Optimization of a magnetic bead-based assay (MAGPIX®-Luminex) for immune surveillance of exposure to malaria using multiple Plasmodium antigens and sera from different endemic settings.

BACKGROUND: Serological markers are potentially useful tools for monitoring the progress of malaria control programs, but a better understanding of antibody response dynamics is necessary. The use of a magnetic bead-based immunoassay (MBA) is advantageous compared to ELISA, due to its multiplexing capacity, but limited information is available on the standardization and validation of this assay. METHODS: Several parameters for multiplex testing of antibodies to Plasmodium antigens were analysed using a set of 4 antigens and 98 sera from Senegalese rural asymptomatic and urban symptomatic individuals. The 4 antigens included Plasmodium falciparum CSP and PfAMA1 peptides, recombinant P. falciparum MSP4p20 and a Plasmodium malariae CSP (PmCSP) peptide. Comparisons with ELISA were done using MSP4p20 and whole schizont extract (SE) antigens. RESULTS: The use of fewer beads (1000 beads per well instead of 2000) and 5 µg of antigen per 106 bead were validated as lower amounts. The use of a carrier protein (BSA) was shown to be critical when using peptides and the effect of a 24 h delayed measures was evaluated (5-25% signal decrease). Analysis of Ab responses showed almost equally high levels and prevalence in all transmission settings. Clear distinctions between rural and urban malaria were noted using PmCSP and SE antigens. CONCLUSIONS: This study underlines the importance of further optimization of the MBA technique and highlights the interest of using multistage/multispecies antigens for surveillance of malaria in endemic settings.

Antibodies to Plasmodium falciparum merozoite surface protein-1p19 malaria vaccine candidate induce antibody-dependent respiratory burst in human neutrophils.

BACKGROUND: Identification of plasmodial antigens targeted by protective immune mechanisms is important for malaria vaccine development. Among functional assays, the neutrophil antibody-dependent respiratory burst (ADRB) induced by opsonized Plasmodium falciparum merozoites has been correlated with acquired immunity to clinical malaria in endemic areas, but the target merozoite antigens are unknown. Here, the contribution of antibodies to the conserved C-terminal domain of the P. falciparum merozoite surface protein-1 (PfMSP1p19) in mediating ADRB was investigated in sera from individuals living in two Senegalese villages with differing malaria endemicity. METHODS: Anti-PfMSP1p19 antibody levels in sera from 233 villagers were investigated and the involvement of anti-PfMSP1p19 antibodies in ADRB was explored in a subset of samples using (1) isogenic P. falciparum parasite clones expressing P. falciparum or Plasmodium chabaudi MSP1p19; (2) PfMSP1p19-coated plaque ADRB; and, (3) ADRB triggering using sera depleted from PfMSP1p19 antibodies by absorption onto the baculovirus recombinant antigen. RESULTS: ADRB activity correlated with anti-PfMSP1p19 IgG levels (P < 10(-3)). A substantial contribution of PfMSP1p19 antibody responses to ADRB was confirmed (P < 10(-4)) in an age-adjusted linear regression model. PfMSP1p19 antibodies accounted for 33.1 % (range 7-54 %) and 33.2 % (range 0-70 %) of ADRB activity evaluated using isogenic merozoites (P < 10(-3)) and depleted sera (P = 0.0017), respectively. Coating of PfMSP1p19 on plates induced strong ADRB in anti-PfMSP1p19-positive sera. CONCLUSION: These data show that naturally acquired P. falciparum MSP1p19 antibodies are potent inducers of neutrophil ADRB and support the development of PfMSP1p19-based malaria vaccine using ADRB assay as a functional surrogate for protection.

Comparative analysis of IgG responses to Plasmodium falciparum MSP1p19 and PF13-DBL1α1 using ELISA and a magnetic bead-based duplex assay (MAGPIX®-Luminex) in a Senegalese meso-endemic community.

