisms and susceptibility to mild malaria in Dogon and Fulani, Mali.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is associated with protection from severe malaria, and potentially uncomplicated malaria phenotypes. It has been documented that G6PD deficiency in sub-Saharan Africa is due to the 202A/376G G6PD A-allele, and association studies have used genotyping as a convenient technique for epidemiological studies. However, recent studies have shown discrepancies in G6PD202/376 associations with severe malaria. There is evidence to suggest that other G6PD deficiency alleles may be common in some regions of West Africa, and that allelic heterogeneity could explain these discrepancies. METHODS: A cross-sectional epidemiological study of malaria susceptibility was conducted during 2006 and 2007 in the Sahel meso-endemic malaria zone of Mali. The study included Dogon (n = 375) and Fulani (n = 337) sympatric ethnic groups, where the latter group is characterized by lower susceptibility to Plasmodium falciparum malaria. Fifty-three G6PD polymorphisms, including 202/376, were genotyped across the 712 samples. Evidence of association of these G6PD polymorphisms and mild malaria was assessed in both ethnic groups using genotypic and haplotypic statistical tests. RESULTS: It was confirmed that the Fulani are less susceptible to malaria, and the 202A mutation is rare in this group (<1% versus Dogon 7.9%). The Betica-Selma 968C/376G (~11% enzymatic activity) was more common in Fulani (6.1% vs Dogon 0.0%). There are differences in haplotype frequencies between Dogon and Fulani, and association analysis did not reveal strong evidence of protective G6PD genetic effects against uncomplicated malaria in both ethnic groups and gender. However, there was some evidence of increased risk of mild malaria in Dogon with the 202A mutation, attaining borderline statistical significance in females. The rs915942 polymorphism was found to be associated with asymptomatic malaria in Dogon females, and the rs61042368 polymorphism was associated with clinical malaria in Fulani males. CONCLUSIONS: The results highlight the need to consider markers in addition to G6PD202 in studies of deficiency. Further, large genetic epidemiological studies of multi-ethnic groups in West Africa across a spectrum of malaria severity phenotypes are required to establish who receives protection from G6PD deficiency.

Antigen-specific IL-2 secretion correlates with NK cell responses after immunization of Tanzanian children with the RTS,S/AS01 malaria vaccine.

RTS,S/AS01, a vaccine targeting pre-erythrocytic stages of Plasmodium falciparum, is undergoing clinical trials. We report an analysis of cellular immune response to component Ags of RTS,S-hepatitis B surface Ag (HBs) and P. falciparum circumsporozoite (CS) protein-among Tanzanian children in a phase IIb RTS,S/AS01(E) trial. RTS,S/AS01 (E) vaccinees make stronger T cell IFN-γ, CD69, and CD25 responses to HBs peptides than do controls, indicating that RTS,S boosts pre-existing HBs responses. T cell CD69 and CD25 responses to CS and CS-specific secreted IL-2 were augmented by RTS,S vaccination. Importantly, more than 50% of peptide-induced IFN-γ(+) lymphocytes were NK cells, and the magnitude of the NK cell CD69 response to HBs peptides correlated with secreted IL-2 concentration. CD69 and CD25 expression and IL-2 secretion may represent sensitive markers of RTS,S-induced, CS-specific T cells. The potential for T cell-derived IL-2 to augment NK cell activation in RTS,S-vaccinated individuals, and the relevance of this for protection, needs to be explored further.

Long-lived antibody and B Cell memory responses to the human malaria parasites, Plasmodium falciparum and Plasmodium vivax.

Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses.

Genetic polymorphisms associated with anti-malarial antibody levels in a low and unstable malaria transmission area in southern Sri Lanka.

