Quantification of malaria antigens PfHRP2 and pLDH by quantitative suspension array technology in whole blood, dried blood spot and plasma.

BACKGROUND: Malaria diagnostics by rapid diagnostic test (RDT) relies primarily on the qualitative detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) and Plasmodium spp lactate dehydrogenase (pLDH). As novel RDTs with increased sensitivity are being developed and implemented as point of care diagnostics, highly sensitive laboratory-based assays are needed for evaluating RDT performance. Here, a quantitative suspension array technology (qSAT) was developed, validated and applied for the simultaneous detection of PfHRP2 and pLDH in a variety of biological samples (whole blood, plasma and dried blood spots) from individuals living in different endemic countries. RESULTS: The qSAT was specific for the target antigens, with analytical ranges of 6.8 to 762.8 pg/ml for PfHRP2 and 78.1 to 17076.6 pg/ml for P. falciparum LDH (Pf-LDH). The assay detected Plasmodium vivax LDH (Pv-LDH) at a lower sensitivity than Pf-LDH (analytical range of 1093.20 to 187288.5 pg/ml). Both PfHRP2 and pLDH levels determined using the qSAT showed to positively correlate with parasite densities determined by quantitative PCR (Spearman r = 0.59 and 0.75, respectively) as well as microscopy (Spearman r = 0.40 and 0.75, respectively), suggesting the assay to be a good predictor of parasite density. CONCLUSION: This immunoassay can be used as a reference test for the detection and quantification of PfHRP2 and pLDH, and could serve for external validation of RDT performance, to determine antigen persistence after parasite clearance, as well as a complementary tool to assess malaria burden in endemic settings.

Multiplexing detection of IgG against Plasmodium falciparum pregnancy-specific antigens

Background: Pregnant women exposed to Plasmodium falciparum generate antibodies against VAR2CSA, the parasite protein that mediates adhesion of infected erythrocytes to the placenta. There is a need of high-throughput tools to determine the fine specificity of these antibodies that can be used to identify immune correlates of protection and exposure. Here we aimed at developing a multiplex-immunoassay to detect antibodies against VAR2CSA antigens. Methods and findings: We constructed two multiplex-bead arrays, one composed of 3 VAR2CSA recombinant-domains (DBL3X, DBL5Ɛ and DBL6Ɛ) and another composed of 46 new peptides covering VAR2CSA conserved and semi-conserved regions. IgG reactivity was similar in multiplexed and singleplexed determinations (Pearson correlation, protein array: R2 = 0.99 and peptide array: R2 = 0.87). IgG recognition of 25 out of 46 peptides and all recombinant-domains was higher in pregnant Mozambican women (n = 106) than in Mozambican men (n = 102) and Spanish individuals (n = 101; p<0.05). Agreement of IgG levels detected in cryopreserved plasma and in elutions from dried blood spots was good after exclusion of inappropriate filter papers. Under heterogeneous levels of exposure to malaria, similar seropositivity cutoffs were obtained using finite mixture models applied to antibodies measured on pregnant Mozambican women and average of antibodies measured on pregnant Spanish women never exposed to malaria. The application of the multiplex-bead array developed here, allowed the assessment of higher IgG levels and seroprevalences against VAR2CSA-derived antigens in women pregnant during 2003-2005 than during 2010-2012, in accordance with the levels of malaria transmission reported for these years in Mozambique.

Changing Trends in P. falciparum Burden, Immunity, and Disease in Pregnancy

Background: Prevention of reinfection and resurgence is an integral component of the goal to eradicate malaria. However, the adverse effects of malaria resurgences are not known. Methods: We assessed the prevalence of Plasmodium falciparum infection among 1819 Mozambican women who delivered infants between 2003 and 2012. We used microscopic and histologic examination and a quantitative polymerase-chain-reaction (qPCR) assay, as well as flow-cytometric analysis of IgG antibody responses against two parasite lines. Results: Positive qPCR tests for P. falciparum decreased from 33% in 2003 to 2% in 2010 and increased to 6% in 2012, with antimalarial IgG antibody responses mirroring these trends. Parasite densities in peripheral blood on qPCR assay were higher in 2010-2012 (geometric mean [±SD], 409±1569 genomes per microliter) than in 2003-2005 (44±169 genomes per microliter, P=0.02), as were parasite densities in placental blood on histologic assessment (50±39% of infected erythrocytes vs. 4±6%, P<0.001). The malaria-associated reduction in maternal hemoglobin levels was larger in 2010-2012 (10.1±1.8 g per deciliter in infected women vs. 10.9±1.7 g per deciliter in uninfected women; mean difference, -0.82 g per deciliter; 95% confidence interval [CI], -1.39 to -0.25) than in 2003-2005 (10.5±1.1 g per deciliter vs. 10.6±1.5 g per deciliter; difference, -0.12 g per deciliter; 95% CI, -0.67 to 0.43), as was the reduction in birth weight (2863±440 g in women with past or chronic infections vs. 3070±482 g in uninfected women in 2010-2012; mean difference, -164.5 g; 95% CI, -289.7 to -39.4; and 2994±487 g vs. 3117±455 g in 2003-2005; difference, -44.8 g; 95% CI, -139.1 to 49.5). Conclusions: Antimalarial antibodies were reduced and the adverse consequences of P. falciparum infections were increased in pregnant women after 5 years of a decline in the prevalence of malaria. (Funded by Malaria Eradication Scientific Alliance and others).