BACKGROUND: Numerous Plasmodium falciparum antigens elicit humoral responses in humans living in endemic areas. Use of multiplex assays is a convenient approach to monitor the antibody response against multiple antigens, but to integrate multiplex assay-derived data with datasets, generated previously using ELISA, comparative studies are needed. This work compares antibody responses to two P. falciparum antigens monitored using both technologies. METHODS: The IgG response against the merozoite surface protein-1 PfMSP1p19 and the PF13-DBL1α1 domain of the P. falciparum Erythrocyte Membrane Protein1, expressed by the rosette-forming parasite 3D7/PF13 (PF13), was investigated using ELISA and a MAGPIX®-Luminex duplex assay. Archived plasma samples collected before the rainy season from 217 villagers living in Ndiop, a Senegalese meso-endemic setting, were studied. ROC analysis was used to define the optimal antibody measure readout. Association of antibody levels with protection against clinical malaria was analysed using Poisson regression in a retrospective study from active case detection records performed during the 5.5-month transmission season that followed blood sampling. RESULTS: There was a strong positive correlation (P<10(-3)) between ELISA and MAGPIX®-Luminex-MFI (median fluorescence intensity) values for antibody to PfMSP1p19 (rho=0.78) and PF13-DBL1α1 (rho=0.89), with a similar degree of concordance in all age groups. Antibody levels to both antigens were high but displayed a different age-associated pattern. Independent age-adjusted Poisson regression analysis showed a significant association with protection only for IgG responses to MSP1p19 (P<0.01 RR=0.71 [0.53-0.93]) measured by ELISA. CONCLUSION: The individual ELISA and duplex-MAGPIX assays provide a concordant evaluation of age-associated antibody responses to MSP1p19 and PF13-DBL1α1, irrespective of the formulation of antibody levels (values, ratios or ROC-adjusted figures) but do diverge with regard to the association of antibody levels with clinical protection in age-adjusted models. This may reflect incomplete overlap of the epitopes presented in the two formats. Further development for multiplex assessment of antibody responses to a larger panel of antigens with the robust and cost effective MAGPIX®-Luminex technology is warranted.

WHO guidance on COVID-19 vaccine trial designs in the context of authorized COVID-19 vaccines and expanding global access: Ethical considerations.

While the degree of COVID-19 vaccine accessibility and uptake varies at both national and global levels, increasing vaccination coverage raises questions regarding the standard of prevention that ought to apply to different settings where COVID-19 vaccine trials are hosted. A WHO Expert Group has developed guidance on the ethical implications of conducting placebo-controlled trials in the context of expanding global COVID-19 vaccine coverage. The guidance also considers alternative trial designs to placebo controlled trials in the context of prototype vaccines, modified vaccines, and next generation vaccines.

Use of HRP-2-based rapid diagnostic test for Plasmodium falciparum malaria: assessing accuracy and cost-effectiveness in the villages of Dielmo and Ndiop, Senegal.