BACKGROUND: The incidence of malaria in Sri Lanka has significantly declined in recent years. Similar trends were seen in Kataragama, a known malaria endemic location within the southern province of the country, over the past five years. This is a descriptive study of anti-malarial antibody levels and selected host genetic mutations in residents of Kataragama, under low malaria transmission conditions. METHODS: Sera were collected from 1,011 individuals residing in Kataragama and anti-malarial antibodies and total IgE levels were measured by a standardized ELISA technique. Host DNA was extracted and used for genotyping of selected SNPs in known genes associated with malaria. The antibody levels were analysed in relation to the past history of malaria (during past 10 years), age, sex, the location of residence within Kataragama and selected host genetic markers. RESULTS: A significant increase in antibodies against Plasmodium falciparum antigens AMA1, MSP2, NANP and Plasmodium vivax antigen MSP1 in individuals with past history of malaria were observed when compared to those who did not. A marked increase of anti-MSP1(Pf) and anti-AMA1(Pv) was also evident in individuals between 45-59 years (when compared to other age groups). Allele frequencies for two SNPs in genes that code for IL-13 and TRIM-5 were found to be significantly different between those who have experienced one or more malaria attacks within past 10 years and those who did not. When antibody levels were classified into a low-high binary trait, significant associations were found with four SNPs for anti-AMA1(Pf); two SNPs for anti-MSP1(Pf); eight SNPs for anti-NANP(Pf); three SNPs for anti-AMA1(Pv); seven SNPs for anti-MSP1(Pv); and nine SNPs for total IgE. Eleven of these SNPs with significant associations with anti-malarial antibody levels were found to be non-synonymous. CONCLUSIONS: Evidence is suggestive of an age-acquired immunity in this study population in spite of low malaria transmission levels. Several SNPs were in linkage disequilibrium and had a significant association with elevated antibody levels, suggesting that these host genetic mutations might have an individual or collective effect on inducing or/and maintaining high anti-malarial antibody levels.

Filter paper collection of Plasmodium falciparum mRNA for detecting low-density gametocytes.

BACKGROUND: Accurate sampling of sub-microscopic gametocytes is necessary for epidemiological studies to identify the infectious reservoir of Plasmodium falciparum. Detection of gametocyte mRNA achieves sensitive detection, but requires careful handling of samples. Filter papers can be used for collecting RNA samples, but rigorous testing of their capacity to withstand adverse storage conditions has not been fully explored. METHODS: Three gametocyte dilutions: 10/µL, 1.0/µL and 0.1/µL were spotted onto Whatman™ 903 Protein Saver Cards, FTA Classic Cards and 3MM filter papers that were stored under frozen, cold chain or tropical conditions for up to 13 weeks . RNA was extracted, then detected by quantitative nucleic acid sequence-based amplification (QT-NASBA) and reverse-transcriptase PCR (RT-PCR). RESULTS: Successful gametocyte detection was more frequently observed from the Whatman 903 Protein Saver Card compared to the Whatman FTA Classic Card, by both techniques (p<0.0001). When papers were stored at higher temperatures, a loss in sensitivity was experienced for the FTA Classic Card but not the 903 Protein Saver Card or Whatman 3MM filter paper. The sensitivity of gametocyte detection was decreased when papers were stored at high humidity. CONCLUSIONS: This study indicates the Whatman 903 Protein Saver Card is better for Pfs25 mRNA sampling compared to the Whatman FTA Classic Card, and that the Whatman 3MM filter paper may prove to be a satisfactory cheaper option for Pfs25 mRNA sampling. When appropriately dried, filter papers provide a useful approach to Pfs25 mRNA sampling, especially in settings where storage in RNA-protecting buffer is not possible.

Effect of the pre-erythrocytic candidate malaria vaccine RTS,S/AS01E on blood stage immunity in young children.

BACKGROUND: RTS,S/AS01(E) is the lead candidate malaria vaccine and confers pre-erythrocytic immunity. Vaccination may therefore impact acquired immunity to blood-stage malaria parasites after natural infection. METHODS: We measured, by enzyme-linked immunosorbent assay, antibodies to 4 Plasmodium falciparum merozoite antigens (AMA-1, MSP-1(42), EBA-175, and MSP-3) and by growth inhibitory activity (GIA) using 2 parasite clones (FV0 and 3D7) at 4 times on 860 children who were randomized to receive with RTS,S/AS01(E) or a control vaccine. RESULTS: Antibody concentrations to AMA-1, EBA-175, and MSP-1(42) decreased with age during the first year of life, then increased to 32 months of age. Anti-MSP-3 antibody concentrations gradually increased, and GIA gradually decreased up to 32 months. Vaccination with RTS,S/AS01(E) resulted in modest reductions in AMA-1, EBA-175, MSP-1(42), and MSP-3 antibody concentrations and no significant change in GIA. Increasing anti-merozoite antibody concentrations and GIA were prospectively associated with increased risk of clinical malaria. CONCLUSIONS: Vaccination with RTS,S/AS01E reduces exposure to blood-stage parasites and, thus, reduces anti-merozoite antigen antibody concentrations. However, in this study, these antibodies were not correlates of clinical immunity to malaria. Instead, heterogeneous exposure led to confounded, positive associations between increasing antibody concentration and increasing risk of clinical malaria.

