Effect of HIV on the anti-CS2_VSA antibody levels in the ‘No Malaria’ histopathology group.

<p>In a univariate analysis of women that showed no evidence of either peripheral or placental malaria infection, using Mann-Whitney ranksum tests, phagocytic antibodies (A) are shown to be significantly decreased by HIV infection (z = −2.17, <i>p</i> = 0.03). On the contrary total IgG to CS2_VSA (B) shows a non-significant decrease in the HIV-positive group (z = −1.35, <i>p</i> = 0.179. Box, median and interquartile range; whiskers, lowest and highest values. P values are shown.</p

Associations of antibodies measured with HIV and infant birth weight.

<p>A) Values for both phagocytic antibodies (circles) and total IgG to CS2_VSA (squares) were plotted according to the women's’ HIV status (HIV negative – full symbols; HIV positive – open symbols). Mann-Whitney rank sum tests were performed on HIV positive vs. negative women. *p = 0.010; n.s. ( p = 0.078). Univariate linear regression of total IgG to CS2_VSA (B) and phagocytic antibodies (C) and infant birth weight. Both p-values are plotted. Only phagocytic antibodies correlated with infant birth weight (r² = 0.025, p = 0.034).</p

Characteristics of the cohort and associations with HIV and placental malaria.

<p>The data represents numbers of patients (percentages of total for each parameter) and means [standard deviations].</p>1<p>Maternal haemoglobin levels.</p>2<p>Birth weight of babies at delivery.</p>3<p>DOUBLE NEGATIVES are women with neither HIV nor malaria.</p>4<p>DOUBLE POSITIVES are women with both HIV and malaria infection. Variables are normally distributed, so Student's t-tests were applied.</p

The placental parameters evaluated by <i>Plasmodium</i> species during infection.

<p>For all placentas, areas of necrosis (B) and intervillous space (C) were measured by overlaying a square grid (A) and counting the number of intersecting points that touched necrotic areas (yellow dots; the white circle indicates an example) or intervillous space areas (blue dots; the black circle indicates an example). The ratios of intervillous space area per necrosis (D) and intervillous space area per placental barrier thickness (E) were calculated. The placentas in the “no plasmodium” group (n = 41; white boxes) appear to have similar necrotic areas and more intervillous space than the placentas in the “P. vivax” group (n = 59; red boxes). The placentas in the “P. falciparum” group (n = 19; grey boxes) exhibited more necrotic areas and less intervillous space. Graphs (B, C, D and E) represent the transformed data. The boxes represent the mean and standard deviation values. The whiskers represent the 5^th and 95^th percentiles. The photograph was taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. Grid overlays and counts were performed using Image J.</p

Results and univariate analysis of the placental parameters, evaluated by histology, according to the species of <i>Plasmodium</i> infection during pregnancy^†.

†<p>Women were grouped according to their malaria diagnoses during pregnancy, based on microscopy data. (IQ) 25^th and 75^th percentile. (n) number of women. (%) percentage (N/A) not applicable. For differences between groups all continuous variables were Ln-transformed and one-way ANOVA tests with Bonferroni post-tests were performed. Chi² tests were used to evaluate differences in categorical variables between groups. Refer to the “methods section” for a full description of how each parameter was measured.</p>*<p><i>P. falciparum</i> vs. No Malaria, P = 0.004.</p>§<p><i>P. falciparum</i> vs. <i>P. vivax</i>, P = 0.005.</p>°<p><i>P. falciparum</i> vs. No Malaria, P = 0.048.</p>#<p>χ² test: <i>P.</i> vivax or <i>P.</i> falciparum, P<0.001.</p

The placental score differentiates the women who were exposed to <i>P. vivax</i> during pregnancy.

<p>A score (termed the ‘vivax-score’) was developed and applied to all of the placental samples in this study (see main text for details). (A) The placental samples from the “no Plasmodium” group (n = 41, white box) revealed a significantly lower score than the placentas from the “P. vivax” group (n = 59, red box) (* Mann-Whitney, P = 0.027). (B) The vivax-score increased significantly (** Cuzick's trend test: z = 2.76, P = 0.006) with increased exposure to <i>P. vivax</i> during pregnancy. “No infection”, n = 41; “1 infection”, n = 39; “2+ infections”, n = 20).</p

Stains and scoring system used to quantify malaria-associated placental parameter.

<p>Stains and scoring system used to quantify malaria-associated placental parameter.</p

The syncytial parameters evaluated by <i>Plasmodium</i> species during infection.

<p>Syncytial knotting (A and B) and syncytial rupture (C and D) were evaluated on H&E-stained slides at 100× magnification. Placental barrier thickness (E and F) was evaluated on Masson's trichrome-stained slides at 1000× magnification after overlaying horizontal lines with 5 µm of interspace (see Methods and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002071#pntd-0002071-t001" target="_blank">Table 1</a>). For all parameters, placentas from the “no plasmodium” group (n = 41; white boxes) had the lowest values, followed by placentas from the “P. vivax” (n = 59; red boxes) and “P. falciparum” (n = 19; grey boxes) groups. Graphs (B, D and F) represent the transformed data. * ANOVA test, P-value≤0,006. The boxes represent the mean and standard deviation values. The whiskers represent the 5^th and 95^th percentiles. Photographs were taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. The grid overlays and counts were conducted using Image J. Arrow heads on A, C and D point to syncytial knots, syncytial rupture and an example of a thickness measurement, respectively.</p

The immune-cell parameters evaluated by <i>Plasmodium</i> species during infection.

<p>The percentage of immune cells present in the intervillous space of the placentas evaluated (A) was calculated after counting a total of 500 intervillous space cells. Total leucocytes percentage (B), mononuclear cells percentage (C) and polymorphonuclear cells percentage (D) were plotted against <i>Plasmodium</i> exposure during pregnancy, assessed by microscopy. The placentas from the “no plasmodium” group (n = 41; white boxes) appear to have less immune cells present in the intervillous space than the placentas from the “P. vivax” group (n = 59; red boxes) and the placentas from the “P. falciparum” group (n = 19; grey boxes). * ANOVA test, P-value = 0,039. Graphs (B, C, and D) represent the transformed data. The boxes represent the mean and standard deviation values. The whiskers represent the 5^th and 95^th percentiles. The photograph was taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. Grid overlays and counts were performed using Image J.</p

Cuzick's trend test analysis of placental changes across ordered groups by number of <i>P. vivax</i> infections during pregnancy^†.

†<p>Women were divided according to the number of <i>P. vivax</i> infections that were diagnosed microscopically during pregnancy.</p>a<p>Cuzick's trend test was performed across the ordered groups: no <i>P. vivax</i> infection (0), one <i>P. vivax</i> infection (1) and two or more <i>P. vivax</i> infections (2+). (z) Measure and direction of tendency.</p