BACKGROUND: In 2006, the Senegalese National Malaria Control Programme (NMCP) has recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria and, in 2007, mandated testing for all suspected cases of malaria with a Plasmodium falciparum HRP-2-based rapid diagnostic test for malaria (RDT(Paracheck). Given the higher cost of ACT compared to earlier anti-malarials, the objectives of the present study were i) to study the accuracy of Paracheck compared to the thick blood smear (TBS) in two areas with different levels of malaria endemicity and ii) analyse the cost-effectiveness of the strategy of the parasitological confirmation of clinically suspected malaria cases management recommended by the NMCP. METHODS: A cross-sectional study was undertaken in the villages of Dielmo and Ndiop (Senegal) nested in a cohort study of about 800 inhabitants. For all the individuals consulting between October 2008 and January 2009 with a clinical diagnosis of malaria, a questionnaire was filled and finger-prick blood samples were taken both for microscopic examination and RDT. The estimated costs and cost-effectiveness analysis were made considering five scenarios, the recommendations of the NMCP being the reference scenario. In addition, a sensitivity analysis was performed assuming that all the RDT-positive patients and 50% of RDT-negative patients were treated with ACT. RESULTS: A total of 189 consultations for clinically suspected malaria occurred during the study period. The sensitivity, specificity, positive and negative predictive values were respectively 100%, 98.3%, 80.0% and 100%. The estimated cost of the reference scenario was close to 700 euros per 1000 episodes of illness, approximately twice as expensive as most of the other scenarios. Nevertheless, it appeared to us cost-effective while ensuring the diagnosis and the treatment of 100% of malaria attacks and an adequate management of 98.4% of episodes of illness. The present study also demonstrated that full compliance of health care providers with RDT results was required in order to avoid severe incremental costs. CONCLUSIONS: A rational use of ACT requires laboratory testing of all patients presenting with presumed malaria. Use of RDTs inevitably has incremental costs, but the strategy associating RDT use for all clinically suspected malaria and prescribing ACT only to patients tested positive is cost-effective in areas where microscopy is unavailable.

Assessment of the relative success of sporozoite inoculations in individuals exposed to moderate seasonal transmission.

BACKGROUND: The time necessary for malaria parasite to re-appear in the blood following treatment (re-infection time) is an indirect method for evaluating the immune defences operating against pre-erythrocytic and early erythrocytic malaria stages. Few longitudinal data are available in populations in whom malaria transmission level had also been measured. METHODS: One hundred and ten individuals from the village of Ndiop (Senegal), aged between one and 72 years, were cured of malaria by quinine (25 mg/day oral Quinimax in three equal daily doses, for seven days). Thereafter, thick blood films were examined to detect the reappearance of Plasmodium falciparum every week, for 11 weeks after treatment. Malaria transmission was simultaneously measured weekly by night collection of biting mosquitoes. RESULTS: Malaria transmission was on average 15.3 infective bites per person during the 77 days follow up. The median reappearance time for the whole study population was 46.8 days, whereas individuals would have received an average one infective bite every 5 days. At the end of the follow-up, after 77 days, 103 of the 110 individuals (93.6%; CI 95% [89.0-98.2]) had been re-infected with P. falciparum. The median reappearance time ('re-positivation') was longer in subjects with patent parasitaemia at enrolment than in parasitologically-negative individuals (58 days vs. 45.9; p = 0.03) and in adults > 30 years than in younger subjects (58.6 days vs. 42.7; p = 0.0002). In a multivariate Cox PH model controlling for the sickle cell trait, G6PD deficiency and the type of habitat, the presence of parasitaemia at enrolment and age >/= 30 years were independently predictive of a reduced risk of re-infection (PH = 0.5 [95% CI: 0.3-0.9] and 0.4; [95% CI: 0.2-0.6] respectively). CONCLUSION: Results indicate the existence of a substantial resistance to sporozoites inoculations, but which was ultimately overcome in almost every individual after 2 1/2 months of natural challenges. Such a study design and the results obtained suggest that, despite a small sample size, this approach can contribute to assess the impact of intervention methods, such as the efficacy vector-control measures or of malaria pre-erythrocytic stages vaccines.

Longitudinal analysis of antibody responses in symptomatic malaria cases do not mirror parasite transmission in peri-urban area of Cote d'Ivoire between 2010 and 2013.

