Jackie Cook

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.

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

BackgroundBlood 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.MethodsBlood 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.ResultsAntibodies 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°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.ConclusionThis 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.

Rapid Assessment of Malaria Transmission Using Age-Specific Sero-Conversion Rates

Background Malaria transmission intensity is a crucial determinant of malarial disease burden and its measurement can help to define health priorities. Rapid, local estimates of transmission are required to focus resources better but current entomological and parasitological methods for estimating transmission intensity are limited in this respect. An alternative is determination of antimalarial antibody age-specific sero-prevalence to estimate sero-conversion rates (SCR), which have been shown to correlate with transmission intensity. This study evaluated SCR generated from samples collected from health facility attendees as a tool for a rapid assessment of malaria transmission intensity. Methodology and Principal Findings The study was conducted in north east Tanzania. Antibodies to Plasmodium falciparum merozoite antigens MSP-119 and AMA-1 were measured by indirect ELISA. Age-specific antibody prevalence was analysed using a catalytic conversion model based on maximum likelihood to generate SCR. A pilot study, conducted near Moshi, found SCRs for AMA-1 were highly comparable between samples collected from individuals in a conventional cross-sectional survey and those collected from attendees at a local health facility. For the main study, 3885 individuals attending village health facilities in Korogwe and Same districts were recruited. Both malaria parasite prevalence and sero-positivity were higher in Korogwe than in Same. MSP-119 and AMA-1 SCR rates for Korogwe villages ranged from 0.03 to 0.06 and 0.07 to 0.21 respectively. In Same district there was evidence of a recent reduction in transmission, with SCR among those born since 1998 [MSP-119 0.002 to 0.008 and AMA-1 0.005 to 0.014 ] being 5 to 10 fold lower than among individuals born prior to 1998 [MSP-119 0.02 to 0.04 and AMA-1 0.04 to 0.13]. Current health facility specific estimates of SCR showed good correlations with malaria incidence rates in infants in a contemporaneous clinical trial (MSP-119 r2 = 0.78, p<0.01 & AMA-1 r2 = 0.91, p<0.001). Conclusions SCRs generated from age-specific anti-malarial antibody prevalence data collected via health facility surveys were robust and credible. Analysis of SCR allowed detection of a recent drop in malaria transmission in line with recent data from other areas in the region. This health facility-based approach represents a potential tool for rapid assessment of recent trends in malaria transmission intensity, generating valuable data for local and national malaria control programs to target, monitor and evaluate their control strategies.

Serologic markers for detecting malaria in areas of low endemicity, Somalia, 2008.

These markers can identify spatial variations in transmission patterns.

Using serological measures to monitor changes in malaria transmission in Vanuatu

BackgroundWith renewed interest in malaria elimination, island environments present unique opportunities to achieve this goal. However, as transmission decreases, monitoring and evaluation programmes need increasingly sensitive tools to assess Plasmodium falciparum and Plasmodium vivax exposure. In 2009, to assess the role of serological markers in evaluating malaria transmission, a cross-sectional seroprevalence study was carried out in Tanna and Aneityum, two of the southernmost islands of the Vanuatu archipelago, areas where malaria transmission has been variably reduced over the past few decades.MethodsMalaria transmission was assessed using serological markers for exposure to P. falciparum and P. vivax. Filter blood spot papers were collected from 1,249 people from Tanna, and 517 people from Aneityum to assess the prevalence of antibodies to two P. falciparum antigens (MSP-119 and AMA-1) and two P. vivax antigens (MSP-119 and AMA-1). Age-specific prevalence was modelled using a simple catalytic conversion model based on maximum likelihood to generate a community seroconversion rate (SCR).ResultsOverall seropositivity in Tanna was 9.4%, 12.4% and 16.6% to P. falciparum MSP-119, AMA-1 and Schizont Extract respectively and 12.6% and 15.0% to P. vivax MSP-119 and AMA-1 respectively. Serological results distinguished between areas of differential dominance of either P. vivax or P. falciparum and analysis of age-stratified results showed a step in seroprevalence occurring approximately 30 years ago on both islands, indicative of a change in transmission intensity at this time. Results from Aneityum suggest that several children may have been exposed to malaria since the 2002 P. vivax epidemic.ConclusionSeroepidemiology can provide key information on malaria transmission for control programmes, when parasite rates are low. As Vanuatu moves closer to malaria elimination, monitoring changes in transmission intensity and identification of residual malaria foci is paramount in order to concentrate intervention efforts.

Serological Markers Suggest Heterogeneity of Effectiveness of Malaria Control Interventions on Bioko Island, Equatorial Guinea