The generation and evaluation of recombinant human IgA specific for Plasmodium falciparum merozoite surface protein 1-19 (PfMSP1 19).

BACKGROUND: Human immunoglobulin G (IgG) plays an important role in mediating protective immune responses to malaria. Although human serum immunoglobulin A (IgA) is the second most abundant class of antibody in the circulation, its contribution, if any, to protective responses against malaria is not clear. RESULTS: To explore the mechanism(s) by which IgA may mediate a protective effect, we generated fully human IgA specific for the C-terminal 19-kDa region of Plasmodium falciparum merozoite surface protein 1 (PfMSP1 19), a major target of protective immune responses. This novel human IgA bound antigen with an affinity comparable to that seen for an epitope-matched protective human IgG1. Furthermore, the human IgA induced significantly higher NADPH-mediated oxidative bursts and degranulation from human neutrophils than the epitope-matched human IgG1 from which it was derived. Despite showing efficacy in in vitro functional assays, the human IgA failed to protect against parasite challenge in vivo in mice transgenic for the human Fcα receptor (FcαRI/CD89). A minority of the animals treated with IgA, irrespective of FcαRI expression, showed elevated serum TNF-α levels and concomitant mouse anti-human antibody (MAHA) responses. CONCLUSIONS: The lack of protection afforded by MSP1 19-specific IgA against parasite challenge in mice transgenic for human FcαRI suggests that this antibody class does not play a major role in control of infection. However, we cannot exclude the possibility that protective capacity may have been compromised in this model due to rapid clearance and inappropriate bio-distribution of IgA, and differences in FcαRI expression profile between humans and transgenic mice.

Correction: Serologically Defined Variations in Malaria Endemicity in Pará State, Brazil.

[This corrects the article DOI: 10.1371/journal.pone.0113357.].

Neutralization of malaria glycosylphosphatidylinositol in vitro by serum IgG from malaria-exposed individuals.

Parasite-derived glycosylphosphatidylinositol (GPI) is believed to be a major inducer of the pathways leading to pathology and morbidity during Plasmodium falciparum infection and has been termed a malaria "toxin." The generation of neutralizing anti-GPI ("antitoxic") antibodies has therefore been hypothesized to be an important step in the acquisition of antidisease immunity to malaria; however, to date the GPI-neutralizing capacity of antibodies induced during natural Plasmodium falciparum infection has not been evaluated. Here we describe the development of an in vitro macrophage-based assay to assess the neutralizing capacity of malarial GPI-specific IgG. We demonstrate that IgG from Plasmodium falciparum-exposed individuals can significantly inhibit the GPI-induced activation of macrophages in vitro, as shown by reduced levels of tumor necrosis factor production and attenuation of CD40 expression. The GPI-neutralizing capacity of individual IgG samples was directly correlated with the anti-GPI antibody titer. IgG from malaria-exposed individuals also neutralized the macrophage-activating effects of P. falciparum schizont extract (PfSE), but there was only a poor correlation between PfSE-neutralizing activity and the anti-GPI antibody titer, suggesting that PfSE contains other macrophage-activating moieties, in addition to GPI. In conclusion, we have established an in vitro assay to test the toxin-neutralizing activities of antimalarial antibodies and have shown that anti-GPI antibodies from malaria-immune individuals are able to neutralize GPI-induced macrophage activation; however, the clinical relevance of anti-GPI antibodies remains to be proven, given that malarial schizonts contain other proinflammatory moieties, in addition to GPI.