BACKGROUND: In the agenda towards malaria eradication, assessment of both malaria exposure and efficacy of anti-vectorial and therapeutic strategies is a key component of management and the follow-up of field interventions. The simultaneous use of several antigens (Ags) as serological markers has the potential for accurate evaluation of malaria exposure. Here we aimed to measure the longitudinal evolution of the background levels of immunity in an urban setting in confirmed clinical cases of malaria. METHODS: A retrospective serological cross-sectional study on was carried out using 234 samples taken from 2010 to 2013 in peri-urban sentinel facility of Cote d'Ivoire. Antibody responses to recombinant proteins or BSA-peptides, 8 Plasmodium falciparum (PfAMA1, PfMSP4, PfMSP1, PfEMP1-DBL1α1-PF13, PfLSA1-41, PfLSA3-NR2, PfGLURP and PfCSP), one P. malariae (PmCSP) and one Anopheles gambiae salivary (gSG6-P1) antigens were measured using magnetic bead-based multiplex immunoassay (MBA). Total anti- P. falciparum IgG responses against schizont lysate from african 07/03 strain (adapted to culture) and 3D7 strain was measured by ELISA. RESULTS: High prevalence (7-93%) and levels of antibody responses to most of the antigens were evidenced. However, analysis showed only marginal decreasing trend of Ab responses from 2010 to 2013 that did not parallel the reduction of clinical malaria prevalence following the implementation of intervention in this area. There was a significant inverse correlation between Ab responses and parasitaemia (P<10-3, rho = 0.3). The particular recruitment of asymptomatic individuals in 2011 underlined a high background level of immunity almost equivalent to symptomatic patients, possibly obscuring observable yearly variations. CONCLUSION: The use of cross-sectional clinical malaria surveys and MBA can help to identify endemic sites where control measures have unequal impact providing relevant information about population immunity and possible decrease of transmission. However, when immunity is substantially boosted despite observable clinical decline, a larger cohort including asymptomatic recruitment is needed to monitor the impact of control measures on level of immunity.

Association of antibodies to Plasmodium falciparum merozoite surface protein-4 with protection against clinical malaria.

Identification of parasite antigens targeted by immune effector mechanisms that confer protection against malaria is important for the design of a multi-component malaria vaccine. Here, the association of antibodies reacting with the Plasmodium falciparum merozoite surface protein-4 (MSP4) with protection against clinical malaria was investigated in a Senegalese community living in an area of moderate, seasonal malaria transmission. Blood samples were collected at the end of an 8-month long dry season without any recorded parasite transmission from 206 residents enrolled in a prospective follow-up study. Active daily clinical monitoring was implemented during the subsequent five months. Entomologic monitoring documented parasite transmission during the first three months of follow-up. Serum IgG levels were determined by ELISA against three MSP4 baculovirus-encoded recombinant protein constructs, namely the full-length MSP4p40, MSP4p30 devoid of a highly polymorphic sequence stretch and the conserved C-terminal EGF-containing MSP4p20, as well as against a merozoite crude extract. Community seroprevalence against all three constructs was quite high, the lowest being against MSP4p30. Seroprevalence and antibody levels against the three MSP4 constructs were age-dependent. IgG1 dominated the anti-MSP4p20 responses, while both IgG1 and IgG3 were observed against MSP4p40. Anti-MSP4 antibodies were associated with the antibody-dependent respiratory burst (ADRB) activity in a functional assay of merozoite phagocytosis by polymorphonuclear cells. Importantly, high antibody levels against each of the three MSP4 constructs at the end of the dry season were associated with reduced morbidity during the subsequent transmission season in an age-adjusted Poisson regression model (IRR = 0.65 [0.50-0.83], P<0.001 for responses over the median values). These data are consistent with a protective role for the naturally acquired anti-MSP4 antibodies and support further development of MSP4 as a candidate component of malaria vaccine.

Serological signatures of declining exposure following intensification of integrated malaria control in two rural Senegalese communities.

