Intestinal barrier disruption with Plasmodium falciparum infection in pregnancy and risk of preterm birth: a cohort study

Abstract

Background: Malaria in early pregnancy is a risk factor for preterm birth and is associated with sustained inflammation and dysregulated angiogenesis across gestation. This study investigated whether malaria is associated with increased gut leak and whether this contributes to systemic inflammation, altered angiogenesis, and preterm birth.

Methods: We quantified plasma concentrations of gut leak markers, soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) from 1339 HIV-negative pregnant Malawians at <24 weeks gestational age. We assessed the relationship of sCD14 and LBP concentrations with markers of inflammation, angiogenesis, and L-arginine bioavailability and compared them between participants with and without malaria, and with and without preterm birth.

Findings: Plasma concentrations of sCD14 and LBP were significantly higher in participants with malaria and were associated with parasite burden (p <0.0001, both analyses and analytes). The odds ratio for preterm birth associated with one log sCD14 was 2.67 (1.33 to 5.35, p = 0.006) and 1.63 (1.07-2.47, p = 0.023) for LBP. Both gut leak analytes were positively associated with increases in proinflammatory cytokines CRP, sTNFR2, IL18-BP, CHI3L1 and Angptl3 (p <0.05, all analytes) and sCD14 was significantly associated with angiogenic proteins Angpt-2, sENG and the sFLT:PlGF ratio (p <0.05, all analytes). sCD14 was negatively associated with L-arginine bioavailability (p <0.001).

Interpretation: Malaria in early pregnancy is associated with intestinal barrier dysfunction, which is linked to an increased risk of preterm birth.

Funding: Open Philanthropy, Canadian Institutes of Health Research, Canada Research Chair program, European and Developing Countries Clinical Trials Partnership, Bill & Melinda Gates Foundation.

Keywords: Gut leak; Inflammation; Intestinal barrier disruption; L-arginine, Plasmodium falciparum; LBP; Malaria in pregnancy; Preterm birth; sCD14.

Fig. 1

Participant and Study Flow Chart. The current study is a secondary analysis of a randomized controlled trial (RCT) cohort. This parent RCT enrolled 1873 participants, of which 1339 met the inclusion criteria for the current study. For the current study, the inclusion criteria were gestational age <24 weeks at enrolment, valid P. falciparum PCR result at enrolment, documented pregnancy outcome, and sufficient volume of stored plasma available for analysis. The parent RCT also included a randomly selected subset of 384 primigravid participants, from whom plasma concentrations of L-arginine and asymmetric dimethyl arginine were previously quantified. Of these, 331 met the inclusion criteria for the current study. RCT: randomized controlled trial. NO: nitric oxide.

Fig. 2

Markers of intestinal barrier dysfunction are associated with P. falciparum infection in early pregnancy. Plasma concentration distributions of sCD14 (left panel) and LBP (right panel), and malaria PCR and infection status were assessed from each participant at gestational age <24 weeks. CD14 and LBP were quantified from plasma using Luminex. P. falciparum PCR status is defined as either negative (yellow) or positive (pink). Comparisons made by Mann–Whitney U tests (A). P. falciparum infection status is defined as either uninfected (PCR negative; yellow), subpatent infection (PCR positive, RDT and microscopy negative; light pink), or patent infection (PCR positive, and RDT and/or microscopy positive; dark pink). Comparisons made by Kruskal–Wallis tests with Dunn's multiple comparisons tests (B). sCD14, soluble CD14; LBP, lipopolysaccharide binding protein; RDT, rapid diagnostic test.

Fig. 3

Markers of intestinal barrier dysfunction in early pregnancy are associated with preterm birth. (A) Plasma concentration distributions of sCD14 (left column) and LBP (right column) measured by luminex in pregnant participants at gestational age <24 weeks who delivered at term vs those who delivered preterm. Statistical analysis by Mann–Whitney U tests. sCD14: soluble CD14; LBP: lipopolysaccharide binding protein; IQR: interquartile range.

Fig. 4

sCD14 and LBP concentrations are associated with preterm birth. Unadjusted (black) and adjusted (pink) odds ratios (squares) with 95% confidence intervals from binary logistic regression models for the associations between log-transformed concentrations of sCD14 and LBP at enrolment and preterm birth. Adjusted models include enrolment log-transformed biomarker concentration, malaria PCR status, gestational age, maternal age, BMI, systolic blood pressure, socioeconomic status. Statistically significant models (p < 0.05) are bolded. sCD14: soluble CD14; LBP: lipopolysaccharide binding protein; OR: odds ratio; CI: confidence interval.

Fig. 5

Mediation analyses of P. falciparum infection on gestational age at delivery through markers of intestinal barrier dysfunction. Panel A + B: Path diagrams for mediation analyses. Regression coefficients a₁, b₁, and c’ with 95% confidence intervals for each step in the mediation analyses independently modelling the effect of log-transformed sCD14 concentrations (A) (n = 1286) and log-transformed LBP concentrations (B) (n = 1285) as mediators of the effect of P. falciparum infection status (PCR positivity) at enrolment on gestational age at delivery (outcome). Indirect (mediation) effects were calculated as the products of coefficients (a₁ x b₁) with bootstrapped confidence intervals. c’ represents the direct effect of P. falciparum infection at enrolment on gestational age at delivery after controlling for CD14 (A) or LBP (B) as mediators. Maternal age, gestational age at enrolment, body mass index, socioeconomic status, and systolic blood pressure at enrolment were included as covariates. n = 1285 ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 for regression coefficients a₁, b₁, and c’. Panel C: Percentage of the total effect of gut-leak marker concentration on gestational age at delivery is calculated as Indirect Effect/Total Effect x 100.