Multiplexed bead-based assays that use Luminex® xMAP® technology have become popular for measuring antibodies against proteins of interest in many fields, including malaria and more recently ...SARS-CoV-2/COVID-19. There are currently two formats that are widely used: non-magnetic beads or magnetic beads. Data are lacking regarding the comparability of results obtained using these two types of beads, and for assays run on different instruments. Whilst non-magnetic beads can only be run on flow-based instruments (such as the Luminex® 100/200™ or Bio-Plex® 200), magnetic beads can be run on both these and the newer MAGPIX® instruments. In this study we utilized a panel of purified recombinant Plasmodium vivax proteins and samples from malaria-endemic areas to measure P. vivax-specific IgG responses using different combinations of beads and instruments. We directly compared: i) non-magnetic versus magnetic beads run on a Bio-Plex® 200, ii) magnetic beads run on the Bio-Plex® 200 versus MAGPIX® and iii) non-magnetic beads run on a Bio-Plex® 200 versus magnetic beads run on the MAGPIX®. We also performed an external comparison of our optimized assay. We observed that IgG antibody responses, measured against our panel of P. vivax proteins, were moderately-strongly correlated in all three of our comparisons (pearson r>0.5 for 18/19 proteins), however higher amounts of protein were required for coupling to magnetic beads. Our external comparison indicated that results generated in different laboratories using the same coupled beads are also highly comparable (pearson r>0.7), particularly if a reference standard curve is used.
Plasmodium vivax remains an important cause of malaria in South America and the Asia-Pacific. Naturally acquired antibody responses against multiple P. vivax proteins have been described in numerous ...countries, however, direct comparison of these responses has been difficult with different methodologies employed. We measured antibody responses against 307 P. vivax proteins at the time of P. vivax infection, and at 2-3 later time-points in three countries. We observed that seropositivity rates at the time of infection were highest in Thailand, followed by Brazil then PNG, reflecting the level of antigenic input. The majority of sero-reactive antigens in all sites induced short-lived antibody responses with estimated half-lives of less than 6 months, although there was a trend towards longer-lived responses in PNG children. Despite these differences, IgG seropositivity rates, magnitude and longevity were highly and significantly rank-correlated between the different regions, suggesting such features are reflective of the individual protein.
Antibody responses as serological markers of Plasmodium vivax infection have been shown to correlate with exposure, but little is known about the other factors that affect antibody responses in ...naturally infected people from endemic settings. To address this question, we studied IgG responses to novel serological exposure markers (SEMs) of P. vivax in three settings with different transmission intensity.
We validated a panel of 34 SEMs in a Peruvian cohort with up to three years' longitudinal follow-up using a multiplex platform and compared results to data from cohorts in Thailand and Brazil. Linear regression models were used to characterize the association between antibody responses and age, the number of detected blood-stage infections during follow-up, and time since previous infection. Receiver Operating Characteristic (ROC) analysis was used to test the performance of SEMs to identify P. vivax infections in the previous 9 months.
Antibody titers were associated with age, the number of blood-stage infections, and time since previous P. vivax infection in all three study sites. The association between antibody titers and time since previous P. vivax infection was stronger in the low transmission settings of Thailand and Brazil compared to the higher transmission setting in Peru. Of the SEMs tested, antibody responses to RBP2b had the highest performance for classifying recent exposure in all sites, with area under the ROC curve (AUC) = 0.83 in Thailand, AUC = 0.79 in Brazil, and AUC = 0.68 in Peru.
In low transmission settings, P. vivax SEMs can accurately identify individuals with recent blood-stage infections. In higher transmission settings, the accuracy of this approach diminishes substantially. We recommend using P. vivax SEMs in low transmission settings pursuing malaria elimination, but they are likely to be less effective in high transmission settings focused on malaria control.