Recent control scale-up has reduced malaria in many areas but new tools are needed to monitor further progress, including indicators of decreasing exposure to parasite infection. Although serology is considered a promising approach in this regard, the serological impact of control interventions has been so far studied using indirect quantification of exposure. Cohort surveys concomitantly recording entomological and malariometric indices have been conducted in two Senegalese settings where supervised control intensification implemented in 2006 shifted malaria from historically holoendemic in Dielmo and mesoendemic in Ndiop to hypoendemic in both settings by 2013. We analyse here serological signatures of declining transmission using archived blood samples. Responses against ten pre-erythrocytic and erythrocytic antigens from Plasmodium falciparum and P. malariae alongside an Anopheles gambiae salivary gland antigen were analysed. Cross-sectional surveys conducted before (2002) and after (2013) control intensification showed a major impact of control intensification in both settings. The age-associated prevalence, magnitude and breadth of the IgG responses to all antigens were village-specific in 2002. In 2013, remarkably similar patterns were observed in both villages, with marginal responses against all parasite antigens in the 0-5y children and reduced responses in all previously seropositive age groups. Waning of humoral responses of individuals who were immune at the time of control intensification was studied from 2006 to 2013 using yearly samplings. Longitudinal data were analysed using the Cochran-Armittage trend test and an age-related reversible catalytic conversion model. This showed that the antigen-specific antibody declines were more rapid in older children than adults. There was a strong association of antibody decline with the declining entomological inoculation rate. We thus identified serological markers of declining exposure to malaria parasites that should help future monitoring of progress towards malaria elimination.

Malaria transmission pattern in an area selected for clinical trials in the sudanian area of senegal (west Africa).

Malaria transmission pattern was studied in 3 villages (Toubanding, Daga Ndoup, and Keur Samba Guèye) situated within an area selected for clinical trials. The study was conducted in the rainy season from July to December 2011. The main objective of this work was to gather baseline data on malaria transmission intensity and other entomological parameters before the advent of clinical trials. Mosquitoes were collected by Human-Landing Collections (HLCs) and by pyrethrum spray catches (PSCs). Five anopheline species were collected, namely, An. arabiensis, An. gambiae, An. funestus, An. pharoensis, and An. rufipes, giving a heterogeneous distribution within the study area. The populations dynamics of the vectors varied temporarily in each village depending on the pattern of the rainy season. Transmission intensity estimated by the entomological inoculation rate (EIR) was measured in each of the three villages with the variations linked to the microecological differences between the villages. Measurements were calculated for August, September, and October and were found to vary between 4 and 30 infected bites per person over the study period with a peak intensity observed in September. These results indicate that epidemiological field trials on malaria could be conducted in this area on the basis of the differences observed with transmission intensity, micro-ecological variations, and the objectives of the trials.

National vitamin A supplementation coverage survey among 6-59 months old children in Guinea (West Africa).

BACKGROUND: Vitamin A deficiency (VAD) is a major contributor to child mortality in sub-Saharan Africa. Sustained control of VAD is essential to meet the Millennium Development Goal (MDG) for reduction of child mortality in Guinea. Since, twice yearly vitamin A supplementation (VAS), either coupled with National Immunization Days or stand-alone has been adopted as a key strategy to combat VAD in 6-59 months old children. OBJECTIVES: The objectives of this survey were to describe national VAS coverage rates and related factors affecting VAS coverage among 6-59 months old Guinean children. METHODS: In July 2003, a VAS coverage survey was implemented in Guinea. A cross-sectional random cluster survey was conducted to select Guinean children (n = 1950, 390 mother-child pairs per zone) aged 6-59 months. The country was divided into four agro-ecological zones with 30 clusters chosen per zone. Within each cluster, a random selection of 13 households with at least one child was carried out with random selection of one child per household. Data on characteristics of children, receipt of VAS, caregivers' socio-economic characteristics, vitamin A knowledge and practices of caregivers were collected by questionnaire. RESULTS: The national coverage rate of 68% is much lower than the official coverage rate of 93%. Middle Guinea, the region most affected by VAD, had the lowest coverage rate (58%). CONCLUSION: In order to increase overall VAS coverage and reduce regional disparities, it is suggested that mass VAS be organized on a regional level, prioritizing rural regions (Middle and Upper Guinea) and the city of Conakry.