Activation of transforming growth factor beta by malaria parasite-derived metalloproteinases and a thrombospondin-like molecule.

Much of the pathology of malaria is mediated by inflammatory cytokines (such as interleukin 12, interferon gamma, and tumor necrosis factor alpha), which are part of the immune response that kills the parasite. The antiinflammatory cytokine transforming growth factor (TGF)-beta plays a crucial role in preventing the severe pathology of malaria in mice and TGF-beta production is associated with reduced risk of clinical malaria in humans. Here we show that serum-free preparations of Plasmodium falciparum, Plasmodium yoelii 17XL, and Plasmodium berghei schizont-infected erythrocytes, but not equivalent preparations of uninfected erythrocytes, are directly able to activate latent TGF-beta (LatTGF-beta) in vitro. Antibodies to thrombospondin (TSP) and to a P. falciparum TSP-related adhesive protein (PfTRAP), and synthetic peptides from PfTRAP and P. berghei TRAP that represent homologues of TGF-beta binding motifs of TSP, all inhibit malaria-mediated TGF-beta activation. Importantly, TRAP-deficient P. berghei parasites are less able to activate LatTGF-beta than wild-type parasites and their replication is attenuated in vitro. We show that activation of TGF-beta by malaria parasites is a two step process involving TSP-like molecules and metalloproteinase activity. Activation of LatTGF-beta represents a novel mechanism for direct modulation of the host response by malaria parasites.

Antibody response to Schistosoma haematobium and other helminth species in malaria-exposed populations from Burkina Faso.

Infection with helminths in sub-Saharan Africa could modulate the immune response towards Plasmodium falciparum as well as susceptibility to malaria infection and disease. The aim of this study is to assess the antibody responses to helminths species in malaria-exposed populations from Burkina Faso. Plasma samples were collected in rural villages inhabited by Fulani, Mossi and Rimaibe communities, and IgG against parasitic helminths were measured by ELISA. The prevalence of IgG against antigens of Strongyloides stercoralis, Wuchereria bancrofti and Schistosoma haematobium (Soluble Egg Antigen, SEA) was 5%, 16% and 63% respectively, in line with estimates of infection prevalence in the region for the three parasites. Anti-SEA IgG prevalence was highest at 10-20 years of age, higher in males than females, and did not show differences between ethnic groups. However, the Fulani showed lower levels of anti-SEA IgG suggesting that lighter S. haematobium infections may occur in the ethnic group known for a marked lower susceptibility to P. falciparum. The present data support the use of serological methods for integrated surveillance of neglected tropical diseases such as soil-transmitted helminths, lymphatic filariasis and bilharzia. Furthermore, as helminth infections might promote downregulation of immune responses against intracellular pathogens, the observation of lower anti-SEA IgG levels in the malaria resistant Fulani population warrants further investigation into the immunological cross-talk between S. haematobium and P. falciparum in this geographical region.

Comparison of surveillance methods applied to a situation of low malaria prevalence at rural sites in The Gambia and Guinea Bissau.

BACKGROUND: Health record-based observations from several parts of Africa indicate a major decline in malaria, but up-to-date information on parasite prevalence in West-Africa is sparse. This study aims to provide parasite prevalence data from three sites in the Gambia and Guinea Bissau, respectively, and compares the usefulness of PCR, rapid diagnostic tests (RDT), serology and slide-microscopy for surveillance. METHODS: Cross-sectional surveys in 12 villages at three rural sites were carried out in the Gambia and Guinea Bissau in January/February 2008, shortly following the annual transmission season. RESULTS: A surprisingly low microscopically detectable parasite prevalence was detected in the Gambia (Farafenni: 10.9%, CI95%: 8.7-13.1%; Basse: 9.0%, CI95%: 7.2-10.8%), and Guinea Bissau (Caio: 4%, CI95%: 2.6-5.4%), with low parasite densities (geometric mean: 104 parasites/microl, CI95%: 76-143/microl). In comparison, PCR detected a more than three times higher proportion of parasite carriers, indicating its usefulness to sensitively identify foci where malaria declines, whereas the RDT had very low sensitivity. Estimates of force of infection using age sero-conversion rates were equivalent to an EIR of approximately 1 infectious bite/person/year, significantly less than previous estimates. The sero-prevalence profiles suggest a gradual decline of malaria transmission, confirming their usefulness in providing information on longer term trends of transmission. A greater variability in parasite prevalence among villages within a site than between sites was observed with all methods. The fact that serology equally captured the inter-village variability, indicates that the observed heterogeneity represents a stable pattern. CONCLUSION: PCR and serology may be used as complementary tools to survey malaria in areas of declining malaria prevalence such as the Gambia and Guinea Bissau.