Plasmodium vivax is the most widespread causative agent of human malaria in the world. Despite the ongoing implementation of malaria control programs, the rate of case reduction has declined over the ...last 5 years. Hence, surveillance of malaria transmission should be in place to identify and monitor areas that require intensified malaria control interventions. Serological tools may offer additional insights into transmission intensity over parasite and entomological measures, especially as transmission levels decline. Antibodies can be detected in the host system for months to even years after parasite infections have been cleared from the blood, enabling malaria exposure history to be captured. Because the Plasmodium parasite expresses more than 5000 proteins, it is important to a) understand antibody longevity following infection and b) measure antibodies to more than one antigen in order to accurately inform on the exposure and/or immune status of populations. This review summarises current practices for surveillance of P. vivax malaria, the current state of research into serological exposure markers and their potential role for accelerating malaria elimination, and discusses further studies that need to be undertaken to see such technology implemented in malaria-endemic areas.
Pregnant women have increased susceptibility to Plasmodium falciparum malaria and acquire protective antibodies over successive pregnancies. Most studies that investigated malaria antibody responses ...in pregnant women are from high transmission areas in sub-Saharan Africa, while reports from Latin America are scarce and inconsistent. The present study sought to explore the development of antibodies against P. falciparum and Plasmodium vivax antigens in pregnant women living in a low transmission area in the Brazilian Amazon.
In a prospective cohort study, plasma samples from 408 pregnant women (of whom 111 were infected with P. falciparum, 96 had infections with P. falciparum and P. vivax, and 201 had no Plasmodium infection) were used to measure antibody levels. Levels of IgG and opsonizing antibody to pregnancy-specific variant surface antigens (VSAs) on infected erythrocytes (IEs), 10 recombinant VAR2CSA Duffy binding like (DBL domains), 10 non-pregnancy-specific P. falciparum merozoite antigens, and 10 P. vivax antigens were measured by flow cytometry, ELISA, and multiplex assays. Antibody levels and seropositivity among the groups were compared.
Antibodies to VSAs on P. falciparum IEs were generally low but were higher in currently infected women and women with multiple P. falciparum episodes over pregnancy. Many women (21%-69%) had antibodies against each individual VAR2CSA DBL domain, and antibodies to DBLs correlated with each other (r ≥ 0.55, p < 0.0001), but not with antibody to VSA or history of infection. Infection with either malaria species was associated with higher seropositivity rate for antibodies against P. vivax proteins, adjusted odds ratios (95% CI) ranged from 5.6 (3.2, 9.7), p < 0.0001 for PVDBPII-Sal1 to 15.7 (8.3, 29.7), p < 0.0001 for PvTRAg_2.
Pregnant Brazilian women had low levels of antibodies to pregnancy-specific VSAs that increased with exposure. They frequently recognized both VAR2CSA DBL domains and P. vivax antigens, but only the latter varied with infection. Apparent antibody prevalence is highly dependent on the assay platform used.
Plasmodium vivax is the most geographically widespread of the malaria parasites causing human disease, yet it is comparatively understudied compared with Plasmodium falciparum. In this article we ...review what is known about naturally acquired immunity to P. vivax, and importantly, how this differs to that acquired against P. falciparum. Immunity to clinical P. vivax infection is acquired more quickly than to P. falciparum, and evidence suggests humans in endemic areas also have a greater capacity to mount a successful immunological memory response to this pathogen. Both of these factors give promise to the idea of a successful P. vivax vaccine. We review what is known about both the cellular and humoral immune response, including the role of cytokines, antibodies, immunoregulation, immune memory and immune dysfunction. Furthermore, we discuss where the future lies in terms of advancing our understanding of naturally acquired immunity to P. vivax, through the use of well-designed longitudinal epidemiological studies and modern tools available to immunologists.
Over the last decades, the number of malaria cases has drastically reduced in Cambodia. As the overall prevalence of malaria in Cambodia declines, residual malaria transmission becomes increasingly ...fragmented over smaller remote regions. The aim of this study was to get an insight into the burden and epidemiological parameters of Plasmodium infections on the forest-fringe of Cambodia.
