MOESM1 of Accuracy of an HRP-2/panLDH rapid diagnostic test to detect peripheral and placental Plasmodium falciparum infection in Papua New Guinean women with anaemia or suspected malaria

Additional file 1: Table S1. Negative qPCR and LM results amongst HRP2-band positive RDT screening episodes

The effect of pooled serum from uninfected and <i>P. falciparum</i> infected women at enrolment on HTR8/SVneo invasion index over 48 h.

<p>Invasion index indicates the proportion of cells invaded through the Matrigel™, normalized to baseline invasion of trophoblast cells treated with complete media (untreated group). Serum from infected women inhibited median (IQR) invasion by 65% (Mann Whitney-U test P = .1) compared to serum from uninfected women, and by 37% compared with complete media treatment (P = .06). The malaria-specific reduction in invasion was comparable to that of 10 ng/ml TGFβ (positive control for invasion inhibition), which inhibited invasion by 31% (Mann Whitney U-test, P = .06). Normal uninfected pregnancy serum enhanced trophoblast invasion by 80% (P = .06) compared with complete media alone. Both viability and invasion assays were repeated three times in triplicate. Data shown are expressed as median and (IQR) of the mean of each of three independent experiments, repeated in triplicate.</p

Concentration of invasion- modulating factors in pooled serum from uninfected and <i>P. falciparum</i> infected women at enrolment.

<p>Note: Mean concentrations (and SD) of invasion stimulatory factors (IGF-I, IGF-II, and IL-8) were lower in pooled serum from women with <i>P. falciparum</i> infection compared to match uninfected controls. In the same pooled serum samples, factors that inhibit trophoblast invasion (hCG, IL-10) were higher in infected women than in uninfected women. IGF- Insulin like growth factor, IL- interleukin, TNF tumor necrosis factor, hCG human chorionic gonadotrophin. (+) indicates known previously reported trophoblast invasion-promoting effects <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Forbes1" target="_blank">[46]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Jovanovic1" target="_blank">[47]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Lockwood1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Huber1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Yagel1" target="_blank">[50]</a>, known trophoblast invasion inhibitory effects are indicated by (−) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Lockwood1" target="_blank">[48]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Huber1" target="_blank">[49]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Roth1" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Yagel2" target="_blank">[52]</a>, reports of both inhibitory and promoting effects are indicated by (+/−) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone.0055269-Jovanovic2" target="_blank">[53]</a>, and (unreported) indicates an absence of previous reports on trophoblast invasion. All analyte concentrations in pooled serum were measured once and are expressed as pg/ml, except hCG (IU/ml). IGF-I, IGF-II and hCG were measured in triplicate (analyzed using the Mann-Whitney test), and all cytokines were measured in duplicate (analyzed using unpaired t test with Welch’s correction).</p

Plasma collected from <i>Plasmodium falciparum</i> infected women in early pregnancy inhibits Swan cell migration at 6 h (A) and 24 h (B).

<p>Median (IQR) percent increase in migration (relative to 0 h) of Swan 71 cells was measured over 24 h in response to control treatments (10% fetal bovine serum [FBS] in serum free medium, untreated [SFM], and 500 ng/ml LPS in SFM, open bars) or with 10% plasma from individuals (black bars) that were malaria-uninfected (n = 13), <i>P. vivax</i>-infected (n = 9) or <i>P. falciparum-</i>infected (n = 13), over 4 independent experiments. (A) At 6 h, plasma collected from women with <i>P. falciparum</i> infection inhibited migration compared with plasma from uninfected women (*P = .01). Treatment with LPS significantly inhibited migration (*P = .03) compared with 10% FBS treatment. There was no difference between uninfected and <i>P. vivax</i> plasma treatments (P = .35). (B) At 24 h, compared with cells treated with uninfected plasma, plasma collected from women with <i>P. falciparum</i> infection significantly reduced migration (**P = .004), while migration with plasma from <i>P. vivax</i> infection was unchanged (P = .64). LPS significantly inhibited migration compared to cells treated with 10% FBS (*P = .03).</p

Migration of Swan cells was determined over time using Wimasis software.