Immunophoretic rapid diagnostic tests as a source of immunoglobulins for estimating malaria sero-prevalence and transmission intensity.

BACKGROUND: Sero-epidemiological methods are being developed as a tool for rapid assessment of malaria transmission intensity. Simple blood collection methods for use in field settings will make this more feasible. This paper describes validation of such a method, by analysing immunoglobulins from blood retained within immunophoretic rapid diagnostic tests (RDTs) for Plasmodium falciparum. RDTs are now widely used for the diagnosis of malaria and estimation of parasite rates, and this method represents a further use for these devices in malaria control. METHODS: Immunoglobulins eluted from RDTs, designed to detect parasite histidine rich protein-2 (HRP-2), were analysed by indirect ELISA for IgG recognizing the P. falciparum blood stage antigens merozoite surface protein-1(19) (MSP-1(19)) and apical membrane antigen-1 (AMA-1). Optimal storage conditions for RDTs were evaluated by comparing antibody responses from RDTs stored in dry or humid conditions at 4 degrees C or at ambient temperature (with or without air-conditioning) for 7, 31 or 70 days. Antibody levels estimated using 3,700 RDT samples from attendees at health facilities in North-eastern Tanzania were compared with contemporaneously collected filter paper blood spots (FPBS) and used to estimate seroconversion rates. RESULTS: Storage of RDTs at 4 degrees C was optimal for immunoglobulin recovery but short-term storage at ambient temperatures did not substantially affect anti-malarial IgG levels. Results from RDTs were comparable with those from FPBSs, for both antigens. RDT-generated titres tended to be slightly higher than those generated from FPBSs, possibly due to greater recovery of immunoglobulins from RDTs compared to filter paper. Importantly, however, RDT-based seroconversion rates, and hence serological estimates of malaria transmission intensity, agreed closely with those from FPBSs. CONCLUSION: RDTs represent a practical option for collecting blood for sero-epidemiological surveys, with potential cost and logistical advantages over filter paper and other blood collection methods. RDT-based seroepidemiology can be incorporated into routine monitoring of malaria endemicity, providing information to supplement parasite prevalence rates and generating rapid, robust assessment of malaria transmission intensity at minimal extra cost.

Alterations of splenic architecture in malaria are induced independently of Toll-like receptors 2, 4, and 9 or MyD88 and may affect antibody affinity.

Splenic microarchitecture is substantially altered during acute malaria infections, which may affect the development and regulation of immune responses. Here we investigated whether engagement of host Toll-like receptor 2 (TLR2), TLR4, TLR9, and the adaptor protein MyD88 is required for induction of the changes and whether antibody responses are modified when immunization takes place during the period of splenic disruption. The alterations in splenic microarchitecture were maximal shortly after the peak of parasitemia and were not dependent on engagement of TLR2, TLR4, or TLR9, and they were only minimally affected by the absence of the MyD88 adaptor molecule. Although germinal centers were formed in infected mice, they did not contain the usual light and dark zones. Immunization of mice with chicken gamma globulin 2 weeks prior to acute Plasmodium chabaudi infection did not affect the quantity or avidity of the immunoglobulin G antibody response to this antigen. However, immunization at the same time as the primary P. chabaudi infection resulted in a clear transient reduction in antibody avidity in the month following immunization. These data suggest that the alterations in splenic structure, particularly the germinal centers, may affect the quality of an antibody response during a malaria infection and could impact the development of immunity to malaria or to other infections or immunizations given during a malaria infection.