Background In order to control and eliminate malaria, areas of on-going transmission need to be identified and targeted for malaria control interventions. Immediately following intense interventions, malaria transmission can become more heterogeneous if interventions are more successful in some areas than others. Bioko Island, Equatorial Guinea, has been subject to comprehensive malaria control interventions since 2004. This has resulted in substantial reductions in the parasite burden, although this drop has not been uniform across the island. Methods/Principal Findings In 2008, filter paper blood samples were collected from 7387 people in a cross-sectional study incorporating 18 sentinel sites across Bioko, Equatorial Guinea. Antibodies were measured to P. falciparum Apical Membrane Antigen-1 (AMA-1) by Enzyme Linked Immunosorbent Assay (ELISA). Age-specific seropositivity rates were used to estimate seroconversion rates (SCR). Analysis indicated there had been at least a 60% decline in SCR in four out of five regions on the island. Changes in SCR showed a high degree of congruence with changes in parasite rate (PR) and with regional reductions in all cause child mortality. The mean age adjusted concentration of anti-AMA-1 antibodies was mapped to identify areas where individual antibody responses were higher than expected. This approach confirmed the North West of the island as a major focus of continuing infection and an area where control interventions need to be concentrated or re-evaluated. Conclusion/Interpretation Both SCR and PR revealed heterogeneity in malaria transmission and demonstrated the variable effectiveness of malaria control measures. This work confirms the utility of serological analysis as an adjunct measure for monitoring transmission. Age-specific seroprevalence based evidence of changes in transmission over time will be of particular value when no baseline data are available. Importantly, SCR data provide additional evidence to link malaria control activities to contemporaneous reductions in all-cause child mortality.

Chapter 5. Potential contribution of sero-epidemiological analysis for monitoring malaria control and elimination: historical and current perspectives.

Anti-malarial antibody responses represent an individuals history of exposure to the disease and, as age sero-conversion rates, reflect cumulative malaria exposure in a population. As such these antibody responses are an alternate measure of malaria transmission intensity and have potential in evaluating changes in exposure. This approach was used in the 1970s to evaluate malaria control and eradication attempts in a variety of different ecological settings. These historical studies provided a wealth of information on how serological data might be used to interpret control measures. However they were limited by a lack of standardized antigens and reproducible high-throughput assays. Current techniques using recombinant antigens with a range of immunogenicities, high-throughput enzyme-linked immunosorbent assays (ELISA) and statistical analysis allow a more robust examination of how serological parameters can be used to evaluate factors affecting malaria transmission. Here we present a review of the historical data and use it to assess the serological contribution to monitoring malaria elimination.

Loop-mediated isothermal amplification (LAMP) for point-of-care detection of asymptomatic low-density malaria parasite carriers in Zanzibar

BackgroundAsymptomatic, low parasite density malaria infections are difficult to detect with currently available point-of-care diagnostics. This study piloted a loop-mediated isothermal amplification (LAMP) kit for field-friendly, high-throughput detection of asymptomatic malaria infections during mass screening and treatment (MSAT) in Zanzibar, a malaria pre-elimination setting.MethodsScreening took place in three known hotspot areas prior to the short rains in November. Finger-prick blood was taken for screening by rapid diagnostic test (RDT) and LAMP and collected on filter paper for subsequent polymerase chain reaction (PCR) analyses. LAMP results were compared to RDT and to PCR using McNemar’s test.ResultsApproximately 1,000 people were screened. RDT detected ten infections (1.0% (95% CI 0.3-1.6)) whilst both LAMP and PCR detected 18 (1.8% (95% CI 0.9-2.6)) infections. However, PCR identified three infections that LAMP did not detect and vice versa. LAMP testing was easy to scale-up in field conditions requiring minimal training and equipment, with results ready one to three hours after screening.ConclusionsDespite lower than expected prevalence, LAMP detected a higher number of infections than the currently used diagnostic, RDT. LAMP is a field-friendly, sensitive diagnostic test that could be useful for MSAT malaria campaigns which require quick results to enable prompt treatment.

Mass Screening and Treatment on the Basis of Results of a Plasmodium falciparum-Specific Rapid Diagnostic Test Did Not Reduce Malaria Incidence in Zanzibar

BACKGROUND Seasonal increases in malaria continue in hot spots in Zanzibar. Mass screening and treatment (MSAT) may help reduce the reservoir of infection; however, it is unclear whether rapid diagnostic tests (RDTs) detect a sufficient proportion of low-density infections to influence subsequent transmission. METHODS Two rounds of MSAT using Plasmodium falciparum-specific RDT were conducted in 5 hot spots (population, 12 000) in Zanzibar in 2012. In parallel, blood samples were collected on filter paper for polymerase chain reaction (PCR) analyses. Data on confirmed malarial parasite infections from health facilities in intervention and hot spot control areas were monitored as proxy for malaria transmission. RESULTS Approximately 64% of the population (7859) were screened at least once. P. falciparum prevalence, as measured by RDT, was 0.2% (95% confidence interval [CI], .1%-.3%) in both rounds, compared with PCR measured prevalences (for all species) of 2.5% (95% CI, 2.1%-2.9%) and 3.8% (95% CI, 3.2%-4.4%) in rounds 1 and 2, respectively. Two fifths (40%) of infections detected by PCR included non-falciparum species. Treatment of RDT-positive individuals (4% of the PCR-detected parasite carriers) did not reduce subsequent malaria incidence, compared with control areas. CONCLUSIONS Highly sensitive point-of-care diagnostic tools for detection of all human malaria species are needed to make MSAT an effective strategy in settings where malaria elimination programs are in the pre-elimination phase.

Effect of the Pre-erythrocytic Candidate Malaria Vaccine RTS,S/AS01E on Blood Stage Immunity in Young Children

(See the article by Greenhouse et al, on pages 19-26.) Background. RTS,S/AS01E 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-142, 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/AS01E or a control vaccine. Results. Antibody concentrations to AMA-1, EBA-175, and MSP-142 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/AS01E resulted in modest reductions in AMA-1, EBA-175, MSP-142, 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.