<p>Swan 71 trophoblast cells were plated, and scratched 12 h later. At the time of treatment (0 h) with either 10% FBS, serum free media (SFM, not shown), or LPS 500 ng/ml in SFM, or with 10% plasma in SFM (not shown); three independent images were taken at 10x magnification. The same frames were imaged subsequently at 6 and 24 h following treatment. Images were up-loaded to <a href="http://www.ibidi.com" target="_blank">www.ibidi.com</a>, and the percent cell coverage (green) compared to the scratched area (black) was determined in each frame using WIMASIS analysis software. The percent increase in migration over time (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone-0055269-g003" target="_blank">Figure 3</a> A and B) was determined by subtracting cell coverage at 0 h from subsequent time points.</p

<i>Plasmodium falciparum</i> infection status does not affect trophoblast viability.

<p>(A) Mean and SD fold change in HTR8/SVneo viability relative to that of cells treated with complete media (open bars) was measured by percent reduction in AB dye after 4 h, following incubation with serum treatments (black bars) for 48 h. Compared with complete medium, complete medium supplemented with 10% volume pooled serum from women with or without infection had no effect on trophoblast viability (ANOVA P = .4). Treatment with SRM (open bar, positive control for a negative effect on trophoblast viability) reduced cell viability compared to trophoblasts treated with complete media (Mann Whitney test P = .06). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055269#pone-0055269-g004" target="_blank">Figure 4A</a> indicates the mean and SD of each of three independent experiments, repeated in triplicate. (B) Median (IQR) fold change in viability of Swan 71 following treatment with plasma from individual women in early pregnancy. Swan 71 cells were treated once in triplicate for 24 h with either complete media (open bar), or serum free media supplemented with 10% plasma from women with (n = 13) and without (n = 11) <i>P. falciparum</i> infection in early pregnancy (black bars). Following treatment, Swan 71 cells were incubated with AB and cell metabolism (as a proxy for viability) was normalized to that of cells treated with complete media. There was no difference in viability between plasma treatments with infection (P = .4), but cells cultured in complete media had relatively higher measure of viability than those treated with 10% plasma (*P = .01 in both cases).</p

Parasite density negatively influences Swan 71 cell migration.

<p>Parasite densities from all <i>P. falciparum and P.vivax</i> infected participants (n = 22) were log transformed, and correlated with respective Swan 71 migration data at 24 h post-treatment. There was a significant negative association between density of infection in early pregnancy with migration (P = .02). No other clinical parameters were found to correlate with Swan 71 cell migration in response to plasma treatment.</p

<i>In vitro</i> production of IFN-γ by peripheral blood mononuclear cells (PBMC) in response to VIR peptide stimulation.

<p>Intracellular production of IFN-γ by PBMCs was analyzed by flow cytometry. Box plots of the percentages of IFN-γ-producing cells from total CD4⁺ (A) or CD8⁺ (B) T cells are shown after PBMCs were cultured in the presence of PvLP1, PvLP2 or medium alone. In C), IFN-γ-producing cells from total CD4⁺ or CD8⁺ T cells are shown after culture in the presence of ant-CD3. IFN-γ production was compared between <i>P</i>. <i>vivax</i> infected (I, N=28) and uninfected (U, N=18) pregnant women; p corresponds to Mann-Whitney test. Median (middle line in white), and 25^th and 75^th percentiles (lower and upper hinge, respectively) are represented in the box. Outside values are excluded.</p

Correlation of IgG antibody responses to VIR antigens at recruitment.

<p>Correlation of IgG antibody responses to VIR antigens at recruitment.</p

Antibody responses to VIR antigens.

<p>Human IgG antibodies against VIR proteins and peptides were detected by Luminex in peripheral blood at different timepoints: A) recruitment (first antenatal visit) (top panel), B) delivery (middle panel) and C) postpartum (after puerperium) (lower panel). Antibody levels are represented as median fluorescence intensity (MFI). Reactivity (MFI) against GST was subtracted from MFI values obtained against individual recombinant proteins. Median (white line), and 25^th and 75^th percentiles (lower and upper hinge respectively) are represented as boxes. Outside values are not displayed in the graph. Numbers below boxes represent percentage of positive responses, calculated as number of plasmas samples with MFI values above the mean plus 3 standard deviations of negative controls (cutoff). Cutoff for India samples was calculated with negative samples analyzed at this site and therefore differed from those used for the other sites. p corresponds to one-way ANOVA plus Bonferroni pairwise correction. Differences only displayed versus PNG. *p<0.05, **p<0.01, ***p<0.001 (adjusted p-values multiple comparisons). Sample size for each country, timepoint and antibody is provided in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005009#pntd.0005009.s006" target="_blank">S4 Table</a>. BR: Brazil; CO: Colombia; GT: Guatemala; IN: India; PNG: Papua New Guinea.</p