Dried blood spots as a source of anti-malarial antibodies for epidemiological studies.

BACKGROUND: Blood spots collected onto filter paper are an established and convenient source of antibodies for serological diagnosis and epidemiological surveys. Although recommendations for the storage and analysis of small molecule analytes in blood spots exist, there are no published systematic studies of the stability of antibodies under different storage conditions. METHODS: Blood spots, on filter paper or glass fibre mats and containing malaria-endemic plasma, were desiccated and stored at various temperatures for different times. Eluates of these spots were assayed for antibodies against two Plasmodium falciparum antigens, MSP-119 and MSP2, and calculated titres used to fit an exponential (first order kinetic) decay model. The first order rate constants (k) for each spot storage temperature were used to fit an Arrhenius equation, in order to estimate the thermal and temporal stability of antibodies in dried blood spots. The utility of blood spots for serological assays was confirmed by comparing antibodies eluted from blood spots with the equivalent plasma values in a series of samples from North Eastern Tanzania and by using blood spot-derived antibodies to estimate malaria transmission intensity in this site and for two localities in Uganda. RESULTS: Antibodies in spots on filter paper and glass fibre paper had similar stabilities but blood was more easily absorbed onto filter papers than glass fibre, spots were more regular and spot size was more closely correlated with blood volume for filter paper spots. Desiccated spots could be stored at or below 4 degrees C for extended periods, but were stable for only very limited periods at ambient temperature. When desiccated, recoveries of antibodies that are predominantly of IgG1 or IgG3 subclasses were similar. Recoveries of antibodies from paired samples of serum and of blood spots from Tanzania which had been suitably stored showed similar recoveries of antibodies, but spots which had been stored for extended periods at ambient humidity and temperature showed severe loss of recoveries. Estimates of malaria transmission intensity obtained from serum and from blood spots were similar, and values obtained using blood spots agreed well with entomologically determined values. CONCLUSION: This study has demonstrated the suitability of filter paper blood spots paper for collection of serum antibodies, and provided clear guidelines for the treatment and storage of filter papers which emphasize the importance of desiccation and minimisation of time spent at ambient temperatures. A recommended protocol for collecting, storing and assaying blood spots is provided.

Serology: a robust indicator of malaria transmission intensity?

To estimate the burden of malarial disease, and evaluate the likely effects of control strategies, requires reliable predictions of malaria transmission intensity. It has long been suggested that antimalarial antibody prevalences could provide a more accurate estimate of transmission intensity than traditional measures such as parasite prevalence or entomological inoculation rates, but there has been no systematic evaluation of this approach. Now, the availability of well characterized malarial antigens allows us to test whether serological measurements provide a practical method for estimating transmission. Here we present a suggested methodology, highlight the advantages and shortcomings of serological measurements of malaria transmission and identify areas in which further work is desirable.

Application of Serological Tools and Spatial Analysis to Investigate Malaria Transmission Dynamics in Highland Areas of Southwest Uganda.

Serological markers, combined with spatial analysis, offer a comparatively more sensitive means by which to measure and detect foci of malaria transmission in highland areas than traditional malariometric indicators. Plasmodium falciparum parasite prevalence, seroprevalence, and seroconversion rate to P. falciparum merozoite surface protein-119 (MSP-119) were measured in a cross-sectional survey to determine differences in transmission between altitudinal strata. Clusters of P. falciparum parasite prevalence and high antibody responses to MSP-119 were detected and compared. Results show that P. falciparum prevalence and seroprevalence generally decreased with increasing altitude. However, transmission was heterogeneous with hotspots of prevalence and/or seroprevalence detected in both highland and highland fringe altitudes, including a serological hotspot at 2,200 m. Results demonstrate that seroprevalence can be used as an additional tool to identify hotspots of malaria transmission that might be difficult to detect using traditional cross-sectional parasite surveys or through vector studies. Our study findings identify ways in which malaria prevention and control can be more effectively targeted in highland or low transmission areas via serological measures. These tools will become increasingly important for countries with an elimination agenda and/or where malaria transmission is becoming patchy and focal, but receptivity to malaria transmission remains high.