950 participants were recruited in the province of Mondulkiri in Cambodia and followed up from 2018 to 2020. Whole-blood samples were processed for Plasmodium spp. identification by PCR as well as for a serological immunoassay. A risk factor analysis was conducted for Plasmodium vivax PCR-detected infections throughout the study, and for P. vivax seropositivity at baseline. To evaluate the predictive effect of seropositivity at baseline on subsequent PCR-positivity, an analysis of P. vivax infection-free survival time stratified by serological status at baseline was performed.
Living inside the forest significantly increased the odds of P. vivax PCR-positivity by a factor of 18.3 (95% C.I. 7.7-43.5). Being a male adult was also a significant predictor of PCR-positivity. Similar risk profiles were identified for P. vivax seropositivity. The survival analysis showed that serological status at baseline significantly correlated with subsequent infection. Serology is most informative outside of the forest, where 94.0% (95% C.I. 90.7-97.4%) of seronegative individuals survived infection-free, compared to 32.4% (95% C.I.: 22.6-46.6%) of seropositive individuals.
This study justifies the need for serological diagnostic assays to target interventions in this region, particularly in demographic groups where a lot of risk heterogeneity persists, such as outside of the forest.
A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study ...developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.
The utilisation of serological surveillance methods for malaria has the potential to identify individuals exposed to
, including asymptomatic carriers. However, the application of serosurveillance ...varies globally, including variations in methodology and transmission context. No systematic review exists describing the advantages and disadvantages of utilising serosurveillance in various settings. Collation and comparison of these results is a necessary first step to standardise and validate the use of serology for the surveillance of
in specific transmission contexts. A scoping review was performed of
serosurveillance applications globally. Ninety-four studies were found that met predefined inclusion and exclusion criteria. These studies were examined to determine the advantages and disadvantages of serosurveillance experienced in each study. If studies reported seroprevalence results, this information was also captured. Measurement of antibodies serves as a proxy by which individuals exposed to
may be indirectly identified, including those with asymptomatic infections, which may be missed by other technologies. Other thematic advantages identified included the ease and simplicity of serological assays compared to both microscopy and molecular diagnostics. Seroprevalence rates varied widely from 0-93%. Methodologies must be validated across various transmission contexts to ensure the applicability and comparability of results. Other thematic disadvantages identified included challenges with species cross-reactivity and determining changes in transmission patterns in both the short- and long-term. Serosurveillance requires further refinement to be fully realised as an actionable tool. Some work has begun in this area, but more is required.
To make progress towards malaria elimination, a highly effective vaccine targeting Plasmodium vivax is urgently needed. Evaluating the kinetics of natural antibody responses to vaccine candidate ...antigens after acute vivax malaria can inform the design of serological markers of exposure and vaccines.
The responses of IgG antibodies to 9 P. vivax vaccine candidate antigens were evaluated in longitudinal serum samples from Brazilian individuals collected at the time of acute vivax malaria and 30, 60, and 180 days afterwards. Antigen-specific IgG correlations, seroprevalence, and half-lives were determined for each antigen using the longitudinal data. Antibody reactivities against Pv41 and PVX_081550 strongly correlated with each other at each of the four time points. The analysis identified robust responses in terms of magnitude and seroprevalence against Pv41 and PvGAMA at 30 and 60 days. Among the 8 P. vivax antigens demonstrating >50% seropositivity across all individuals, antibodies specific to PVX_081550 had the longest half-life (100 days; 95% CI, 83-130 days), followed by PvRBP2b (91 days; 95% CI, 76-110 days) and Pv12 (82 days; 95% CI, 64-110 days).
This study provides an in-depth assessment of the kinetics of antibody responses to key vaccine candidate antigens in Brazilians with acute vivax malaria. Follow-up studies are needed to determine whether the longer-lived antibody responses induced by natural infection are effective in controlling blood-stage infection and mediating clinical protection.
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